Poster Abstracts
Visualization of Protein Quality Control Processes at Aggregate Interfaces
Presenting author:
Institut für Biochemie und Pathobiochemie, Ruhr-Universität Bochum, Department of Molecular Cell Biology, Universitätsstrasse 150, 44801 Bochum [DE], verian.bader@rub.de
Author(s):
Verian Bader, Nikolas Furthmann, Jörg Tatzelt, Konstanze F. Winklhofer
Degradation of dysfunctional, damaged, or misfolded proteins is crucial for maintaining cellular proteostasis. Both proteasomal and autophagosomal/lysosomal degradation use the diversity of the ubiquitin code for selecting and targeting substrates. Our previous work revealed that linear ubiquitylation is involved in the quality control of misfolded proteins linked to neurodegenerative diseases. Linear ubiquitin chains are generated exclusively by the E3 ubiquitin ligase HOIP, part of the linear ubiquitin chain assembly complex (LUBAC). LUBAC has primarily been studied in immune signaling, NF-κB activation, and cell death regulation. In these contexts, NEMO is significant, acting both as an interactor of linear ubiquitin chains and a substrate of HOIP. Using structured-illumination microscopy and biochemical approaches, we discovered an NF-κB-independent function of LUBAC in clearing protein aggregates. HOIP is recruited to misfolded proteins by VCP/p97 and helps coat protein aggregates with ubiquitin. NEMO amplifies linear ubiquitylation and reshapes the aggregate surface by co-condensing with linear ubiquitin chains and the autophagy cargo receptor p62, facilitating autophagic degradation of protein aggregates. Supporting the physiological relevance, a patient with a NEMO variant defective in binding linear ubiquitin chains developed widespread lethal brain proteinopathy, including ⍺-synuclein, tau, and TDP-43 pathology.
__________Fast, long-time STED imaging of endoplasmic reticulum dynamics living cells
Presenting author:
Goethe - Universität Frankfurt, Institute of Physical and Theoretical Chemistry, Max-von-Laue str. 7, 60438 Frankfurt am Main [DE], balakrishnan@chemie.uni-frankfurt.de
Author(s):
Ashwin Balakrishnan, Johanna Rahm, Alexandra Kaminer, Mike Heilemann
Super-resolution microscopy techniques have been crucial in recent years in realizing structural cell biology a reality.However, biomolecules in a cell are highly dynamic and in order to assess these dynamics, two challenges need to be adressed: first, photobleaching limits the information that can be extracted from a cell. Second, phototoxicity leads to cell damage. Here, we demonstrate long-time STED microscopy with fast temporal resolution in living cells. We achieve this by employing very low irradiation intensities, and restore information by using convolutional neural network-based image denoising. We demonstrate live-cell imaging for hours with a temporal resolution of seconds. We use this approach to study the dynamics of the endoplasmic reticulum (ER) in healthy and autophagy-induced cells. By extending this further with a neural network-based segmentation, we quantify morphological rearrangements. We find that the ER is highly dynamic and visualize morphological changes under stress-induced conditions that have proved elusive before. In summary, we present an experimental approach that can be transferred to other live-cell super-resolution organelle dynamics.
__________Distinct types of intramitochondrial protein aggregates protect mitochondria against proteotoxic stress
Presenting author:
University of Kaiserslautern, RPTU, Cell Biology, Erwin-Schrödinger-Str. 13, 67663 Kaiserslautern [DE], lbertgen@rptu.de
Author(s):
Lea Bertgen, Johannes M. Herrmann, Markus Räschle, Jan-Eric Bökenkamp
Mitochondria consist of hundreds of proteins, most of which are long-lived and inaccessible to the proteasomal quality control system of the cytosol. How cells stabilize the mitochondrial proteome during challenging conditions is poorly understood. Here we show that mitochondria form spatially defined protein aggregates as a stress-protecting mechanism. Therefore, different types of intramitochondrial protein aggregates can be distinguished. The mitoribosomal protein Var1 (uS3m) undergoes a stress-induced transition from a soluble, chaperone-stabilized protein that is prevalent under benign conditions to an insoluble aggregated form upon acute stress. The formation of Var1 bodies leads to a transient change of available chaperones and the folding capacity of the matrix. The AAA chaperone Hsp78 is associated with a different type of intramitochondrial aggregates. These mitochondrial quality control compartments sequester matrix proteins to promote their subsequent folding and impede Pim1-mediated degradation. Our study shows that mitochondrial proteins actively control the formation of different types of intramitochondrial protein aggregates, which cooperate to stabilize the mitochondrial proteome during stress conditions.
__________High-resolution structures of the UapA elevator-type purine transporter rationalize the importance of the cytosolic N-terminus in trafficking and transport activity
Presenting author:
Uni Münster, Center for Sofr Nanoscience, Busso-Peus-Straße 10, 48149 Münster [DE], broutzak@uni-muenster.de
Author(s):
George Broutzakis, Yiannis Pyrris, Ifigeneia Akrani, Emmanuel Mikros, Alexander Neuhaus, George Diallinas, Christos Gatsogiannis
UapA is an elevator-type purine transporter from Aspergillus nidulans. Functional studies and determination of a 3.6Å inward-facing crystal structure, together with molecular dynamics (MD) and mutational analysis have led to speculative models of its mechanism of action and determination of substrate specificity. Here we report high-resolution full-length cryo-EM structures of UapA in new inward-facing apo- and substrate-loaded conformations at 2.05-3.60 Å in detergent and lipid nanodiscs. The structures explain functional data in an unprecedented level of detail, including the role of water molecules and lipids in ligand binding and dimerization. The cytoplasmic N-tail domain is involved in multiple dynamic interactions with both the core and dimerization domains, stabilizing the transporter in the inward-open conformation. This structural framework together with mutational analysis, in vivo functional studies and MD reveal how the N-tail may influence both the biogenesis and transport activity of UapA by wrapping the transporter in a stable conformation necessary for membrane trafficking, but also control the elevator movement of the transport domain to achieve substrate translocation once UapA has integrated into the plasma membrane. These findings provide important insights into how the evolution of specific functional elements in the cytosolic tails of eukaryotic transporters might regulate and link proper subcellular trafficking with transport catalysis.
__________A systematic bi-genomic split-GFP assay illuminates the mitochondrial matrix proteome and protein targeting routes
Presenting author:
RPTU, Cell Biology, Erwin-Schrödinger-Str. 13, 67633 Kaiserslautern [DE], yury.bykov@rptu.de
Author(s):
Yury Bykov
The majority of mitochondrial proteins are encoded in the nuclear genome and often lack clear targeting signals. Therefore, what constitutes the entire mitochondrial proteome is still unclear. We here build on our previously developed bi-genomic (BiG) split-GFP assay (Bader et al. 2020) to solidify the list of matrix and inner membrane mitochondrial proteins. The assay relies on one fragment (GFP1-10) encoded in the mitochondrial DNA enabling specific visualization of only the proteins tagged with a smaller fragment, GFP11, and localized to the mitochondrial matrix or the inner membrane. We used the SWAp-Tag (SWAT) strategy to tag every protein with GFP11 and mated them with the BiG GFP strain. Imaging the collection in six different conditions allowed us to visualize almost 400 mitochondrial proteins, 50 of which were never visualized in mitochondria before, and many are poorly studied dually localized proteins. We also show how this data can be applied to study mitochondrial inner membrane protein topology and sorting. This work brings us closer to finalizing the mitochondrial proteome and the freely distributed library of GFP11-tagged strains will be a useful resource to study protein localization, biogenesis and interactions.
__________Expansion microscopy and MALDI Imaging approaches to investigate inter-organelle communication
Presenting author:
University Hospital Düsseldorf, Heinrich Heine University Düsseldorf , Klinik für Herzchirurgie, Moorenstraße 5, 40225 Düsseldorf [DE], margret.buelow@hhu.de
Author(s):
Margret Bülow, Marie König
Organelle contact sites mediate the exchange of material, such as lipids, between cellular compartments. We focus on the impact of organelle contact sites on the function of neurons in vivo. We found that phospholipid transfer at ER-mitochondria contact sites drives H2O2 formation and is required for dopaminergic neuron function and thereby locomotion in Drosophila (Paradis et al., 2022). For further analysis of neuronal organelle contact sites, we employed Expansion Microscopy coupled to Airyscan confocal microscopy of peptidergic neurons. We identified an interaction of peroxisomes with the Golgi apparatus that plays an important role in the trafficking and secretion of dense-core vesicles. Peroxisome loss in mutants for peroxisome biogenesis factors leads to block of insulin-like peptide secretion upon a nutrient stimulus.
We described previously how peroxisome function affects the fatty acid profile (Bülow et al., 2018; Sellin et al; 2018) and how lipids are allocated by lipid receptor – lipoprotein interplay (Carrera et al., 2024). To investigate how peroxisome-Golgi interaction and loss of peroxisomes affect organismal lipid metabolism and distribution, we employed MALDI Imaging and found that peroxisome-Golgi interaction in peptidergic neurons affects the lipid composition of peripheral organs. Thereby we show how intracellular processes determine systemic physiology.
__________Imaging Beyond The Visible: advantages of the Shortwave-Infrared spectral range for confocal microscopy and Raman scattering imaging.
Presenting author:
NCT / DKFZ Heidelberg, Department of Functional Imaging in Surgical Oncology, Im Neuenheimer Feld 280, 69120 Heidelberg [DE], andriy.chmyrov@nct-dresden.de
Author(s):
Andriy Chmyrov, Bernardo Arus, Jakob Lingg, Alexander Bartelt, Tulio A. Valdez, Christopher J. Rowlands, Ellen Sletten, Olver Bruns
In the realm of biomedical imaging, the utilization of the short wave infrared (SWIR) range of the electromagnetic spectrum offers substantial advantages, including deeper tissue penetration and reduced autofluorescence. These properties significantly enhance the clarity and accuracy of biological imaging. Historically, access to SWIR detectors was restricted to ordinary users due to military regulations on dual-use technologies. However, this landscape has shifted a bit more than a decade ago, with SWIR cameras and detectors becoming deregulated. Major market players, such as Sony, have started producing InGaAs (Indium Gallium Arsenide) sensors at more affordable prices. This newfound accessibility paves the way for leveraging SWIR technology in bioimaging applications, including fluorescence microscopy and label-free methods. We will report about:
1) a SWIR line-scan one-photon confocal microscope that is capable of deep imaging of biological specimens, as demonstrated by visualization of labelled glomeruli in a fixed uncleared kidney at depths up to 400µm.
2) benefits of SWIR range for wide field spontaneous Raman imaging on a scale of a whole small animal. With fields of view surpassing 50 cm2, we showcase the versatility of SWIR Raman imaging by monitoring body composition dynamics in living mice, non-invasively detecting liver lipid content in metabolically challenged mice, and identifying calcified areas and lipid-rich deposits in human atherosclerotic plaques.
Imaging of membrane-bound Y5 RNA using (bi)RhoBAST Fluorescent Light Up Aptamers
Presenting author:
Heidelberg Universität, IPMB , INF 364, 69120 Heidelberg [DE], alempijevic@uni-heidelberg.de
Author(s):
Andrijana Crenner, Andres Jaeschke
The goal of this work is to develop a genetically encodable system for imaging of Y5 RNAs on membrane surface. For that, fluorescent light up aptamers (FLAPs) RhoBAST and biRhoBAST (Sunbul et al, 2021 and Buehler et al. 2023) previously developed in our group are cloned in different Tornado constructs (Litke and Jaffrey, 2019) allowing the overexpression and imaging of Y5-FLAP tagged constructs. The overexpressed constructs are further studied by Northern Blot to quantify and compare the transfected and non-transfected cells on one side, and to assess the formation of segmented, stress-associated Y5 RNA fragments (Y5sRNA) on the other (Hall, 2013). When it comes to imaging, the results are compared to FISH under different fixation conditions. The development and fine tuning of tools for Y5 RNA imaging is of great interest for exploring of other membrane bound RNAs. Furthermore, Y5 RNAs have recently been reported as highly enriched in newly discovered glycoRNAs (Flynn et al, 2021), consequently the tools developed for RNA imaging could be further functionalized for labelling and detecting of the glycosylated versus non glycosylated fraction of Y5 on cell membrane.
__________TRC40 serves as a redox-regulated chaperone and interacts with protein quality control systems to cope with oxidative unfolding stress
Presenting author:
University of Cologne, Institut für Biochemie, Zülpicher Str. 47, 50674 Köln [DE], bdempsey@uni-koeln.de
Author(s):
Bianca Dempsey, Kathrin Ulrich, Risai Dubrall
Exposure of cells to oxidative stress can cause a significant drop in intracellular ATP levels and promote protein unfolding and aggregation. The depletion of cell energy levels will reduce the activity of ATP-dependent chaperone systems and the proteasome, which turns protein mis- and unfolding into a serious challenge. It has been shown that cells activate under these conditions with ATP-independent chaperones to prevent irreversible protein aggregation. We discovered that the ATP-dependent targeting factor TRC40 becomes activated as an ATP-independent chaperone upon ATP-depleting, oxidative stress. Controlled by a highly conserved redox switch, TRC40 forms chaperone-active tetramers and high-molecular complexes, preventing unfolding protein aggregation. In HeLa cells expressing the Grx1-roGFP2 sensor, we observed that the recovery from hydrogen peroxide treatment is strongly reduced in TRC40-depleted cells, suggesting a crucial role of TRC40 in oxidative stress response. Under these conditions, TRC40 forms reversible cytosolic foci that colocalize with HSP70 and HSP105/110. Our results further show that TRC40 facilitates the clearance of mis- and unfolded proteins during stress recovery. Altogether, our work provides novel insights into how mammalian cells deal with proteotoxic stress and that TRC40 plays a crucial role in cell survival and proteostasis maintenance.
__________Why is mtDNA lost in cells lacking an active mitochondrial fusion machinery?
Presenting author:
Institute of Cell Biology, University of Tübingen, Molecular Cell Biology, Auf der Morgenstelle 15, 72076 Tübingen [DE], lisa.dengler@uni-tuebingen.de
Author(s):
Lisa Dengler, Francesco Padovani, Bianca Lemke, Boris Maček, Kurt Schmoller, Jennifer Ewald
Mitochondria constantly fuse and divide to maintain their health. Impairing the fusion machinery by deleting the yeast mitofusin Fzo1, causes fragmentation of mitochondria and loss of mtDNA. It is known that the morphological changes are primary, however it is unclear why mtDNA is lost in these cells. Here, we inducibly deplete Fzo1 to unravel how the phenotype develops and mtDNA is lost. We found that a knockout-like phenotype devoid of mtDNA is reached within 10 doublings. The mtDNA is lost proportionally with each cell division. Using live cell imaging we observed that during cell division mitochondrial mass is unequally distributed: while daughters obtain high concentration of mitochondria their mothers retain only a low concentration. Quantification of the mitochondrial localization of proteins whose import is differentially dependent on the mitochondrial membrane potential (MMP) revealed a large variation of the MMP within the population. Single cell analysis showed that cells with a low mitochondrial content exhibit a lower MMP and slower doubling times. We suggest that these cells with less mitochondrial mass are more susceptible to loss of mtDNA, which we are currently investigating by analysing a fluorescently tagged mitochondrial encoded protein.
__________Restoring ER morphology and lysosome dynamics as therapeutic targets for axonopathies
Presenting author:
Leibniz Institute on Aging - Fritz Lipmann Institute (FLI), , Beutenbergstraße 11, 07745 Jena [DE], William.Durso@leibniz-fli.de
Author(s):
William Durso, Torsten Kroll, Christoph Kaether
The endoplasmic reticulum (ER) is the cell's largest organelle, comprising sheets, a tubular network, and three-way junctions (3WJ). Atlastins (ATLs), GTPases regulating ER fusion, are crucial for ER structure. The ATL3 Y192C mutation causes hereditary sensory and autonomic neuropathy (HSAN1), disrupting ER membrane tethering and tubule organization. We developed a live-cell imaging assay using a U2OS cell line expressing an ER marker, mRFP-KDEL, and inducible GFP-ATL3 Y192C, which reduces ER network complexity and 3WJs. The analysis pipeline quantifies the changes in ER morphology via branch points, a feature related to 3WJ. Screening a Dharmacon siRNA library identified genes that restore or worsen ER morphology defects caused by mutant ATL3 Y192C. We are also screening compounds to find small molecules that restore the ER network with therapeutic potential. Using single particle tracking, we measured lysosome motility in U2OS cells expressing GFP-ATL3 Y192C, finding decreased active transport and increased subdiffusive motion compared to the wild-type. Interestingly, the autophagy inducer Torin-1 rescued lysosome dynamics. Investigating lysosome dynamics in primary cortical mouse neurons expressing GFP-ATL3 showed that the mutated form hinders lysosomal transport in neurites. Preliminary data suggest mutated ATL3 reduces lysosomal motility in both polarized primary neurons and non-polarized cells, prompting further investigation into the physiological consequences.
__________Correlative X-Ray and Volume Electron Microscopy to Study Cerebral Malaria Pathogenesis in a 3D Blood Brain Barrier Model
Presenting author:
EMBL Heidelberg, , Meyerhofstr. 1, 69117 Heidelberg [DE], hannah.fleckenstein@embl.es
Author(s):
Hannah Fleckenstein, Inés Romero Brey, Jonas Albers, Rory Long, Livia Piatti, Sílvia Sanz Sender, François Korbmacher, Alina Batzilla, Elizabeth Duke, Yannick Schwab, Maria Bernabeu
Cerebral malaria (CM) is one of the deadliest complications of malaria. A hallmark of pediatric CM is cytoadhesion of Plasmodium falciparum infected RBCs (iRBCs) to the brain microvasculature and brain swelling, likely resulting from the disruption of the blood-brain barrier (BBB). BBB breakdown can be caused by the disruption of endothelial junctions and/or increased trafficking across cells. Both cellular events occur at the nanoscale and require high-resolution microscopy.
Here, we use a bioengineered 3D-BBB model to get a deeper understanding of the interactions between iRBCs and endothelial cells, and its consequences for BBB integrity. One of the main challenges is efficiently targeting the cytoadhesion events within the non-fluorescent 3D-BBB models. Therefore, we have developed a correlative X-Ray and electron microscopy (CXEM) workflow encompassing microCT, synchrotron-based X-Ray imaging, guided ultramicrotomy and volume EM. We have precisely targeted 20 iRBCs attached to endothelial cells and imaged them by focused ion beam scanning EM (FIB-SEM). One of the most striking features we observe is that endothelial cells actively wrap around the attached iRBCs. Segmentation and quantitative analysis will complement this analysis.
Our CXEM workflow provides a unique opportunity to explore tissue organization and parasite-BBB interactions from the mesoscale to micro- and nanoscale and could be applied for other bioengineered devices or imaging of blood vessels in tissues.
__________MitoTraP: Mitochondria triage precursor proteins into the intermembrane space to protect the cellular proteome from orphaned ribosomal proteins
Presenting author:
RPTU Kaiserslautern-Landau, Zellbiologie, Erwin-Schrödinger-Straße 13, 67663 Kaiserslautern [DE], tflohr@rptu.de
Author(s):
Tamara Flohr, Markus Räschle, Johannes M. Herrmann
Most mitochondrial proteins are synthezised in the cytosol and have to be imported into mitochondria. Targeting information signals guide the precursor proteins into their respective destination within the organelle. We used a proximity-labeling based assay to monitor the proteomes of the two mitochondrial subcompartments, the matrix and the intermembrane space (IMS). We confirmed that in healthy cells, mitochondria have a clear seperation of the proteomes of the two subcompartments. However, upon mitochondrial dysfunction such as upon inactivation of the ATPase, many matrix proteins were found to be mislocalized to the IMS. The Mitochondrial Triage of Precursor proteins (MitoTraP) have been particularly seen for a defined subgroup of matrix proteins, namely mitoribosomal proteins (MRPs). We propose that trapping MRPs in the IMS could prevent cytosolic accumulation of non-imported proteins and thereby prevent MRPs to interfere with the assembly of the cytosolic ribosome. Thus, MitoTrap represents a novel mechanism of the quality control network that protects the cellular proteome against non-imported mitochondrial precursor proteins.
__________Towards imaging of thin Drosophila cells using cryo-transmission electron tomography
Presenting author:
Forschungszentrum Juelich GmbH, Structural Biology ER-C-3, Forschungszentrum Jülich GmbH, 52425 Jülich [DE], t.goetze@fz-juelich.de
Author(s):
Tom Goetze, Thomas Klein, Carsten Sachse
Cryo-electron tomography (cryoET) of single cells is a powerful technique to study cellular processes in situ. One of the main limitations of cryoET is the natural thickness of eukaryotic cells which are often impenetrable for electrons, difficult to vitrify and require extensive lamella formation down to a few hundred nanometers. This time-demanding process forms one of the many bottlenecks in cellular tomography and requires highly specialized equipment such as focused ion beam (FIB) milling systems. Certain eukaryotic cell types can undergo large morphological changes that overcome this limitation and may allow cryoET of unaltered, near-native cells. Macrophage cells from Drosophila melanogaster are able to adapt a “pancake-like” morphology with long protrusions reaching thicknesses of up to 50 nanometers, which allows them to be directly imaged under an electron microscope. This minimizes the risk of ice contamination and sample devitrification, and allows cellular processes to be studied under almost native conditions. Our work suggests that Drosophila melanogaster macrophages may serve as a new model system for cryo-electron tomography of eukaryotic cells that eliminate the need for extensive lamella preparation.
__________Label-free Infrared Imaging for Cancer Diagnostics in Computational Pathology
Presenting author:
Ruhr- Universitaet Bochum, Zentrum für Proteindiagnostik (PRODI) - Biospektroskopie, Gesundheitscampus 4, 44801 Bochum [DE], frederik.grosserueschkamp@ruhr-uni-bochum.de
Author(s):
Frederik Großerüschkamp, Klaus Gerwert
Label-free infrared (IR) imaging allows the analysis of unstained tissue slides. Morphology and molecular composition are determined by spatially resolved IR spectra. By using artificial intelligence (AI), disease-specific information can thus be obtained. This has already been successfully used for various cancers, e.g. colon (CC) and lung cancer (LC).1,2 Unstained cancer tissue slides were analyzed (avg. 30 min/slide). Cancer regions were first identified based on AI (CompSegNet). Subsequently, the lesions were analyzed in more depth using further AI models to identify molecular changes. Endpoints were always area under receiver operating characteristic (AUROC) and area under precision recall curve (AUPRC). In our most comprehensive study 547 patients (validation n=147) with CC were analyzed to identify microsatellite instability (MSI). The MSI classification reached an AUROC of 0.9 and AUPRC of 0.74 (sensitivity 85%, specificity 84%).1 IR imaging achieves comparable results to the current gold standard staining. It shows its potential for diagnostics and could facilitate the development of prognostic/predictive classifiers in the setting of randomized controlled trials. The unstained tissue samples are still available after analysis for further investigations. We expect our approach to be a broadly applicable diagnostic tool in the future.
1 Gerwert, K., Schörner, S. et al., Eur J Cancer, 2023, 182, 122-131
2 Goertzen, N. et al., Am J Pathol, 2021, 191, 7, 1269-1280
__________NUCLEAR LOCALIZATION OF NON-IMPORTED MITOCHONDRIAL PROTEINS
Presenting author:
RPTU Kaiserslautern-Landau, DEPARTMENT OF CELL BIOLOGY, Erwin-Schrödinger-Straße, Room 449 , 67663 kaiserslautern [DE], n.gupta@rptu.de
Author(s):
Nikita Gupta, Johannes M. Herrmann
Defects in the import of proteins into mitochondria leads to the accumulation of mitochondrial precursor proteins in the cytosol. In most cases, accumulating precursors are rapidly degraded by the ubiquitin proteasome system or sequestered into cytosolic aggregates called MitoStores. However, a small subgroup of precursor proteins was reported to be rerouted into the nuclear lumen if they fail to enter mitochondria (Shakya et al., 2021. eLife 10:e61230). In that study, it was proposed that the targeting into the nucleus might accelerate the proteasomal degradation of these proteins.
In my project, I characterize the fate of the mito-to-nucleus targeted proteins in baker’s yeast. Most of these proteins belong to one metabolic pathway that mediates the synthesis of branched-chain amino acids. I developed a system to monitor the targeting of these proteins into the nucleus upon clogging of the mitochondrial TOM complex. My first results indicate that these precursors are very stable in the nucleus and are exempt from proteolytic degradation. On my poster, I will present results about the mechanisms and the physiological implications that underly the mito-to-nucleus targeting of proteins.
__________Characterization of interphase nuclear pore complex assembly
Presenting author:
Uniklinik Aachen, Biochemie (AG Antonin), Pauwelsstraße 30, 52074 Aachen [DE], mhamed@ukaachen.de
Author(s):
Mohamed Ismail Hamed
Nuclear pore complexes (NPCs) are the gateways of the nuclear envelope that control the transport of molecules such as proteins, RNAs and ribonucleoprotein complexes between the nucleus and cytoplasm. In animal cells, the NPCs assemble and integrate into the nuclear envelope by two defined modes: at mitotic exit and during interphase. Mitotic assembly is a well-studied pathway that initiates at the decondensed chromatin. The newly reforming NPCs and nuclear envelope establish the transport function between the nucleus and cytoplasm in a few minutes. On the contrary, interphase NPC assembly is less studied. It is initiated by a different set of nucleoporins that scaffold the inner nuclear membrane rather than binding to the chromatin and last over a period of several hours.
Preliminary data suggest that different nucleoporins have variable levels of contribution to interphase NPC assembly. Depletion of some nucleoporins inhibits the assembly process while depletion of other nucleoporins has no effect on interphase NPC assembly. Interestingly, some nucleoporins are dispensable for interphase but not mitotic NPC assembly and vice versa.
Using confocal and high resolution STED imaging, I will decipher and differentiate interphase NPC assembly, at a molecular level, from mitotic NPC assembly. Understanding the underlying mechanics of NPC assembly is a major step toward a clear distinction of the two assembly pathways and the role of individual nucleoporins in the assembly processes.
__________Fluorescence and electron microscopy study to elucidate the structure of Arf1-coated membrane tubules
Presenting author:
Martin-Luther University Halle, Charles-Tanford Protein Centre, Institute of Chemistry, Kurt-Mothes-Str. 3A, 06120 Halle [DE], caroline.haupt@chemie.uni-halle.de
Author(s):
Caroline Haupt, Dmitry A. Semchonok, Ambroise Desfosses, Sebastian Daum, Sarah Neudorf, Farzad Hamdi, Panagiotis Kastritis, Milton T. Stubbs, Kirsten Bacia
Curvature of biological membranes underpins many cellular processes. Membrane-associated small GTPases are key regulators of intracellular trafficking in cells by recruiting additional coat proteins. However, the structural mechanisms underlying membrane constriction remain poorly understood. Here, we focus on Arf1 as a small GTPase, which is involved not only in the retrograde vesicular coat protein transport machinery COPI, but was also reported to shape tubular membrane carriers in the transport towards the plasma membrane and in intracisternal Golgi transport. Combining cryo-fluorescence microscopy and cryo-electron microscopy studies, we investigated the structure and function of Arf1 in reshaping membranes into tubular structures. We solved the cryo-EM structure of Arf1-coated membrane tubules at 3.1 Å resolution. The structure demonstrates that Arf1 polymerises into a stable tubular scaffold of Arf1 molecules with a consistent back-to-face orientation. We elucidated the functional protein-protein contacts within the regular helical lattice, conserved across tubules of varying diameter. These findings underscore the potential role of a minimal protein network maintaining curved membrane structures across diverse cellular processes.
__________Unraveling the consequences for cellular rescue and protein degradation responses upon allotopic expression
Presenting author:
RPTU Kaiserslautern-Landau, Standort Kaiserslautern, AG Zellbiologie, Erwin-Schrödinger-Straße 13, 67663 Kaiserslautern [DE], ecker@rhrk.uni-kl.de
Author(s):
Anna-Lena Heußer
Mitochondria are essential organelles of eukaryotic cells. They consist of hundreds of nuclear encoded proteins, but also harbor a small genome as a remnant of a bacterial ancestor. Mitochondrial genomes encode a small number of very hydrophobic proteins. Why the genes of these proteins were not transferred into the nucleus is not well understood.
To elucidate the molecular consequences of such mitochondria-to-nucleus gene transfer reactions, we allotopically expressed the model protein Cox3 with mitochondrial targeting sequences in the cytosol of yeast cells. The fusion protein is not imported into mitochondria but rather accumulate on the cytosolic surface of the outer membrane translocase. The highly hydrophobic character of this protein presumably prevents efficient translocation through the TOM complex.
These stalled translocation intermediates are efficiently removed by proteolysis, specifically by components of the cytosolic ubiquitin-proteasome system (UPS). Mutants in the UPS which prevent the efficient degradation of these proteins lead to growth defects and induce cell death.
Thus, the protein quality system on the mitochondrial surface is important for cellular functionality, however, it prevents the productive gene transfer from mitochondria to the nucleus and forces eukaryotic cells to maintain the genes of a core set of highly aggregation-prone proteins.
__________Characterizing the role of TRIB3 in fine-tuning chronic mtUPR mediated cell death transition.
Presenting author:
Goethe Universitaet Frankfurt am Main, Institut für Biochemie II, Theodor-Stern-Kai 7, 60590 Frankfurt [DE], N.Ho@med.uni-frankfurt.de
Author(s):
Napyin Ho, FX Reymond Sutandy, Nikita Verheyden, Christian Münch
Mitochondria are crucial organelles that play a pivotal role in a myriad of cellular processes. A decline in mitochondrial health, such as through disruption of protein homeostasis (proteostasis) in mitochondria, can impair cellular function and survival.
In recent years, a specific stress response activated during accumulation of unfolded proteins in mitochondria (mtUPR) has been discovered and characterized. At its core, the mtUPR is a mitoprotective response that aims to restore mitochondrial health by upregulating mitochondria proteostasis. However, under prolonged or severe stress conditions, the mtUPR transitions from a survival-promoting response towards a cell death inducing response. The precise molecular mechanism governing the transition remains elusive.
Our work aims at providing further insights to this transition process. Using mitochondrial specific chaperone inhibitors, we chronically induce mtUPR and investigate relevant downstream changes using genomic and proteomic approaches. At current, our findings suggest apoptosis as the main driving cell death mechanism. Furthermore we identified superoxide and the ISR as two essential signaling pathways for chronic mtUPR apoptosis. Surprisingly, both components are induced independent of each other but are both required for apoptosis. We further identified TRIB3 as a potentially relevant factor and are currently investigating the fine-tuning role of TRIB3 in chronic mtUPR apoptosis.
Porous flow across nuclear pores and the in situ NPC structure of Dictyostelium in response to osmotic stress
Presenting author:
Max Planck Institut für Biophysik, Molekulare Soziologie, Max-von-Laue-Str. 3, 60438 Frankfurt [DE], patrick.hoffmann@biophys.mpg.de
Author(s):
Patrick Hoffmann, Hyuntae Kim, Agnieszka Obarska-Kosinska, Gerhard Hummer, Martin Beck
Osmotic stress causes solvent flux into or out of cells as a result of changing environmental conditions. Cellular volume adjustments occur on short second to minute time scales. How cells cope with the mechanical impact on the nucleus during these changes remains underexplored. Using in situ electron cryo-tomography, we visualize the reorganization of cytosolic and nuclear supramolecular architecture in Dictyostelium discoideum, and quantify cellular crowding as well as nuclear membrane changes during acute osmotic stress. We reveal a unique D. discoideum NPC architecture with three concentric Y complex rings at the nuclear side, providing additional rigidity to the scaffold during demanding environmental conditions. In addition, we integrate the results of nuclear and cellular size changes during acute osmotic stress, which are experimentally determined from live cell fluorescence microscopy and microfluidics, into mathematical models. In this way we highlight a neglected role of NPCs in its permeability to cellular solvents and directional solvent flux through NPCs during changing osmotic conditions. We demonstrate that altered NPC permeability correlates with constriction and dilation of the NPCs.
__________The reHaloTag: Reversible labeling for live cell long-term high- and superresolution imaging
Presenting author:
Universität Osnabrück, Biology, Barbarastraße 11, 49076 Osnbarück [DE], mholtmannspo@uni-osnabrueck.de
Author(s):
Michael Holtmannspötter, Eike Wienbeuker, Timo Dellmann, Isabelle Watrinet, Anna Garcia-Sáez, Kai Johnsson, Rainer Kurre, Jacob Piehler
Self-labeling enzymes (SLE) such as the SNAPtag and HaloTag are powerful tools for visualizing cellular processes with high spatiotemporal resolution. However, covalent labeling via SLEs faces the issue of a finite pool of fluorescent labels, which irreversibly photo-bleaches over time, limiting microscopic approaches. We addressed this by engineering two HaloTag variants, reHaloTagS and reHaloTagF, restoring the original dehalogenase functionality to enable ligand exchange over time. Labeling experiments in cells and kinetic studies in vitro confirmed efficient, yet reversible labeling of both variants. Our reHaloTagS and reHaloTagF variants showed different turnover kinetics (~0.006 s⁻¹ and ~0.055 s⁻¹, respectively), making them suitable for various time windows. By combining our mutants with fluorogenic substrates, we demonstrated prolonged time-lapse imaging by confocal and STED microscopy, achieving up to three-fold enhanced photostability. Live cell two- and three-dimensional single molecule tracking and localization microscopy (TALM) benefited from reHaloTag labeling by enabling robust control of labeling density over extended time periods. The combination of TALM with structured illumination microscopy allowed simultaneous superresolved visualization of single molecule diffusion and organellar dynamics. These applications highlight the potential of reHaloTag labeling for advancing fluorescence microscopy techniques.
__________Actin reorganization controls different phases of Plasma Membrane Repair
Presenting author:
University Clinic Muenster - UKM, , Von-Esmarch-Straße 56, 48149 Münster [DE], jekabson@uni-muenster.de
Author(s):
Rafaella Jekabson, Felix Uecker, Christian Schuberth, Roland Wedlich-Söldner
Plasma membrane repair (PMR) plays a critical role in the protection of cells from a variety of damage sources during tissue homeostasis but also in pathological conditions such as cancer. Traditionally, PMR has been studied in a variety of cellular model systems and with a wide range of methodologies. This has led to the identification of several core PMR pathways but with sometimes ambivalent findings. For example, cortical actin has been suggested to have both negative (barrier to vesicle fusion) and positive (facilitator of membrane remodeling) functions in PMR. Here, we employ a combination of 2-photon laser ablation and live fluorescent reporters to study PMR in mammalian cells. We found that actin reorganization following PM damage varied depending on wound severity. In smaller wounds, the response remained locally restricted. In contrast, larger wounds with more pronounced calcium influx first led to global reorganization of actin from cortex to cell interior and subsequent accumulation at the wound site. Importantly, actin remodeling during PMR was dependent on the calcium regulated inverted formin 2 (INF2). The cytosolic INF2 isoform was recruited to the wound site by a short peptide within its C-terminus. Wound localization of INF2 occurred simultaneously with the known PMR effector Annexin A2. We propose using actin reorganization as sensitive readout for PMR and introduce a novel PM wound-specific marker for future studies.
__________SELF-QUENCHED FLUOROPHORE LABELS FOR DNA-PAINT AND STED MICROSCOPY
Presenting author:
Johann Wolfgang Goethe-University, , 7 Max-von-Laue-Straße, 60438 Frankfurt am Main [DE], lkessler@chemie.uni-frankfurt.de
Author(s):
Laurell Kessler, Tanja Menche, Dongni Wang, Catarina Sabino, Ashwin Balakrishnan
Abstract:
Exchangeable fluorophore labels have enriched the landscape of deterministic and stochastic super-resolution microscopy techniques. One prominent method for achieving weak affinity, transient binding is by exploiting DNA hybridisation1. Used in DNA-PAINT2 and STED-PAINT3, such labels have the advantage of multi-target imaging that can be extended to 10s of targets and are an integral part of adding knowledge to optical cell biology. However, these systems suffer from low signal to background ratio due to an increased background contribution from fluorophore-coupled unbound DNA-strands. We circumvent this by utilising short-distance quenching mechanisms between fluorophore dimers4. We designed DNA imager strands carrying the same fluorophore at both termini that are compatible with pre-existing docking strand bound targets. We show that fluorophore dimers across a spectrum from 560 to 640 nm exhibit reduced background fluorescence signal in their unbound state as well as an increased photon output in their bound state thereby achieving a fluorogenicity of up to 5x. We characterize the spectroscopic and thermodynamic properties of fluorophore-dimers and the mechanism behind their reduced background fluorescence. Finally, we show such fluorophore dimers at work in super-resolution imaging using both STED and SMLM with background reduction and an increased spatial resolution.
__________Live-cell super resolution microscopy of mitochondrial dynamics
Presenting author:
ETH Zuerich, Department of Biology, Institute of Biochemistry, Otto-Stern-Weg 3, 8093 Zürich [DE], tatjana.kleele@biol.ethz.ch
Author(s):
Tatjana Kleele
Mitochondria perform a multitude of metabolic, biosynthetic and signaling functions. While initially perceived as isolated, static organelles, convergent advances in light microscopy revealed that mitochondria form an interconnected network, communicate with other organelles and rapidly modulate their structure. Mitochondrial morphology is shaped by opposing events of fission and fusion, which are tightly regulated. Disruptions in these processes can lead to bioenergetic defects and are linked to a variety of human diseases. But despite the inevitable link between mitochondrial structure and function, it remains unclear how these organelles integrate cellular cues to remodel their network structure, consequently adjusting their function. Broadly, our group investigates how mitochondria are dynamically remodeled to support cellular metabolism and tissue specific functions. For this we employ a range of super-resolution microscopy techniques, automated AI-based data analysis, combined with different model systems, including bone marrow derived macrophages and iPSC derived neurons. Using this approach, we investigate how mitochondria are dynamically remodeled in different metabolic conditions, during cell state transitions as well as during neuronal differentiation and maturation. Moreover, we are currently developing a super-resolution based imaging screen to identify novel protein kinases regulating mitochondrial fission and fusion.
__________Analysing nuclear foci and their subnuclear localisation using CellProfiler
Presenting author:
University Hospital Aachen, RWTH Aachen University, Biochemistry and Molecular Biology, Auf der Hörn 106, 52074 Aachen [DE], sarah.knapp@rwth-aachen.de
Author(s):
Sarah Knapp, Maud Verheirstraeten, Ani Sabcheva, Barbara Lippok, Bernhard Lüscher, Patricia Korn
Nuclear foci are distinct, localised clusters of proteins and/or nucleic acids, observed in various biological imaging experiments. Foci can be associated with different nuclear processes, for example replication, transcription or splicing events. While some foci are present under normal conditions, others form specifically in response to cellular stress. This process allows cells to defend against various forms of insult, such as DNA damage or pathogen infection.
The size, shape and, in particular, the localisation of nuclear foci are important features, as they can provide valuable insights into the biological role of their components. In this project, we utilised CellProfiler to analyse nuclear foci formed by a GFP-fusion protein in confocal microscopy images with DNA staining.
By identifying the cell nucleus and nucleoli we were able to map the subnuclear localisation of these foci, distinguishing between nucleoli, the nucleolar periphery and the nucleoplasm. Dependent on different treatment conditions we identified either GFP signals ubiquitously distributed in the nucleoplasm or localised to distinct foci. We compared control cells with foci stabilising treatment and foci dissolving treatment. This pipeline is a valuable tool for studies ranging from basic cell biology to disease-related research, where localisation of nuclear foci plays a central role.
Keywords: nuclear foci, image analysis, confocal microscopy, localisation
__________Elucidating the Roles of Glutathione and Reactive Oxygen Species in Basal Land Plant Development through Redox Sensor Microscopy
Presenting author:
University Osnabrück, Botany, Barbarastr. 11, 49076 Osnabrück [DE], cilikock@uni-osnabrueck.de
Author(s):
Cilian Kock, Judith Helmig, Nora Gutsche, Sabine Zachgo
The roGFP2-hGRX1 sensor has been used to study GSH redox potentials in the model angiosperm species Arabidopsis thaliana. A. thaliana establishes redox gradients with a more reduced state in the stem cell area. We used this reporter to analyze and compare redox potentials in the bryophyte M. polymorpha that does not form complex organs and belongs to an evolutionary informative, early diverging liverwort land plant lineage. Microscopic visualization was conducted to investigate redox gradients in M. polymorpha tissues and shed light on the crucial roles of GSH in developmental processes.
Additionally, the HyPer7 biosensor was established in M. polymorpha to visualize H2O2 dynamics in meristematic and mature tissue. This approach in a bryophyte enables analyses of the distribution and possible regulatory activities of H2O2 known to regulate stem cell activity and differentiation processes in angiosperms. Treatment of M. polymorpha with the MpCLE2 peptide hormone induces an enlargement of the meristematic zone by increasing stem cell activities. Microscopic analysis of treated plants revealed an altered ROS pattern, supporting crucial function of ROS and redox gradients in meristem regulation and thus body plan formation.
Together, the establishment of redox sensors and ratiometric microscopy techniques in M. polymorpha unveils novel insights into the crucial roles of redox gradients, GSH and ROS in regulating stem cell activity and differentiation processes in basal land plants.
__________Live-cell STED super-resolution nanoscopy reveals modulation of cristae dynamics in bioenergetically compromised mitochondria
Presenting author:
University Hospital Düsseldorf, Heinrich Heine University Düsseldorf , Institute of Biochemistry and Molecular Biology I, Universitätsstrasse 1, 40225 Düsseldorf [DE], kondadi@hhu.de
Author(s):
Mathias Golombek, Thanos Tsigaras, Yulia Schaumkessel, Sebastian Hänsch, Stefanie Weidtkamp-Peters, Ruchika Anand, Andreas Reichert, Arun Kumar Kondadi
Recently, we and others showed that cristae membranes undergo intramitochondrial remodeling events. The bioenergetic and metabolic factors regulating them are not yet understood. We investigated whether cristae dynamics are dependent on optimum functioning of oxidative phosphorylation (OXPHOS) complexes, the mitochondrial membrane potential (ΔΨm), and the ADP/ATP nucleotide translocator. Advanced live-cell STED nanoscopy was employed to analyse cristae morphology and dynamics after treatment of mammalian cells with rotenone, antimycin A, oligomycin A, and CCCP. This led to formation of enlarged mitochondria along with reduced cristae density but did not impair cristae dynamics. Inhibition of OXPHOS complexes was accompanied by reduced ATP levels but did not affect cristae dynamics. CCCP treatment leading to ΔΨm abrogation even enhanced cristae dynamics showing its ΔΨm-independent nature. However, inhibition of ADP/ATP exchange led to aberrant cristae morphology and impaired cristae dynamics in a mitochondrial subset indicating that cristae form and function are connected. Overall, we provide data supporting an important interplay between OXPHOS, metabolite exchange, and cristae membrane dynamics.
__________Development of a ratiometric biosensor to measure ciliary cAMP levels and to elucidate Paladin1 accumulation in the primary cilia
Presenting author:
Universitätsklinikum des Saarlandes, Medizinische Biochemie und Molekularbiologie, Campus Homburg Geb. 45, 66421 Homburg [DE], leakunz@yahoo.com
Author(s):
Lea Kunz, Patrick Schuster, Elena May, Matea Ivanicic, Oliver Griesbeck, David Mick
The primary cilium is a highly specialized organelle that serves a crucial sensory role, particularly in the transduction of the Hedgehog signaling pathway. A time-resolved cilia proteome analysis using proximity labeling revealed Paladin1 as a component of the signaling pathway. It has been shown that Paladin1 accumulates in the cilium not only upon pathway activation but moreover when Hedgehog-independent receptors are activated.
Given that the ciliary cAMP level drops both when the Hedgehog pathway is activated and when cilia-localized SSTR3 receptors are stimulated by somatostatin, we hypothesize that the accumulation of Paladin1 in the primary cilium is attributed to the decreasing cAMP concentration within cilia. By manipulating the cAMP level using pharmacology, we can show that Paladin1 undergoes cAMP-dependent translocation, which supports our hypothesis. To further elucidate Paladin1 accumulation and to investigate cAMP dynamics in the primary cilium we developed a ratiometric cAMP-biosensor targeted to the primary cilium. This biosensor is based on a blue fluorescent protein and R-FlincA, a circularly permuted red fluorescent protein inserted into the cAMP binding motif of the regulatory subunit of PKA.
__________Aneuploidy-induced protein aggregation impairs mitochondria
Presenting author:
RPTU Rheinland-Pfälzische Technische Universität Kaiserslautern, Molecular Genetics, Paul-Ehrlich-Straße, Gebäude 24, 67663 Kaiserslautern [DE], olhakurpa01@gmail.com
Author(s):
Olha Kurpa, Prince Saforo Amponsah, Zuzana Storchova
Aneuploidy, a chromosome number abnormality that mostly occurs in cancer, is linked to protein imbalance, impaired protein folding and proteotoxic stress. Hence, aneuploid cells elevate protein degradation pathways and accumulate cytosolic deposits positive for the autophagy receptor SQSTM1/p62. Yet, the nature of these deposits remains unclear. Using confocal microscopy, we show that the deposits are protein aggregates sequestered by p62. By immunoprecipitation and proximity biotinylation-based proteomics, we show that the aggregates are enriched with mitochondrial proteins that accumulate in the cytosol due to import delays. Notably, compared to their isogenic diploids, aneuploid cells harbor more defective mitochondria to which p62 colocalizes. Improving protein folding via overexpression of chaperones and mitigating mitochondrial import defect via overexpression of the import factor MIA40 alleviates p62-positive protein aggregates formation as well as p62-mitochondria colocalization in aneuploid cells. Our work elucidates the proteostasis burden of aneuploidy and links cytosolic protein imbalance to mitochondrial homeostasis.
__________Modular dual-color BiAD sensors for locus-specific readout of epigenome modifications in single cells
Presenting author:
University of Stuttgart Germany, Institute of Biochemistry and Technical Biochemistry, Department of Biochemistry, Allmandring 31, 70569 Stuttgart [DE], anja.koehler@ibtb.uni-stuttgart.de
Author(s):
Anja R. Köhler, Pavel Bashtrykov, Albert Jeltsch
The precise spatio-temporal expression of genes in mammalian cells is regulated by epigenomic modifications on DNA and histones. To understand the dynamic changes in these modifications that occur during differentiation and disease onset in live cells, we developed epigenetic sensors to visualize locus-specific epigenome modifications via fluorescence microscopy [1]. The Bimolecular Anchor Detector (BiAD) technology combines an sgRNA/dCas9 complex as a programmable DNA-binding anchor with chromatin reader domains binding specific epigenome modifications as detector modules. Both modules are fused to complementary parts of a split fluorophore, reconstituting a full fluorophore upon binding in close proximity. To further enhance the sensors’ sensitivity towards a broader range of endogenous target loci, we recruited a second, full fluorophore to the sgRNA to mark the genomic locus of interest [2]. This novel dual-color readout, combined with an expanded detector module palette, enabled the quantitative analysis of various key epigenome modifications. We showcased applications of the dual-color BiAD technology in development and disease research by detecting dynamic changes in locus-specific DNA methylation upon inhibitor treatment and dissecting the enzymatic network responsible for the deposition of a critical repressive histone modification at a specific locus on the female inactive X chromosome.
[1] Lungu et al., 2017, Nat. Comm.
[2] Koehler et al., 2024, Cell Reports Methods
__________Dysferlin recruits Vesicles to Calcium Release Units at Endomembrane Tubules in Cardiomyocytes
Presenting author:
, Cardiology and Pneumology, Robert-Koch-Str. 42a, Göttingen [DE], slehnart@med.uni-goettingen.de
Author(s):
Stephan E. Lehnart, Nora Josefine Paulke, Carolin Fleischhacker, Justus B. Wegener, Gabriel C. Riedemann, Constantin Cretu, Mufassra Mushtaq, Nina Zaremba, Wiebke Möbius, Yannik Zühlke, Jasper Wedemeyer, Lorenz Liebmann, Anastasiia A. Gorshkova, Daniel Kownatzki-Danger, Eva Wagner, Tobias Kohl, Carolin Wichmann, Olaf Jahn, Henning Urlaub, Gerd Hasenfuß, Tobias Moser, Julia Preobraschenski, Christof Lenz, Eva A. Rog-Zielinska, Sören Brandenburg
We hypothesized that the type II transmembrane protein Dysferlin, a Ca2+-binding multi-C2-domain protein for membrane repair, maintains the integrity of Ca2+ release unit (CRU) membrane contact nanodomains in cardiomyocytes. A key CRU component, the electrically excitable transverse-axial tubule (TAT) endomembrane network is subject to cycles of significant membrane deformations during cardiac contraction and relaxation. STED microscopy and electron tomography of mouse cardiomyocytes identified a previously unknown reservoir of membrane vesicles decorated by Dysferlin clusters in CRU vicinity at Ryanodine Receptor type 2 (RyR2) Ca2+ release channel clusters in the SER membrane. LC-MS/MS identified a novel Dysferlin interactor, Juntophilin-2, a RyR2 interactor tail-anchored in the SER membrane. Dysferlin knockout mice subjected to transverse aortic constriction (TAC)-induced pressure overload developed significantly less left-ventricular hypertrophy compared to wild-type littermates significantly increasing Dysferlin protein expression. Live-cell nanoscopy showed a profound reorganization of the TAT network 4 weeks post-TAC with predominant proliferation of axial tubule components; this was accompanied by increased axial Dysferlin clustering. We conclude that Dysferlin facilitates the recruitment of vesicles to CRUs through interactions with Junctophilin-2 during increased left-ventricular afterload, contributing to hypertrophic TAT network reorganization in cardiomyocytes.
__________Single-cell imaging identifies cGMP signalling heterogeneity in living VSMCs
Presenting author:
Univerersität Tübingen, Interfakultäres Institut für Biochemie - AG Feil, Auf der Morgenstelle 34, 72076 Tübingen [DE], moritz.lehners@uni-tuebingen.de
Author(s):
Moritz Lehners, Hannes Schmidt, Susanne Feil, Robert Feil
The second messenger cyclic guanosine monophosphate (cGMP) modulates cardiovascular homeostasis in humans. Vascular smooth muscle cells (VSMCs) can generate cGMP via three guanylyl cyclases that are stimulated by NO, atrial natriuretic peptide (ANP), or C-type natriuretic peptide (CNP). It is not known why VSMCs generate cGMP via several pathways. One theory is that, within a cell, local cGMP microdomains are generated by the different cyclases. Considering that VSMCs can change their phenotype upon environmental changes like development of atherosclerosis, another possibility is that individual VSMCs differ in their cGMP generation pathways.
To analyse a potential cGMP signalling heterogeneity between VSMCs, we used transgenic cGMP sensor mice expressing a fluorescent cGMP biosensor. We performed real-time cGMP imaging with single-cell resolution in primary VSMCs and intact murine arteries. By subsequent immunofluorescence staining of marker proteins, we discovered a phenotype-dependent cGMP signalling heteorgeneity between individual VSMCs. While contractile VSMCs showed mainly ANP-dependent cGMP generation, modulated VSMCs exhibited CNP-dependent cGMP signalling. Most strikingly, we identified a similar shift from ANP-dependent cGMP signalling in healthy arteries (contractile VSMCs) towards CNP-induced cGMP signalling in atherosclerotic arteries (modulated VSMCs).
These imaging data suggest a context-dependent cGMP signalling heterogeneity between healthy and diseased VSMCs.
__________Establishing a cytosolic version of the mitochondrial processing peptidase to study mitochondrial protein import
Presenting author:
, , , Kaiserslautern [DE], lenhard@rhrk.uni-kl.de
Author(s):
Svenja Lenhard, Johannes Herrmann
Mitochondria consist of many hundreds of different proteins that are synthesized on cytosolic ribosomes. Mitochondrial protein import mechanisms have been extensively studied in the past. Aminoterminal presequences ensure the reliable targeting of client proteins into mitochondria. Subsequently to the import of these proteins, the presequences are proteolytically removed in the mitochondrial matrix by the mitochondrial processing peptidase, MPP. Strikingly, the processes occurring right before the translocation of a polypeptide remain unclear. In order to better understand the timing of the synthesis and import of precursor proteins, we engineered a yeast strain which expresses MPP in the cytosol. Expression of this cytosolic MPP (cytoMPP) is highly toxic as MPP cleavage in the cytosol obviously competes with mitochondrial import of precursor proteins. Establishment of this tool is expected to provide novel insights into (1) how different precursors are sequestered to the mitochondrial surface, (2) the determinants of post- or co-translational protein import, (3) which proteins are particularly sensitive to cleavage by cytoMPP and (4) which factors determine the import efficiency into mitochondria. Furthermore, we aim to characterize the conserved C-terminus of the MPP α-subunit concerning thus far unknown structure-function relationships. Therefore, this study aims to investigate both, endogenous as well as cytosolic MPP.
__________The N terminus of the Oxoeicosanoid receptor 1–does its length affect the receptor’s localisation?
Presenting author:
Universität Leipzig / Medizinische Fakultät, Rudolf-Schönheimer Institut für Biochemie, Johannisallee 30, 04103 Leipzig [DE], aenne-dorothea.liebing@medizin.uni-leipzig.de
Author(s):
Aenne-Dorothea Liebing, Claudia Stäubert
G protein-coupled receptors (GPCRs) are the largest family of transmembrane receptors and transduce a plethora of stimuli into an intracellular response. The classical view includes receptor expression at the plasma membrane and recognition of extracellular agonists. However, in the last 20 years, evidence accumulated for the presence and signaling of GPCRs from intracellular compartments, like endosomes, the Golgi-apparatus, the endoplasmic reticulum, mitochondria or even the nucleus. This is highly fascinating in the context of metabolite-sensing GPCRs, activated by intra- and extracellular metabolites. It becomes increasingly apparent that receptor internalization and location itself play a key role in the regulation of signaling kinetics.
We used confocal imaging to investigate (sub)cellular localization of the oxoeicosanoid receptor 1 (OXER1) with various marker proteins targeting ER, endosomal compartments and mitochondria. OXER1 is activated by arachidonic acid metabolites and is highly expressed in eosinophils, the liver and the kidney. We found that the receptor's N terminus length significantly influences its localization. Image-based analyses using the Celigo Imaging Cytometer further revealed that a shorter OXER1 N terminus increases expression whereas total receptor amount is decreased in the full-length construct. Combined with classical signal transduction assays such as cAMP inhibition assays, imaging provides a deeper understanding of OXER1 signaling dynamics.
__________Canonical and non-canonical integrin-based adhesions dynamically interconvert
Presenting author:
Technische Universität Kaiserslautern, , Paul-Ehrlich-Strasse 23, 67633 Kaiserslautern [DE], flukas@rptu.de
Author(s):
Fabian Lukas, Claudia Matthaeus, Tania López-Hernández, Ines Lahmann, Nicole Schultz, Martin Lehmann, Dmytro Puchkov, Jan Pielage, Volker Haucke, Tanja Maritzen
Cell-matrix adhesions are critical for anchoring cells in their environment, as signaling platforms and for cell migration. In line with these diverse functions, different types of cell-matrix adhesions exist. Best-studied are the canonical integrin-based focal adhesions. In addition, non-canonical integrin adhesions lacking focal adhesion proteins have been discovered. These include reticular adhesions, which are enriched in clathrin and other endocytic proteins, as well as extensive adhesion networks and retraction fibers. How these different adhesion types that share a common integrin backbone are related and whether they can interconvert is largely unknown. Employing multi-color live cell microscopy techniques, particularly total internal reflection fluorescence (TIRF) microscopy and fluorescence recovery after photobleaching (FRAP) in combination with genome-edited cells, we performed a detailed investigation of adhesion dynamics. Our study identifies stonin1 as a marker for non-canonical αVβ5 integrin-based adhesions and reveals that canonical and non-canonical adhesions can interconvert by selectively exchanging components on a stable αVβ5 integrin scaffold.
__________Imaging conformational rearrangements on the extracellular region of the neuropeptide Y Y5 receptor
Presenting author:
Leipzig University, Institute of Biochemistry, Brüderstraße 34, 04103 Leipzig [DE], antonio.lopez_sierra@uni-leipzig.de
Author(s):
Antonio López Sierra, Irene Coin
The neuropeptide Y receptors are a family of class A GPCRs activated by the related peptides NPY, PYY and PP. They are involved in the control of appetite, circadian rhythm and anxiety. For the Y1, Y2 and Y4 receptors there are cryoEM structures published; however, there is no structure available for the Y5 receptor. On top of that, the role of the N-termini in ligand binding and receptor activation is not clear yet. In this body of work, we aim to study the dynamics of the binding and activation processes on the Y5 receptor via fluorescence microscopy. Through genetic incorporation of unnatural amino acids carrying anchors for biorthogonal chemistry, we can perform post-translational GPCR labeling by the desired organic dyes in intact living cells. This technique is minimally invasive and allows labeling interesting regions of the receptor without leading to detrimental effects on receptor function. We define a sensor as a position on the receptor where we can observe a clear fluorescence change in single cell imaging upon ligand addition, performed using a perfusion system. We can calculate the amplitude of the fluorescence change and dynamic parameters of each sensor. This procedure allows us to map the speed of the conformational changes that the receptor undergoes. Interestingly, the receptor dynamics data collected indicate not a binding mechanism where the ligand is first recruited by the N-terminus, but a concerted movement of the N-terminus and the receptor core.
__________Canonical and non-canonical integrin-based adhesions dynamically interconvert
Presenting author:
RPTU Kaiserslautern-Landau, Nanophysiologie, Paul-Ehrlich-Str. 23, 67663 Kaiserslautern [DE], maritzen@rptu.de
Author(s):
Tanja Maritzen, Fabian Lukas, Claudia Matthaeus, Tania López-Hernández, Ines Lahmann, Nicole Schultz, Martin Lehmann, Dmytro Puchkov, Jan Pielage, Volker Haucke
Cell-matrix adhesions are critical for anchoring cells in their environment, as signaling platforms and for cell migration. In line with these diverse functions, different types of cell-matrix adhesions exist. Best-studied are the canonical integrin-based focal adhesions. In addition, non-canonical integrin adhesions lacking focal adhesion proteins have been discovered. These include reticular adhesions, which are enriched in clathrin and other endocytic proteins, as well as extensive adhesion networks and retraction fibers. How these different adhesion types that share a common integrin backbone are related and whether they can interconvert is largely unknown. Employing multi-color live cell microscopy techniques, particularly total internal reflection fluorescence (TIRF) microscopy and fluorescence recovery after photobleaching (FRAP) in combination with genome-edited cells, we performed a detailed investigation of adhesion dynamics. Our study identifies stonin1 as a marker for non-canonical αVβ5 integrin-based adhesions and reveals that canonical and non-canonical adhesions can interconvert by selectively exchanging components on a stable αVβ5 integrin scaffold.
__________LiveCellMiner: A new tool to analyze mitotic progression
Presenting author:
Medical School, RWTH Aachen University, Institute of Biochemistry and Molecular Cell Biology, Pauwelsstraße 30, 52074 Aachen [DE], dmoreno@ukaachen.de
Author(s):
Daniel Moreno-Andrés, Anuk Bhattacharyya, Anja Scheufen, Johannes Stegmaier
Live-cell imaging has become state of the art to accurately identify the nature of mitotic and cell cycle defects. Low- and high-throughput microscopy setups yield huge data amounts of cells recorded in different experimental and clinically relevant situations. Tailored semi-automated and automated image analysis approaches allow the analysis of high-content screening data sets, saving time and avoiding bias. However, they are mostly designed for very specific experimental setups, which restricts their flexibility and usability. The general need for dedicated experiment-specific user-annotated training sets and experiment-specific user-defined segmentation parameters remains a major bottleneck for automating the analysis process. In this work we present LiveCellMiner, a highly flexible open-source software tool to automatically extract, analyze and visualize both aggregated and timeresolved
image features with potential biological relevance. LivCellMiner allows analysis across high-content data sets obtained in different platforms, in a quantitative and unbiased manner. As proof of principle application, we analyze here the dynamic chromatin features in human cells passing through mitosis, highlighting the versatile and flexible potential of this tool set.
Investigating the Cytoskeletal Interactions with the Golgi Apparatus: A Quantitative Ultrastructural Study
Presenting author:
CERBM Gie, IGBMC, 1 rue Laurent Fries, 67404 Illkirch [FR], delnianazari@gmail.com
Author(s):
Delnia NAZARI BANYARANI, Amina Sabar, Chantal Weber, Florian Faessler
The Golgi apparatus plays a vital role in protein modification and intercellular communication,and its defects are linked to cancer and inflammation.This organele consists of stacked flattened membrane disks called cisternae.In most vertebrate,the Golgi apparatus comprises a unique structure where the stacks are connected laterally to form the Golgi ribbon,positioned next to the centrosome,and attached to microtubules.This arrangement creates a single microtubule organizing center,which is crucial for directional secretion and persistent cell migration.In addition to MT,actin assemblies are involved in linking of Golgi stacks into a ribbon.Consequently,the position and morphology of the ribbons depend upon interactions with microtubules and the actin cytoskeleton.In contrast to microtubules and actin,less is known about the association of the Golgi complex with another cytoskeleton element,the intermediate filaments.A commonality among these three cytoskeleton types and their assemblies at the Golgi,have not been well described at the ultrastructural levels.We will provide insights into this matter employing cryo-FIB milling supported by CLEM and cryo-electron tomography(cryo-ET) to study cytoskeleton in the context of the Golgi in situ.We will derive quantitative ultrastructural from tomograms acquired combining segmentation,vectorization and geometrical analysis which guide us to employ subtomogram averaging for detailed characterization of abundant cytoskeletal organizers
__________
Developping new concepts for drug discovery using high-content imaging
Presenting author:
Applied University Darmstadt, Biochemistry, Stephanstrasse 7, 64295 Darmstadt [DE], petra.neumann-staubitz@h-da.de
Author(s):
Petra Neumann-Staubitz, Heinz Neumann
The safe supply of the population with established and newly developed drugs is essential for a functioning healthcare system and is currently the focus of public interest due to supply bottlenecks. We are researching the development of new concepts for the discovery and production of active ingredients using a high-content, high-throughput microscope. This will enable us to optimise new methods for drug screening, expand other methods that are important for clinical feasibility, such as organ-on-a-chip systems, and enable the systematic, standardised characterisation of biomolecular condensates. For example, in order to link protein localisation with the protein function, we use cell painting under different cellular stress conditions and in different cell types. In addition, biomolecular condensates are currently the focus of biochemical and cell biological research. Many processes appear to be regulated by this form of compartmentalisation in cells. However, there is disagreement about the relevance of biochemical observations for cell biological processes. This could be improved by standardising and systematically analysing the conditions under which the condensates form. We want to contribute to this by creating a platform for the study of biomolecular condensates with high-throughput microscopy that is available to the entire scientific community.
__________Discovering the early stages of mitochondrial carrier biogenesis: a proximity-based labeling approach
Presenting author:
RPTU Rheinland-Pfälzische Technische Universität Kaiserslautern-Landau, Zellbiologie, Erwin-Schrödinger-Str. 13, 67663 Kaiserslautern [DE], anutz@rptu.de
Author(s):
Annika Nutz, Büsra Kizmaz, Hyun-Woo Rhee, Johannes Herrmann
Mitochondrial carrier proteins are integral to the inner mitochondrial membrane and are key elements of cellular transport of various metabolites to and from the mitochondrial matrix. They lack mitochondrial matrix-targeting sequences and instead contain at least one internal signal sequence that leads them along the distinct TIM22 pathway into mitochondria. The recognition of these carriers by Tom70 on the mitochondrial surface, their translocation by the TOM complex across the outer membrane, guidance by the small Tim proteins through the intermembrane space, and insertion by the TIM22 complex into the inner membrane are well-studied processes. However, the mechanisms that govern the initial stages of carrier biogenesis remain ambiguous. Due to the hydrophobic nature of carrier proteins, a complex network of chaperones and targeting factors is likely required.
To address this knowledge gap, we have adopted a proximity-based labeling approach an advanced version of the known Turbo-ID. This approach enables us to identify transient interactions with carriers in the cytosol by labeling with the biotin-derivative dethiobiotin. Affinity-based purification and competitive elution with biotin allows specific identification of labeled proteins over endogenously biotinylated proteins via a proteomics approach.
__________Insights into caveolae-mediated lipid transport
Presenting author:
University of Potsdam, Institute of Nutritional Sciences, cellular physiology of nutrition (AG Matthaeus), Karl-Liebknecht-Str. 24/25, 14476 Potsdam [DE], esther.ocket@uni-potsdam.de
Author(s):
Esther Ocket, Claudia Matthaeus
Caveolae are specialized invaginations of the plasma membrane that play a role in cellular lipid uptake and transport, signal transduction and endocytosis. Neutral lipids are stored intracellularly in organelles called lipid droplets. Previous studies showed that caveolae proteins can accumulate at the lipid droplets when cells are treated with lipids. However, it is currently not understood how caveolae facilitate lipid uptake and trafficking from the plasma membrane to lipid droplets. Neither do we know how caveolae mechanistically transport lipids or how this trafficking is regulated. This study aims to investigate the role of caveolae in lipid transport to lipid droplets. Here, we demonstrate the trafficking of caveolin proteins to lipid droplets and provide insights into the specific trafficking routes. Using confocal microscopy, we visualize caveolin proteins and lipid droplets, revealing that caveolin-1 and -2 relocate from the plasma membrane to lipid droplets within 3 hours. Additionally, we use specific inhibition of the lysosome, as well as dynein inhibition to get a better idea of the intracellular trafficking route. Our findings provide initial molecular details on the specific intracellular route of caveolae. Understanding the mechanism of caveolae-mediated lipid transport through advanced imaging techniques such as STED and TIRF can help us investigate the impact on overall lipid metabolism and better understand conditions where lipid metabolism is disrupted.
__________Misfolded ER membrane proteins partition into the Reticulated Quality control compartment
Presenting author:
ZMBH - Heidelberg University, , Im Neuenheimer Feld 345, D-69120 Heidelberg [DE], c.peselj@zmbh.uni-heidelberg.de
Author(s):
Carlotta Peselj
The spatial sequestration of excess misfolded proteins into distinct subcellular deposits has
emerged as integral part of quality control in the cytoplasm. Though the endoplasmic reticulum
(ER) serves as biogenesis hub for one third of a cell’s proteome, a role for spatial quality control
in ER proteostasis remains unclear. Here, we report that loss of the ER-resident protein Snd3
unveils the Reticulated Quality control compartment (ReQ) that sequesters aggregated
membrane proteins. ReQ is decorated by the disaggregase Hsp104. Further increasing the
protein misfolding load by inactivation of ER quality control pathways, such as ER-associated
degradation, resulted in massive ReQ formation and widespread cell death. Enabling ER
membrane expansion reduced protein aggregation, compacted ReQ and restored cellular
viability. Thus, ReQ safeguards ER proteostasis by partitioning misfolded ER membrane
proteins into a spatial quality control compartment.
Structural and functional interaction of IRAG1 with IP₃R1
Presenting author:
University Regensburg, Chemistry and Pharmacology, Universitätsstraße 31, 93053 Regensburg [DE], robert.peter@ur.de
Author(s):
Robert Peter, Leonhard Jakob, Jens Schlossmann
The NO/cGMP signalling is important in various physiological processes such as smooth muscle relaxation and hemostasis. These effects are mediated by cGMP-dependent protein kinases - like cGMP-dependent protein kinase 1β (cGK1β), which selectively phosphorylate proteins. One of these substrates is the IP3R1-associated cGMP kinase substrate protein (IRAG1), which forms a macro complex with cGK1β and the intracellular calcium channel inositol trisphosphate receptor type I (IP3R1). Activation of cGK1β, which phosphorylates IRAG1 at serine 683&696, ultimately reduces calcium efflux. So far, it has been shown that IRAG1 interacts with IP3R1 via its coiled-coil domain. However, it is still unclear which region of IP3R1 interacts with IRAG1 and which molecular mechanisms underlie this interaction.
Our results show using Strep-Tactin XT pulldown an interaction of IRAG1 with IP3R1 in the N-terminal region of amino acids 1581-1903. In contrast, 8-AET-cGMP pulldown shows an interaction of IP3R1 with IRAG1 both in N-terminal and C-terminal regions. Hence, cGMP stimulation may cause the macro complex to undergo a certain conformational change, resulting in the formation of new interaction regions.
These data suggest a possible interaction of IRAG1 with IP3R1 in the N-terminal region. In addition, activation of NO-cGMP signalling could lead to the formation of a further interaction region through molecular mechanisms such as conformational changes.
__________Investigation of the S. cerevisiae Start checkpoint and its regulation under acute carbon starvation
Presenting author:
Universität Tübigen, Cell Biology, Auf der Morgenstelle 15, 72076 Tübingen [DE], anastasia.petropoulou@student.uni-tuebingen.de
Author(s):
Anastasia Petropoulou, Deniz Irvali, Jennifer Ewald
G1 cells must decide whether to initiate a new division cycle, at a G1/S checkpoint termed Start in yeast and Restriction Point in mammals. Whether a cell begins a new round of division is determined in part by nutrient availability. When cells face nutrient starvation, the G1/S checkpoint prohibits transition into S phase and cells arrest at G1. In Saccharomyces cerevisiae the critical point for a cell to pass Start is nuclear export of the inhibitor Whi5. Once 50% of Whi5 has been exported, the cell is considered irreversibly committed to a new cycle. What happens however if a cell after Start is faced with acute starvation?
To answer this question, we use a microfluidics system for live cell imaging of asynchronous cells, to track fluorescently tagged proteins involved in the regulation of Start, in nutrient-rich and starvation conditions.
With this setup, we recently showed that post-Start cells facing acute carbon starvation within 20 minutes after Start can re-import Whi5 to the nucleus. These cells return to a pre-Start state, become sensitive to mating pheromone again and re-activate CDK when nutrients are available once more. However, through which mechanism cells interrupt the G1/S transition and re-import Whi5 is still unclear. We are now developing single-cell reporters to uncover the upstream signals.
__________Characterization of spindle ER determinants
Presenting author:
Heidelberg University Biochemistry Center, Biochemie-Zentrum, Im Neuenheimer Feld 328, 69120 Heidelberg [DE], martijn.plug@bzh.uni-heidelberg.de
Author(s):
Martijn Plug, Anne-Lore Schlaitz
The endoplasmic reticulum (ER) is a large single organelle that spans across the entire cell, enclosing the chromatin and forming membrane contact sites with other organelles. During mitosis we have observed ER tubules dynamically enter and exit the organelle-free spindle area. We have termed this subdomain of the ER spindle ER. Spindle ER forms during metaphase, and often approaches the kinetochore, the attachment site for microtubules on the chromatin. This led us to hypothesize that spindle ER may interface with the kinetochore, potentially to assess mitotic progression and/or to deliver and remove proteins from the kinetochore. To uncover proteins involved in spindle ER emergence, I screened a couple of candidates through knockdown essays. These candidates are chromatin associated proteins, or nuclear envelope proteins enriched on spindle ER. From these, spindly depletion showed the strongest reduction of spindle ER formation, and is a promising target to further study the ER-kinetochore interaction. We also hypothesized that the Ran-GTP gradient could play a role in spindle ER induction. Disruption of the Ran-GTP gradient altered overall ER structure, possibly through the perturbation of the mitotic spindle organization. Lastly, BioID or other proximity assays could help determine if there is a direct interaction between the kinetochore and the ER, and which factors are involved. This way we hope to unravel the interplay between the ER and the mitotic spindle in mitosis.
__________Optimized ultrathin embedding protocol to trace rare trafficking organelles in neuronal and other cells.
Presenting author:
, , , [0], Puchkov@fmp-berlin.de
Author(s):
Max Lucht, Alexander Stockhammer, Sila Rizalar, Francesca Bottanelli, Martin Lehmann, Volker Haucke, Dmytro Puchkov
Extensive developments in fluorescently tagged proteins allowed to study intracellular trafficking in amazing detail and dynamics. Combining light microscopy data with subsequent FIBSEM imaging, allows to complement different protein localizations with subcellular and compartment ultrastructure context delivered by electron microscopy observation. Here we would like to present our approach for correlative light electron microscopy. Our adapted ultrathin embedding protocol and internal fiducial selection has delivered very reliable navigation and alignment results. Thus, we could successfully track individual transport compartments responsible for axonal anterograde transport (Rizalar et al., 2023) as well as to characterize arf1 positive compartment acting upstream of trans-Golgi network (Stockhammer et al., 2023). Presented CLEM workflow, although is hunted by some limitations associated with fixation and imaging methods used per ce, represents a straightforward CLEM approach, affordable to most of cellular imaging facilities.
Rizalar et al., 2023
https://www.science.org/doi/10.1126/science.adg1075
Stockhammer et al., 2023
https://www.biorxiv.org/content/10.1101/2023.10.27.564143v1
doi: https://doi.org/10.1101/2023.10.27.564143
__________
A modular cloning (MoClo) toolkit for reliable intracellular protein targeting in Saccharomyces cerevisiae
Presenting author:
RPTU Kaiserslautern-Landau, Standort Kaiserslautern, AG Zellbiologie, Erwin-Schrödinger-Straße 13, 67663 Kaiserslautern [DE], simakin@rhrk.uni-kl.de
Author(s):
Pavel Simakin, Christian Koch, Johannes M. Herrmann
The method of Modular Cloning (MoClo) allows the combinatorial assembly of plasmids from standardized genetic parts. It is a very powerful strategy which enables highly flexible expression patterns without the need of repetitive cloning procedures. In this study, we describe an advanced MoClo toolkit that is designed for the yeast Saccharomyces cerevisiae and optimized for the targeting of proteins of interest to specific cellular compartments. We designed signals directing proteins to all of the mitochondrial subcompartments with a high degree of specificity by comparing various targeting sequences. Furthermore, we optimized the subcellular targeting by controlling expression levels using a collection of different promoter cassettes. the MoClo strategy allows it to generate arrays of expression plasmids in parallel to optimize gene expression levels and reliable targeting for each given protein and cellular compartment. Consequently, the MoClo strategy enables the generation of protein-expressing yeast plasmids that accurately target proteins of interest to various cellular compartments.
__________Illuminating biology with fluorescent RNAs
Presenting author:
Heidelberg Universität, Institute of Pharmacy and Molecular Biotechnology, Im Neuenheimer Feld 364, 69120 Heidelberg [DE], msunbul@uni-heidelberg.de
Author(s):
Murat Sünbül
We have recently developed RhoBAST1, a genetically encoded fluorescent RNA tag that binds fluorogenic rhodamine dyes2 with exceptionally rapid association and dissociation kinetics. These quick binding and unbinding events cause intermittent fluorescence emission (blinking), facilitating super-resolution imaging of RNAs tagged with RhoBAST by single-molecule localization microscopy (SMLM).1,2 Furthermore, the fast and continuous dye exchange is particularly beneficial for STED microscopy, which commonly faces issues with fluorophore photobleaching.2 The rapid exchange kinetics allow bleached dyes to be replaced by fresh ones within seconds (or less), thereby restoring the fluorescence signal.
Here, I will demonstrate the remarkable potential and versatility of RhoBAST technology for imaging and tracking3 not only RNA but also DNA and proteins using various super-resolution imaging techniques.4
[1] Sunbul et al. “Super-resolution RNA imaging using a rhodamine-binding aptamer with fast exchange kinetics” Nature Biotechnology, 2021, 39, 686
[2] Englert et al. “Fast-exchanging spirocyclic rhodamine probes for aptamer-based super-resolution RNA imaging” Nature Communications, 2023, 14, 3879
[3] Bühler et al. “Avidity-based bright and photostable light-up aptamers for single-molecule mRNA imaging” Nature Chemical Biology, 2023, 19, 478
[4] Grün et al. “Super-resolved protein imaging using bifunctional light-up aptamers” bioRxiv, 2024, 2024.01.26.577321
__________Cryo-EM reveals the compositional landscape of the mammalian mitochondrial respiratory supercomplexes
Presenting author:
Forschungszentrum Jülich GmbH, Ernst Ruska Centre For Microscopy and Spectroscopy with Electrons, Wilhelm-Johnen-Straße , 52428 Jülich [DE], i.vercellino@fz-juelich.de
Author(s):
Irene Vercellino, Leonid Sazanov
The mitochondrial respiratory chain (MRC) is central to mammalian metabolism. The MRC complexes are functional in isolation but associate in the membrane in high-order structures named supercomplexes. Important open questions regarding the assembly and composition of supercomplexes need to be addressed to elucidate the physiological role of supercomplexes in metabolism. Our cryo-EM work tackled the role that the supercomplex associated factor SCAF1 plays in supercomplexes assembly using murine mitochondria as mammalian model. SCAF1 is required for the assembly of supercomplex CIII2CIV, while its role in the formation of respirasome (supercomplex CICIII2CIV), featuring CIII2CIV alongside CI, is unclear. SCAF1 expression has been linked to differential fitness in humans and mice and to cancer, making it a relevant target for human health. Our structures showed that SCAF1 N-terminus inserts into CIII2, while the C-terminus binds CIV, keeping CIII2 and CIV together during the CIII2CIV assembly and in the mature form and biochemical assays proved that CIII2 and CIV gain catalytic advantage when assembled in the supercomplex. We also confirmed that SCAF1 is not present in the canonical respirasome, but we found it bound to a newly identified respirasome, explaining why SCAF1 is biochemically detected in respirasome-like species. Thus, our structures reveal the SCAF1-driven compositional landscape of mammalian supercomplexes and suggest how they can fine-tune mammalian metabolism.
__________Identification of candidates from high-throughput proteomic profiling of PROTAC-induced degradation
Presenting author:
Goethe Universitaet Frankfurt, Molecular Systems Medicine, Theodor-Stern-Kai 7, 60596 Frankfurt am Main [DE], a.verheyden@med.uni-frankfurt.de
Author(s):
Nikita Verheyden, Giulio Ferrario, Jasmin Schäfer, Bhavesh Parmar, Christian Münch
Advances in mass spectrometry-based high-throughput proteomics have made it possible to perform rapid quantitative proteomic screens for small molecules in pharmacology. Their effects are sometimes difficult to predict and many variants can be designed and produced cheaply. A specific application is their use in targeted proteolysis by facilitating proximity between the E3 ligase complex and the target protein. This leads to ubiquitination and subsequent degradation of the target protein. These molecules are known as proteolysis targeting chimeras (PROTACs).
To determine both degradation levels and translational effects, we employ multiplexed enhanced protein dynamics (mePROD) using SILAC labels. Using a 96-well plate to screen multiple compounds allows us to rapidly generate proteome-wide data of both steady-state and newly translated protein levels for a large number of candidates.
However, this large amount of data requires an effective processing pipeline to find relevant effects within the screening data. Changes in effects are visualized using complex plots, such as circus or hive plots, to show overall trends without losing much detail. However, to improve the process of discovering relevant effects in our datasets, we developed a scoring system to identify new candidates. This score includes several layers of information, such as fold changes for all proteins across samples, and determines direct and indirect effects of the PROTAC.
Microglia signs to distinct intracellular events upon acute inflammation
Presenting author:
Heidelberg University, Centre for Organismal Studies, Im Neuenheimer Feld 230, 69120 Heidelberg [DE], venera.weinhardt@cos.uni-heidelberg.de
Author(s):
Anthoula Chatzimpinou, Ayse Erozan, Venera Weinhardt
Neurodegenerative diseases concern each of us. To tackle this fundamental problem, we need to understand how the immune cells commonly orchestrate the events that prevent neuronal damage. Microglia are such cells that get involved in acute pro- or anti-inflammation, which have been linked to neuronal degeneration or restoration due to different cytokine expressions. Of this involvement, they have been named as pro-inflammatory (M1) or anti-inflammatory (M2a) microglia. The intracellular drive for switching microglia to each of these states is not yet known in the overview of their cell anatomy. We hypothesize that specific cell anatomical rearrangements signify a transient activation of M1 or M2a microglia. In this work, we examine the different organelle contents inside single cells in 3D, at a 50 nm spatial resolution, by using quantitative soft X-ray tomography. Based on the follow-up analysis, we connect these intracellular changes with the M1 and M2a-activated cell proportions by correlative light microscopy. Deepening our understanding of microglia switchability between states, we propose a common mechanistic pathway that describes these processes with the respective type of activation as a potential target for precise immunotherapy.
__________Soft X-ray tomography reaches its full potential
Presenting author:
Heidelberg University, Centre for Organismal Studies, Im Neuenheimer Feld 230, 69120 Heidelberg [DE], venera.weinhardt@cos.uni-heidelberg.de
Author(s):
Venera Weinhardt
Soft X-ray tomography (SXT) is a unique imaging technique that visualizes entire mammalian cells at nanometer resolution with minimal sample preparation. Using soft X-rays (284-543 eV), SXT provides quantitative protein content information in cells' native state without fixation or staining. SXT microscopes have evolved to offer higher resolutions, switchable modes, and alternative acquisition schemes to accommodate diverse biological samples. Factors affecting imaging capabilities include X-ray transmission, depth of field, and field of view. Recent advancements allow imaging of cells up to 18µm thick. A "half-acquisition" strategy has extended SXT capabilities, enabling imaging of whole cells in pathological conditions at twice the previously reported size. This advancement visualizes cellular remodeling and structural changes associated with various pathological states, providing insights into disease mechanisms at the subcellular level. SXT imaging allows researchers to investigate pathology-induced changes in host cells and understand cellular remodeling quantitatively. While access to full-rotation SXT microscopes is limited, laboratory-based systems are expanding accessibility. Ongoing developments in SXT technology at synchrotron and laboratory sources are paving the way for high-throughput 3D imaging of cells in healthy and pathological conditions, offering unprecedented opportunities to study cellular mechanisms in various disease states.
__________Structural insights into the activation mechanism of antimicrobial GBP1
Presenting author:
Max Delbrück Center for Molecular Medicine, , Robert-Rössle-Straße 10, 13125 Berlin [DE], marius.weismehl@mdc-berlin.de
Author(s):
Marius Weismehl, Xiaofeng Chu, Miriam Kutsch, Paul Lauterjung, Christian Herrmann, Misha Kudryashev, Oliver Daumke
The dynamin-related human guanylate-binding protein 1 (GBP1) mediates host defense against microbial pathogens. Upon GTP binding and hydrolysis, auto-inhibited GBP1 monomers dimerize and assemble into soluble and membrane-bound oligomers, which are crucial for innate immune responses. How higher-order GBP1 oligomers are built from dimers and how assembly is coordinated with nucleotide-dependent conformational changes has remained elusive. We present cryo-electron tomography data of the membrane-bound GBP1 oligomer demonstrating that GBP1 assembles in an outstretched dimeric conformation. By combining new and published structural insights with biochemical, mechanistic, and pathogen-based data, our study provides the molecular basis for understanding GBP-mediated antimicrobial functions. Our results reveal a sophisticated activation mechanism for GBP1 in which nucleotide-dependent structural changes coordinate dimerization, oligomerization, and membrane binding to allow encapsulation of pathogens with an antimicrobial protein coat. In this way, our structure-function study deepens our understanding of the underlying molecular coupling of the GTPase cycle and oligomerization which is crucial for GBP’s antimicrobial functions.
__________The cellular fate of RNA-nanoparticles - uptake, release & translation
Presenting author:
BioNTech SE, Analytical Development, An der Goldgrube 12, 55131 Mainz [DE], Anke.Westenberger@biontech.de
Author(s):
Anke Westenberger
The detailed cellular mechanisms of RNA-nanoparticle uptake, endosomal escape, RNA release, processing, and translation are not fully understood to this day. One major bottleneck for the efficient translation of nanoparticle-delivered mRNA is the uptake and endocytic escape. The aim of this project is to gain a deeper understanding of the underlying uptake and endosomal escape mechanisms of different RNA-nanoparticle delivery systems currently in use to potentially improve their effectiveness. This approach implements super resolution (STORM) and high content screening microscopy (confocal imaging) of different cell systems including target primary cells like dendritic cells. With these tools we want to analyse the localisation of nanoparticles during uptake and endosomal escape at a nanoscale level. Live-cell microscopy and multi-colour imaging complement these investigations. To quantify colocalization between nanoparticle components and cellular compartments that indicate relevant points of endocytosis, a fully automated and KI-based software is established within the analytical imaging pipeline. Detailed time-resolved studies demonstrate the route of the nanoparticle through different endosomal compartments, ending with either endosomal escape or lysosomal degradation. By tracing the intracellular pathways, we aim to explain variations in the efficiencies of different nanoparticle systems.
__________A new task for eIF4A1: A RNA helicase in chromatin decondensation
Presenting author:
RWTH Aachen, Biochemistry and Molecular Cell Biology, Pauwelsstraße 30, 52074 Aachen [DE], sabwiesmann@ukaachen.de
Author(s):
Sabine Wiesmann, Ramona Jühlen, Wolfram Antonin
During mitosis, chromatin undergoes tremendous changes, starting with condensation into tightly packed chromosomes. During mitotic exit, decondensation is essential to reestablish the nuclear structure and transcription. A search for chromatin decondensation factors in a cell-free assay identified the eukaryotic translation initiation factor eIF4A1. Depletion of eIF4A1 and its paralogue eIF4A2 blocks mitotic chromatin decondensation, whereas the addition of recombinant eIF4A1 or eIF4A2 rescues the phenotype. HeLa cells were analysed using high-throughput or high-resolution confocal live cell imaging. CellCognintion or Live Cell Miner analysis revealed prolonged telophase and impaired chromatin decondensation upon eIF4A1/2 knockdown. Forced localization of eIF4A1 to mitotic chromatin enhances decondensation.
Mitotic chromosomes are covered by a layer containing nuclear and nucleolar proteins and RNAs. This chromosomal periphery is progressively displaced and rearranged into pre-nucleolar bodies during mitotic exit. eIF4A1/2 RNAi prolonged RNA retention on chromosomes at mitotic exit. In vitro, eIF4A1 disassembled RNA-protein condensates and eIF4A1 RNA binding but not ATP hydrolysis, is critical for this. In the cell-free decondensation assay, eIF4A1 requires similarly RNA binding but not ATPase activity. We propose that, during mitotic exit, eIF4A1 acts as an RNA chaperone, resolving RNA condensates on the surface of chromosomes, which is important for chromatin decondensation.
__________Poster Teasers
1. Caroline Haupt /Halle
Fluorescence and electron microscopy study to elucidate the structure of Arf1-coated membrane tubules
2. Cilian Kock /Osnabrück
Elucidating the Roles of Glutathione and Reactive Oxygen Species in Basal Land Plant Development through Redox Sensor Microscopy
3. Yury Bykov /Kaiserslautern
A systematic bi-genomic split-GFP assay illuminates the mitochondrial matrix proteome and protein targeting routes ()
4. Dmytro Puchkov /Berlin
Optimized ultrathin embedding protocol to trace rare trafficking organelles in neuronal and other cells
5. George Broutzakis /Münster
High-resolution structures of the UapA elevator-type purine transporter rationalize the importance of the cytosolic N-terminus in trafficking and transport activity
6. Margret Bülow /Düsseldorf
Expansion microscopy and MALDI Imaging approaches to investigate inter-organelle communication
7. William Durso /Jena
Restoring ER morphology and lysosome dynamics as therapeutic targets for axonopathies
8. Laurell Kessler /Frankfurt
Self-quenched fluorophore labels for DNA-paint and STED microscopy
9. Michael Holtmannspötter /Osnabrück
The reHaloTag: Reversible labeling for live cell long-term high- and superresolution imaging
10. Rafaella Jekabson /Münster
Actin reorganization controls different phases of Plasma Membrane Repair
11. Arun Kumar Kondadi /Düsseldorf
Live-cell STED super-resolution nanoscopy reveals modulation of cristae dynamics in bioenergetically compromised mitochondria
12. Lea Kunz /Homburg
Development of a ratiometric biosensor to measure ciliary cAMP levels and to elucidate Paladin1 accumulation in the primary cilia
13. Moritz Lehners /Tübingen
Single-cell imaging identifies cGMP signalling heterogeneity in living VSMCs
14. Fabian Lukas /Kaiserslautern
Canonical and non-canonical integrin-based adhesions dynamically interconvert
15. Verian Bader /Bochum
Visualization of Protein Quality Control Processes at Aggregate Interfaces
16. Marius Weismehl /Berlin
Structural insights into the activation mechanism of antimicrobial GBP1
Abstracts
Ada Yonath Lecture:
Title to be announced
Stefan Hell
Göttingen, Germany
Abstract not submitted yet
Abstracts
Recording the physiological history of cells with fluorescence labeling
Kai Johnsson
Heidelberg, Germany
Recordings of the physiology of cells provide insights into biological processes, yet obtaining such recordings is a challenge. To address this challenge, I will discuss a method to record transient cellular events for later analysis. The method is based on designed proteins that become labeled in the presence of both a specific physiological activity and a fluorescent substrate. The recording period is set by the presence of the substrate, whereas the physiological activity controls the degree of the labeling. The use of substrates of different colors enables the recording of successive periods of activity. Applications include the recording of protein-protein interactions, receptor activation and elevations in intracellular calcium. The recording of elevated calcium levels can be used for the sorting of cells from heterogenous populations according to their calcium levels for transcriptomic analysis, or for the tracking of neuronal activities in vivo.
Abstracts
Modular dual-color BiAD sensors for locus-specific readout of epigenome modifications in single cells
Anja R. Köhler
University of Stuttgart Germany, Institute of Biochemistry and Technical Biochemistry, Department of Biochemistry, Allmandring 31, 70569 Stuttgart [DE], anja.koehler@ibtb.uni-stuttgart.de
Author(s):
Anja R. Köhler, Pavel Bashtrykov, Albert Jeltsch
The precise spatio-temporal expression of genes in mammalian cells is regulated by epigenomic modifications on DNA and histones. To understand the dynamic changes in these modifications that occur during differentiation and disease onset in live cells, we developed epigenetic sensors to visualize locus-specific epigenome modifications via fluorescence microscopy [1]. The Bimolecular Anchor Detector (BiAD) technology combines an sgRNA/dCas9 complex as a programmable DNA-binding anchor with chromatin reader domains binding specific epigenome modifications as detector modules. Both modules are fused to complementary parts of a split fluorophore, reconstituting a full fluorophore upon binding in close proximity. To further enhance the sensors’ sensitivity towards a broader range of endogenous target loci, we recruited a second, full fluorophore to the sgRNA to mark the genomic locus of interest [2]. This novel dual-color readout, combined with an expanded detector module palette, enabled the quantitative analysis of various key epigenome modifications. We showcased applications of the dual-color BiAD technology in development and disease research by detecting dynamic changes in locus-specific DNA methylation upon inhibitor treatment and dissecting the enzymatic network responsible for the deposition of a critical repressive histone modification at a specific locus on the female inactive X chromosome.
[1] Lungu et al., 2017, Nat. Comm.
[2] Koehler et al., 2024, Cell Reports Methods
Abstracts
Illuminating biology with fluorescent RNAs
Murat Sünbül
Heidelberg Universität, Institute of Pharmacy and Molecular Biotechnology, Im Neuenheimer Feld 364, 69120 Heidelberg [DE], msunbul@uni-heidelberg.de
Author(s):
Murat Sünbül
We have recently developed RhoBAST1, a genetically encoded fluorescent RNA tag that binds fluorogenic rhodamine dyes2 with exceptionally rapid association and dissociation kinetics. These quick binding and unbinding events cause intermittent fluorescence emission (blinking), facilitating super-resolution imaging of RNAs tagged with RhoBAST by single-molecule localization microscopy (SMLM).1,2 Furthermore, the fast and continuous dye exchange is particularly beneficial for STED microscopy, which commonly faces issues with fluorophore photobleaching.2 The rapid exchange kinetics allow bleached dyes to be replaced by fresh ones within seconds (or less), thereby restoring the fluorescence signal.
Here, I will demonstrate the remarkable potential and versatility of RhoBAST technology for imaging and tracking3 not only RNA but also DNA and proteins using various super-resolution imaging techniques.4
[1] Sunbul et al. “Super-resolution RNA imaging using a rhodamine-binding aptamer with fast exchange kinetics” Nature Biotechnology, 2021, 39, 686
[2] Englert et al. “Fast-exchanging spirocyclic rhodamine probes for aptamer-based super-resolution RNA imaging” Nature Communications, 2023, 14, 3879
[3] Bühler et al. “Avidity-based bright and photostable light-up aptamers for single-molecule mRNA imaging” Nature Chemical Biology, 2023, 19, 478
[4] Grün et al. “Super-resolved protein imaging using bifunctional light-up aptamers” bioRxiv, 2024, 2024.01.26.577321
Abstracts
Live-cell super resolution microscopy of mitochondrial dynamics
Tatjana Kleele
ETH Zuerich, Department of Biology, Institute of Biochemistry, Otto-Stern-Weg 3, 8093 Zürich [CH], tatjana.kleele@biol.ethz.ch
Author(s):
Tatjana Kleele
Mitochondria perform a multitude of metabolic, biosynthetic and signaling functions. While initially perceived as isolated, static organelles, convergent advances in light microscopy revealed that mitochondria form an interconnected network, communicate with other organelles and rapidly modulate their structure. Mitochondrial morphology is shaped by opposing events of fission and fusion, which are tightly regulated. Disruptions in these processes can lead to bioenergetic defects and are linked to a variety of human diseases. But despite the inevitable link between mitochondrial structure and function, it remains unclear how these organelles integrate cellular cues to remodel their network structure, consequently adjusting their function. Broadly, our group investigates how mitochondria are dynamically remodeled to support cellular metabolism and tissue specific functions. For this we employ a range of super-resolution microscopy techniques, automated AI-based data analysis, combined with different model systems, including bone marrow derived macrophages and iPSC derived neurons. Using this approach, we investigate how mitochondria are dynamically remodeled in different metabolic conditions, during cell state transitions as well as during neuronal differentiation and maturation. Moreover, we are currently developing a super-resolution based imaging screen to identify novel protein kinases regulating mitochondrial fission and fusion.
Abstracts
FEBS National Award:
Discoveries in cellular architecture by correlative microscopy
Wanda Kukulski
Bern, Switzerland
Abstract not submitted yet
Abstracts
In situ structural analysis of the nuclear pore complex
Martin Beck
Frankfurt am Main, Germany
Abstract not submitted yet
Abstracts
From DNA Nanotechnology to biomedical insight: Towards single-molecule spatial omics
Ralf Jungmann
Martinsried, Germany
Abstract not submitted yet
Abstracts
Cryo-EM reveals the compositional landscape of the mammalian mitochondrial respiratory supercomplexes
Irene Vercellino
Forschungszentrum Jülich GmbH, Ernst Ruska Centre For Microscopy and Spectroscopy with Electrons, Wilhelm-Johnen-Straße , 52428 Jülich [DE], i.vercellino@fz-juelich.de
Author(s):
Irene Vercellino, Leonid Sazanov
The mitochondrial respiratory chain (MRC) is central to mammalian metabolism. The MRC complexes are functional in isolation but associate in the membrane in high-order structures named supercomplexes. Important open questions regarding the assembly and composition of supercomplexes need to be addressed to elucidate the physiological role of supercomplexes in metabolism. Our cryo-EM work tackled the role that the supercomplex associated factor SCAF1 plays in supercomplexes assembly using murine mitochondria as mammalian model. SCAF1 is required for the assembly of supercomplex CIII2CIV, while its role in the formation of respirasome (supercomplex CICIII2CIV), featuring CIII2CIV alongside CI, is unclear. SCAF1 expression has been linked to differential fitness in humans and mice and to cancer, making it a relevant target for human health. Our structures showed that SCAF1 N-terminus inserts into CIII2, while the C-terminus binds CIV, keeping CIII2 and CIV together during the CIII2CIV assembly and in the mature form and biochemical assays proved that CIII2 and CIV gain catalytic advantage when assembled in the supercomplex. We also confirmed that SCAF1 is not present in the canonical respirasome, but we found it bound to a newly identified respirasome, explaining why SCAF1 is biochemically detected in respirasome-like species. Thus, our structures reveal the SCAF1-driven compositional landscape of mammalian supercomplexes and suggest how they can fine-tune mammalian metabolism.
Abstracts
Microscopy-based systematic approaches to identify new players in lipid droplet biology
Maria Bohnert
Münster, Germany
Lipid droplets (LDs) are ubiquitous organelles that fulfil central roles in lipid storage and metabolism. LD dysfunction is related to human diseases including lipodystrophy, obesity, diabetes, fatty liver, and cardiovascular diseases. Despite their (patho-)physiological relevance, key LD biology processes including organelle formation, degradation, motility, protein targeting, and communication with the rest of the cell are incompletely understood. My lab uses microscopy-based genome-wide screening approaches in the yeast Saccharomyces cerevisiae to identify unknown players in organelle biology. We identify molecular mechanisms underlying the life cycle of the LD.
Abstracts
Soft X-ray tomography reaches its full potential
Venera Weinhardt
Heidelberg University, Centre for Organismal Studies, Im Neuenheimer Feld 230, 69120 Heidelberg [DE], venera.weinhardt@cos.uni-heidelberg.de
Author(s):
Venera Weinhardt
Soft X-ray tomography (SXT) is a unique imaging technique that visualizes entire mammalian cells at nanometer resolution with minimal sample preparation. Using soft X-rays (284-543 eV), SXT provides quantitative protein content information in cells' native state without fixation or staining. SXT microscopes have evolved to offer higher resolutions, switchable modes, and alternative acquisition schemes to accommodate diverse biological samples. Factors affecting imaging capabilities include X-ray transmission, depth of field, and field of view. Recent advancements allow imaging of cells up to 18µm thick. A "half-acquisition" strategy has extended SXT capabilities, enabling imaging of whole cells in pathological conditions at twice the previously reported size. This advancement visualizes cellular remodeling and structural changes associated with various pathological states, providing insights into disease mechanisms at the subcellular level. SXT imaging allows researchers to investigate pathology-induced changes in host cells and understand cellular remodeling quantitatively. While access to full-rotation SXT microscopes is limited, laboratory-based systems are expanding accessibility. Ongoing developments in SXT technology at synchrotron and laboratory sources are paving the way for high-throughput 3D imaging of cells in healthy and pathological conditions, offering unprecedented opportunities to study cellular mechanisms in various disease states.
Abstracts
Not submitted yet
Bonnie Murphy
Max Planck Institute of Biophysics
Abstract not submitted yet
Abstracts
Fast, long-time STED imaging of endoplasmic reticulum dynamics living cells
Ashwin Balakrishnan
Goethe - Universität Frankfurt, Institute of Physical and Theoretical Chemistry, Max-von-Laue str. 7, 60438 Frankfurt am Main [DE], balakrishnan@chemie.uni-frankfurt.de
Author(s):
Ashwin Balakrishnan, Johanna Rahm, Alexandra Kaminer, Mike Heilemann
Super-resolution microscopy techniques have been crucial in recent years in realizing structural cell biology a reality.However, biomolecules in a cell are highly dynamic and in order to assess these dynamics, two challenges need to be adressed: first, photobleaching limits the information that can be extracted from a cell. Second, phototoxicity leads to cell damage. Here, we demonstrate long-time STED microscopy with fast temporal resolution in living cells. We achieve this by employing very low irradiation intensities, and restore information by using convolutional neural network-based image denoising. We demonstrate live-cell imaging for hours with a temporal resolution of seconds. We use this approach to study the dynamics of the endoplasmic reticulum (ER) in healthy and autophagy-induced cells. By extending this further with a neural network-based segmentation, we quantify morphological rearrangements. We find that the ER is highly dynamic and visualize morphological changes under stress-induced conditions that have proved elusive before. In summary, we present an experimental approach that can be transferred to other live-cell super-resolution organelle dynamics.
Abstracts
Visualizing the structure and dynamics of mitochondria at the nanoscale
Till Stephan
Frankfurt am Main, Germany
Abstract not submitted yet
Abstracts
Highly Multiplexed Imaging with Speed and Fluorogenic DNA-PAINT (preliminary)
Jörg Bewersdorf
New Haven, USA
Abstract not submitted yet
GBM Compact - Workshops
Workshop 1:
Research Data Management for Microscopy and BioImage Analysis
- Introduction to BioImaging Research Data Management, NFDI4BIOIMAGE and I3D:bio
Christian Schmidt /DKFZ Heidelberg - OMERO as a tool for bioimaging data management
Tom Boissonnet /Heinrich-Heine Universität Düsseldorf - Reproducible image analysis workflows with OMERO software APIs
Michele Bortolomeazzi /DKFZ Heidelberg - Publishing datasets in public archives for bioimage data
Ksenia Krooß /Heinrich-Heine Universität Düsseldorf
Date & Venue:
Thursday, Sept. 26, 5.30 p.m.
Haus 22 / Paul Ehrlich Lecture Hall (H22-1)
Workshop 2:
Choosing the Most Suitable Imaging Approach: Towards an Application Guideline
- From high resolution to high content - how imaging techniques help us to explore cellular structures, dynamics and function
Stefanie Weidtkamp-Peters /Heinrich-Heine Universität Düsseldorf &
Thomas Zobel /Universität Münster
Date & Venue:
Thursday, Sept. 26, 5.30 p.m.
Haus 22 / Franz Volhard Lecture Hall (H22-2)
GBM Young Investigator Networking Event
Join us for the first in-person meeting for all AK Young Investigators! Seize the opportunity to meet your peers, network, and chat about science and life as an group leader - and – enjoy complimentary drinks and snacks!
All members of the GBM AK Young Investigators are welcome, also if you did not register for GBM compact.
Please let us know whether you would like to attend (for free!) by sending an email to young.investigators@gbm-online.de
We look forward to seeing all of you there!
Date & Venue:
University Hospital Frankfurt
Theodor-Stern-Kai 7
60598 Frankfurt am Main
Haus 22 / Paul Ehrlich Lecture Hall (H22-1)
Thursday, Sept. 26, 7 p.m.
Funding Opportunities for Postdocs
The newly founded interest group ‘GBM Postdocs’ aims to build a strong network for postdocs to facilitate exchange and support during this fascinating and challenging phase of the career.
Our first event will take place as a satellite event at the GBM Compact Meeting.
We have organized two talks on ‘Funding Opportunities for Postdocs’ provided by the Graduate Academy Frankfurt and the DFG, with plenty of time for questions and discussions. PhD students, who are at the end of their doctorate and are looking for the next step are welcome as well.
Registration for the conference is not mandatory, but is advantageous as places for the event are limited and conference participants will be given priority, if the event is overbooked
The event is free of charge, but registration using the following form is necessary.
Venue & Date:
University Hospital Frankfurt
Theodor-Stern-Kai 7
60598 Frankfurt am Main
Haus 22 / Paul Ehrlich Lecture Hall (H22-1, The room may still change. Please check this website on the day of the event)
Friday, Sept. 27, 2 p.m.
Abstracts
M 05 - Aerts, Jordan
G 11 - Astorga, Jose
G 04 - Bender, Julian
G 14 - Bourke, Ashley
G 18 - Bozkurt, Süleyman
M 11 - Cabrera-Orefice, Alfredo
G 13 - Cavalcanti Franco, Pedro Henrique
G 25 - Chakraborty, Rahul
G 24 - Desch, Kristina
O 01 - Ecker, Anna-Lena
G 02 - Eirich, Juergen
O 05 - Farges, Frederic
G 22 - Fehmer, Martin
M 09 - Gerwien, Maximilian
G 12 - Gupta, Nikita
C 01 - Hamed, Mohamed Ismail
S 04 - Hesselbarth, Julia
M 12 - Hofacker, Daniel
G 03 - Höhlschen, Julia
O 04 - Jooyeh, Bahareh
M 04 - Kaspar-Schoenefeld, Stephanie
G 15 - Kohli, Aneesha
G 17 - Kokoli, Marianna
G 09 - Kotnik, Florian
M 03 - Kühl, Toni
G 05 - Lenhard, Svenja
G 29 - Lenz, Thomas
S 01 - Lermyte, Frederik
M 02 - Li, Chengkang
G 08 - Li, Mujia
G 21 - Lichtner, Simone
M 06 - Link, Martin
G 26 - Lucht, Max Thomas
G 28 - Meier-Credo, Jakob
M 08 - Mick, David
G 16 - Mohseni, Farbod
G 10 - Müller, Torsten
M 01 - Omage, Sylvia
O 03 - Pandrea, Maria
C 04 - Pauls, Stella
B 01 - Rrustemi, Trendelina
G 06 - Russo, David A.
M 10 - Schmidt, Andreas
G 23 - Schmidt, Jonas
M 13 - Schroda, Michael
C 03 - Schwarzmüller, Luisa
O 02 - Simakin, Pavel
M 07 - Tascher, Georg
G 19 - van der Laan, Martin
G 30 - van Oostrum, Marc
G 01 - Vu, Van Loi
G 20 - Waselenchuk, Quinn
S 03 - Weir, John
M 09 - Welter, Anna Sophie
C 02 - Werner, Tilman
S 02 - Wittig, Ilka
G 27 - Wohlgemuth, Ingo
G 07 - Wolfgramm, Hannes
O 05 - Zöller, Jonathan
B 01 - Rrustemi, Trendelina
C 01 - Hamed, Mohamed Ismail
C 02 - Werner, Tilman
C 03 - Schwarzmüller, Luisa
C 04 - Pauls, Stella
G 01 - Vu, Van Loi
G 02 - Eirich, Juergen
G 03 - Höhlschen, Julia
G 04 - Bender, Julian
G 05 - Lenhard, Svenja
G 06 - Russo, David A.
G 07 - Wolfgramm, Hannes
G 08 - Li, Mujia
G 09 - Kotnik, Florian
G 10 - Müller, Torsten
G 11 - Astorga, Jose
G 12 - Gupta, Nikita
G 13 - Cavalcanti Franco, Pedro Henrique
G 14 - Bourke, Ashley
G 15 - Kohli, Aneesha
G 16 - Mohseni, Farbod
G 17 - Kokoli, Marianna
G 18 - Bozkurt, Süleyman
G 19 - van der Laan, Martin
G 20 - Waselenchuk, Quinn
G 21 - Lichtner, Simone
G 22 - Fehmer, Martin
G 23 - Schmidt, Jonas
G 24 - Desch, Kristina
G 25 - Chakraborty, Rahul
G 26 - Lucht, Max Thomas
G 27 - Wohlgemuth, Ingo
G 28 - Meier-Credo, Jakob
G 29 - Lenz, Thomas
G 30 - van Oostrum, Marc
M 01 - Omage, Sylvia
M 02 - Li, Chengkang
M 03 - Kühl, Toni
M 04 - Kaspar-Schoenefeld, Stephanie
M 05 - Aerts, Jordan
M 06 - Link, Martin
M 07 - Tascher, Georg
M 08 - Mick, David
M 09 - Welter, Anna Sophie
M 09 - Gerwien, Maximilian
M 10 - Schmidt, Andreas
M 11 - Cabrera-Orefice, Alfredo
M 12 - Hofacker, Daniel
M 13 - Schroda, Michael
O 01 - Ecker, Anna-Lena
O 02 - Simakin, Pavel
O 03 - Pandrea, Maria
O 04 - Jooyeh, Bahareh
O 05 - Farges, Frederic
O 05 - Zöller, Jonathan
S 01 - Lermyte, Frederik
S 02 - Wittig, Ilka
S 03 - Weir, John
S 04 - Hesselbarth, Julia
M 05
Comparison of HPLC and Capillary Electrophoresis for Hydrogen Exchange Mass Spectrometry
Presenting author:
Uppsala universitet, Pharmaceutical Biosciences, husargatan 3, 751 24 Uppsala [SE], jordan.aerts@uu.se
Author(s):
Jordan Aerts, Jonathan Zöller, Julian Langer, Erik Jansson
Hydrogen deuterium exchange mass spectrometry (HDX-MS) has been a valuable tool for structural proteomics studies for more than 30 years. Labeling of proteins with deuterium in solution is a straightforward experiment, but downstream sample handling steps should be conducted under quench conditions (low temperature and pH) to maximize the structural information obtained from protein and peptide measurement. Traditional HDX-MS workflows utilize low-temperature liquid chromatography (LC) with short gradients for peptide separations. However, operating an LC system at low temperatures generally suffers from increased sample carry-over, and the need for expensive system components. Cold capillary electrophoresis (CE) separations offer a low cost method of separating peptides at quench conditions for HDX-MS workflows. Here we present a direct comparison at the peptide level using bovine hemoglobin analyzed on both a laboratory-built CE platform and a fully automated Waters HDX-2 system, both measured with a Waters Synapt G2-Si. The quenched, and digested protein (10,000 fmol on column for LC, 50 fmol on capillary for CE) was measured after labeling in D₂O for 0, 50, 500, 5000, and 50,000 s. Preliminary results demonstrate similar deuterium uptake curves for proteolytic peptides detected across both separation methods, at significantly lower sample amounts. These findings validate the use of cold capillary electrophoresis as an alternative to HPLC in HDX-MS workflows.
Short talk 5
Investigation of the human lysosomal proteome by targeted proteomics
Presenting author:
University of Bonn, , Nussallee11, 53115 Bonn [DE], darora@uni-bonn.de
Author(s):
Dhriti Arora, Stephanie Kaspar-Schoenefeld, Andreas Schmidt, Dominic Winter
Lysosomes, the main lytic organelles of mammalian cells, play a vital role in cellular homeostasis. This is facilitated by ~340 lysosomal-related proteins whose loss of function can result in a variety of disorders. To study diseases and cellular processes related to lysosomes, reproducible quantification of these proteins is crucial. However, the low abundance of the organelle makes it difficult to quantify these proteins using untargeted proteomics. Therefore, DIA and targeted approaches such as PRM are the most effective methods for the detection of lysosomal proteins. In this study, we investigated the lysosomal proteome of four human cell lines by dia-PASEF, merging the benefits of DIA with the advantages of ion mobility in proteomics. To investigate the coverage of lysosomal proteins in whole-cell lysates and lysosome-enriched fractions, we used a targeted data processing library consisting of 297 manually selected lysosomal proteins. To assess the quantitative performance a lysosome-enriched sample was spiked into the whole cell lysate to simulate the constitutive upregulation of lysosomal proteins. A total of 165 lysosomal proteins showed significantly higher abundance indicating that dia-PASEF is well suited for analysing the lysosomal proteome, providing both good coverage and quantitative reproducibility of the targeted lysosomal proteins. Finally, we developed prm-PASEF assays based on our dia-PASEF analyses to enable targeted analysis of lysosomal proteins
G 11
Protein Synthesis in Autism Spectrum Disorder
Presenting author:
Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany, Proteomics , Blankestr 8A, 13403 Berlin [DE], jose.astorga@mdc-berlin.de
Author(s):
Jose Astorga
Autism spectrum disorder (ASD) is a complex, lifelong and highly prevalent neurodevelopmental disorder. ASD exhibits significant heterogeneity in genotypes and phenotypes. In recent years, dysregulation of protein synthesis has emerged as a convergent mechanism underlying ASD. The goal of this project is to better understand the pathophysiology of ASD by investigating clinically relevant mutations associated with protein synthesis. To achieve this goal, ASD-related mutations were introduced into induced pluripotent stem cells (iPSCs) derived from healthy donors. These iPSCs were then differentiated into neurons and their proteome were analysed by mass spectrometry. Given the highly polarised nature of neurons, investigating the local proteome of these cells is of great interest. For this purpose, neurons will be cultured using inserts that allow the soma to be separated from the neurites, which facilitates the analysis of the cell's local proteome. In addition, a pulsed stable isotope stable amino acid labelling in cell culture (SILAC) technique will be used to quantify changes in protein synthesis. Once these methodologies are established, cell lines carrying PTEN and TSC2 mutations, which are clinically relevant in ASD, will be incorporated into the study. Consequently, the results of this research are expected to elucidate the molecular underpinnings of ASD, encompassing not only case-specific mutations, but also alterations shared between different ASD-related mutations.
G 04
Autoprot: A Modular Package for Processing, Analysis and Visualization of Complex Proteomics Data in Python
Presenting author:
Würzburg University, Chair of Biochemistry II, Am Hubland, 97074 Würzburg [DE], julian.bender@uni-wuerzburg.de
Author(s):
Julian Bender, Wignand W. D. Mühlhäuser, Johannes P. Zimmermann, Friedel Drepper, Bettina Warscheid
The rising complexity of mass spectrometry (MS) data sets in proteomics research requires standardized and reliable data analysis workflows. Python-based software tools, particularly jupyter notebooks, provide a simple yet powerful solution for this. However, there is only a limited repertoire of Python software available for standardised and easy-to-use MS data analysis. This software is often restricted to algorithms developed in Python while excluding existing and well-tested software developed in other programming languages, such as R. Furthermore, current Python software frequently lacks interactive data visualization for improved and convenient exploratory inquiries and sharing of results with collaboration partners. We developed autoprot, a Python module for analysing MS-based proteomics search results generated with the MaxQuant software. Autoprot offers access to functions in Python and R for statistical testing and data transformation. Furthermore, it generates dynamic javascript-based charts that can be integrated into interactive web applications. We show the application of autoprot using publicly available MS datasets, highlight functions of the submodules for data preprocessing, analysis and visualisation and showcase interactive plots generated with the software. In summary, autoprot provides standardised, fast, and reliable proteomics data analysis while ensuring a high customisability needed to tailor the analysis pipeline to specific experimental strategies.
G 14
Using quantitative proteomics to uncover ribosome heterogeneity in neurons
Presenting author:
Max Planck Institute for Brain Research, Department of Synaptic Plasticity, Max-von-Laue Str. 4, 60438 Frankfurt am Main [DE], ashley.bourke@brain.mpg.de
Author(s):
Ashley Bourke, Kristina Desch, Claudia Fusco, Sara Mota, Julian Langer, Erin Schuman
Customized remodeling of synaptic proteomes is essential for proper neuronal function. During brain development and plasticity, local protein synthesis is differentially regulated in individual synaptic compartments to control synapse formation and strength (Bernard et al., 2022; Hafner et al., 2019), however the repertoire of molecular mechanisms used is not well understood. A promising mechanism for sculpting synapse-specific proteomes is selective mRNA translation by 'specialized ribosomes' - ribosomes with different subunit compositions or associated proteins. This possibility is supported by recent findings of synaptic mRNA translation on 80S monosomes (Biever et al., 2020) and context-dependent ribosome remodeling in dendrites (Fusco et al., 2021), however the extent to which neuronal ribosomes are compositionally distinct remains unknown. Here, we use two MS-based approaches to identify ribosome-associated proteins at specific subcellular regions and across ribosomal subcomplexes. In the first approach, we combine proximity labeling, ribosome purification, and label-free quantification to map the protein interactomes of ribosomes in the nucleus (nuclear membrane), cytoplasm, and dendritic spines of rat hippocampal neurons. In the second approach, we use polysome proteome profiling (Imami et al., 2018), which couples sucrose gradient fractionation with SILAC-based proteomics, to identify the interactors of monosomes, polysomes, and other ribosomal subcomplexes.
G 18
The Hidden Role of Reactive Oxygen Species in Modulating Mitochondrial-Processing Peptidase
Presenting author:
Institute of Biochemistry II, , Theodor-Stern-Kai 7, 60590 Frankfurt am Main [DE], bozkurt@med.uni-frankfurt.de
Author(s):
Süleyman Bozkurt, Doha Boutguetait, FX Reymond Sutandy, Christian Münch
Most mitochondrial proteins, encoded in the nucleus, require accurate translocation into mitochondria. The mitochondrial-processing peptidase (MPP), critical for precursor protein processing, ensures their proper function. Any disruption in this process can lead to protein dysfunction, causing diseases. Reactive Oxygen Species (ROS), including H2O2, are small, reactive molecules produced within cells, particularly in mitochondria. ROS regulate growth and stress response. Despite being a ROS, H2O2 is a vital cellular signaling molecule due to its permeability through cell membranes. We employed a peroxisomal enzyme, D-alanine oxidase (DAO), to study ROS signaling. DAO catalyzes D-amino acids into pyruvate, ammonia, and H2O2. We targeted DAO to mitochondrial matrix, and conducted biochemical experiments, including proteomics. H2O2 production increased with D-alanine treatment, verified with Hyper7 probe. Longer treatment caused mitochondrial stress; reducing MMP, protein import, leading to precursor protein accumulation and OMA1 activation. Shorter treatment induced precursor protein accumulation in the mitochondrial matrix, suggesting the malfunction of MPP. In conclusion, mitochondrial proteins must be accurately imported and processed by MPP for proper function. Disruptions to MPP can lead to unprocessed protein accumulation in mitochondria, triggering serious cellular complications and potentially contributing to diseases.
M 11
Let’s make it clear: systematic exploration of mitochondrial DNA-/RNA-protein complexes by complexome profiling
Presenting author:
Goethe Universität Frankfurt, Institute for Cardiovascular Physiology, Theodor-Stern-Kai 7, Haus 26, 5th floor, 60590 Frankfurt am Main [DE], alfredbiomed@gmail.com
Author(s):
Alfredo Cabrera-Orefice, Alisa Potter, Johannes N. Spelbrink
To synthesize the mtDNA-encoded proteins, both strands of the circular mtDNA are transcribed into polycistronic RNAs, which are processed and maturated generating tRNAs, rRNAs and mRNAs for mitochondrial translation. The mtDNA replication and gene expression machinery are tightly regulated by specific sets of nuclear-encoded proteins. Although the roles of the major mitochondrial nucleic acid-interacting proteins have been described, a lot of interactors remain unverified or unknown. We have improved native gel electrophoresis-based complexome profiling (CP) for examination of mitochondrial DNA-/RNA-protein complexes. Our adaptations enabled the systematic exploration of mtDNA- and RNA-protein interactions in human mitochondria, thereby unlocking the comprehensive analysis of a near-complete mitochondrial complexome. To illustrate the applicability of our approach, we performed a proof-of-principle experiment using inhibition and recovery of transcription with a transient ethidium bromide treatment, identified and validated many of the known mitochondrial protein-RNA interactions involved in, for instance, mitoribosome biogenesis. Thus, our method not only helps validate and unveil proteins involved in mitochondrial DNA-/RNA-related processes, but also offers a convenient and systematic way to analyse these interactions that can be virtually applied to investigate any kind of nucleic acid-protein complexes.
G13
A study on small proteins present in terminal cytochrome oxidases
Presenting author:
Max-Planck Institut für Biophysik, Mass Spectrometry and Proteomics, Max-von-Laue Straße 3, 60438 Frankfurt [DE], pedrofrancobh@gmail.com
Author(s):
Pedro Henrique Cavalcanti Franco, Rilee Zeinert, Imke Wüllenweber, Gisela Storz, Julian Langer
Small proteins, < 50 amino acids in length, have been shown to regulate cellular processes such as antibiotic resistance and cell development. Despite their perspective as therapeutics, their characterization remains limited due to insufficient gene annotation and challenges associated with their characterization. In E. coli, CydH (29 aa) and CydX (37 aa) bind the cytochrome bd-I oxidase and AppX (30 aa) binds the cytochrome bd-II oxidase. However, their specific function within the complexes and potential interaction with additional complexes remain unclear. In this study we investigated the binding partners of CydX and CydH during aerobic and anaerobic growth. To identify conditions for interaction studies we monitored expression levels in these conditions and examined the impact of their deletion on E. coli growth. We find that all three small proteins are induced in anaerobic conditions, being CydX expression the most abundant relative to the others. We performed immunoprecipitation assays to look for interacting partners of CydH and CydX in aerobic vs anaerobic growth and current work is aimed at globally identifying putative partners using LC-MS/MS. The results thus far suggest these small proteins might be most important during anaerobic growth or transitions between aerobic and anaerobic growth, conditions for which the roles of small proteins have not been explored.
G 25
Investigating novel functions of Rab24 in mitochondrial fission and protein secretion
Presenting author:
LMU, Munich Cluster of system Neurology, Feodor-Lynen Str. 17, 81377 Munich [DE], rahulchakraborty725@gmail.com
Author(s):
Rahul Chakraborty, Syed Qaaifah Gillani, Anja Zeigerer, Christian Behrends
Globally, the incidence of non-alcoholic fatty liver disease (NAFLD), a crucial factor in type 2 diabetes caused by obesity, is rising. More severe liver damage, such as cirrhosis and hepatocellular carcinoma, arise because there are currently no robust treatment options. Here, we explore an unanticipated role for the small Rab GTPase Rab24, an intracellular trafficking regulator, in mitochondrial fission and activation, which directly affects hepatic and systemic energy homeostasis. RAB24 has previously been demonstrated to be significantly elevated in livers of obese individuals with NAFLD and to have a strong positive correlation with increased body fat in humans. This atypical GTPase has recently been discovered as a novel interactor of mitochondrial fission protein Fis1 in the liver. Split GFP protein complementation assays and APEX2-based proximity labeling approaches are now employed to identify Rab24-Fis1 regulating and scaffolding proteins. In both strategies, candidate interacting or neighboring proteins are enriched by affinity purification and subsequently identified by mass spectrometry. Complementarily, we are performing whole cell protein abundance profiling in conditional Rab24 or Fis1 knockout cells using DDA- and DIA-based mass spectrometry to uncover factors that operate downstream of the Rab24-Fis1 interaction. Together, these efforts will help to decipher the functional role of Rab24 in the context of Fis1.
G 24
Proteomic profiling of sex- and oestrus-cycle specific changes in the midbrain
Presenting author:
Max-Planck-Institute for Brain Research, Synaptic Plasticity, Max-von-Laue-Straße 4, 60438 Frankfurt [DE], kristina.desch@brain.mpg.de
Author(s):
Kristina Desch, Elena Kutsarova, Petros Chalas, Genesis Rosiles, Vanessa Stempel, Julian Langer
While instinctive behaviors are evolutionarily conserved, they can be adapted to different environments and internal states of animals. Despite its involvement in many of these behaviors, the midbrain periaqueductal gray (PAG) has been mostly considered a simple relay station. However, expression of certain candidate proteins suggests that it may be able to confer behavioral flexibility. To understand how individual behavior is modulated in the PAG and if it allows for context-dependent adaptations, we characterized its proteomic composition and investigated possible sex- and estrous-cycle-specific changes using DIA-LC-MS. Initial results revealed comprehensive proteomic coverage with ~7,300 proteins per sample. Proteins associated with synaptic plasticity were abundantly identified suggesting that the PAG has the potential to undergo plasticity-induced changes. While the overall proteomic composition was highly similar among all animals, several extracellular matrix proteins implicated in Alzheimer's disease and synaptic remodeling showed differential abundance between male and female mice. Interestingly, no reliable changes during different stages of the estrous cycle were observed. In conclusion, the proteomic compositions of the male and female mouse PAG are largely similar with a few proteins showing differential abundance. For further characterization and to overcome dilution effects from bulk tissue analysis, future studies may require cell-type-specific labeling.
O 01
Why do mitochondria still contain a genome? Mechanistic insights from allotopically expressed proteins
Presenting author:
RPTU Kaiserslautern-Landau, Standort Kaiserslautern, AG Zellbiologie, Erwin-Schrödinger-Straße 13, 67663 Kaiserslautern [DE], ecker@rhrk.uni-kl.de
Author(s):
Anna-Lena Ecker, Johannes M. Herrmann
Mitochondria are essential organelles of eukaryotic cells. They consist of hundreds of nuclear encoded proteins, but also harbor a small genome as a remnant of a bacterial ancestor. Mitochondrial genomes encode a small number of very hydrophobic proteins. Why the genes of these proteins were not transferred into the nucleus is not well understood. To elucidate the molecular consequences of such mitochondria-to-nucleus gene transfer reactions, we allotopically expressed the model proteins Cox3 and Atp6 with mitochondrial targeting sequences in the cytosol of yeast cells. The fusion proteins are not imported into mitochondria but rather accumulate on the cytosolic surface of the outer membrane translocase. The highly hydrophobic character of these proteins presumably prevents efficient translocation through the TOM complex. These stalled translocation intermediates are efficiently removed by proteolysis, specifically by components of the cytosolic ubiquitin-proteasome system (UPS). Mutants in the UPS which prevent the efficient degradation of these proteins lead to growth defects and induce cell death. Thus, the protein quality system on the mitochondrial surface is important for cellular functionality, however, it prevents the productive gene transfer from mitochondria to the nucleus and forces eukaryotic cells to maintain the genes of a core set of highly aggregation-prone proteins.
G 02
The interplay of posttranslational protein modifications in Arabidopsis leaves during photosynthesis induction
Presenting author:
University of Muenster, Institute of Plant Biology and Biotechnology, Schlossplatz 7, 48149 Muenster [DE], juergen.eirich@wwu.de
Author(s):
Juergen Eirich, Jonas Giese, Iris Finkemeier
Diurnal dark to light transition causes profound physiological changes in plant metabolism. These changes require distinct modes of regulation as a unique feature of photosynthetic lifestyle. The activities of several key metabolic enzymes are regulated by light-dependent post-translational modifications (PTM). A global picture of the light-dependent PTMome dynamics was lacking so far. Here we investigated the light-dependent proteome changes in Arabidopsis leaves in a time-resolved manner to dissect global phosphorylation, lysine acetylation, and cysteine-based redox switches using different quantification strategies, including DiMethyl- and iodoTMT labeling. Of over 24,000 PTM sites that were detected on an Orbitrap Q Exactive HF, more than 1,700 were changed during the transition from dark to light. While the first changes, as measured 5 min after the onset of illumination, occurred mainly in the chloroplasts, PTM changes at proteins in other compartments coincided with the full activation of the Calvin-Benson cycle and the synthesis of sugars at later timepoints. Our data reveals connections between metabolism and PTM-based regulation throughout the cell. The comprehensive multiome profiling analysis provides unique insights into the extent by which photosynthesis re-programs global cell function and adds a powerful resource for the dissection of diverse cellular processes in the context of photosynthetic function.
G 22
Quantitative Secretome Kinetics
Presenting author:
Institute of Biochemistry II, , Theodor-Stern-Kai 7, 60590 Frankfurt am Main [DE], fehmer@med.uni-frankfurt.de
Author(s):
Martin Fehmer
Secreted proteins play a central role in coordinating both basic biological functions such as cell growth, division and differentiation as well as complex cellular programs including apoptosis and signaling. It is estimated that about 15% of the human genome encode factors that are putatively secreted, with about roughly a third of these factors acting locally in a tissue- or microenvironment-specific manner. Alterations in the cellular secretome composition have been associated with several malignancies, including the development of chemoresistance, the progression and modulation of infectious diseases, as well as mast cell dysfunction. Mass spectrometry-based proteomics have been successfully integrated into mapping the cellular secretome, providing insights into both the fundamental composition as well as disease associated changes of the extracellular environment. These studies are comprised of either stand-alone proteomic datasets or encompass a comparative analysis between one or several conditions to a control without assessing the accumulation of secretory proteins in a time-resolved manner. Using a SILAC-TMT-based approach following the mePROD method developed by Klann et al. in 2020, we set out to establish cellular secretion kinetics across the secretome. We then strive to apply this method towards the investigation of secretory disease models as well as delineating the route different components may take along both canonical and non-canonical secretory pathways.
Short talk 4
A toolbox for systematic discovery of stable and transient protein interactors in baker’s yeast
Presenting author:
Weizmann Institute of Science, Molecular Genetics, 234 Herzl Street, 7610001 Rehovot [IL], emma.fenech@weizmann.ac.il
Author(s):
Emma Fenech, Maya Schuldiner
Identification of both stable and transient interactions is essential for understanding protein function and regulation. While assessing stable interactions is more straightforward, capturing transient ones is challenging. In recent years, sophisticated tools have emerged to improve transient interactor discovery, with many harnessing the power of evolved biotin ligases for proximity labelling. However, biotinylation-based methods have lagged behind in the model eukaryote, Saccharomyces cerevisiae, possibly due to the presence of several abundant, endogenously biotinylated proteins. In this study, we optimised robust biotin-ligation methodologies in yeast and increased their sensitivity by creating a bespoke technique for downregulating endogenous biotinylation which we term ABOLISH (Auxin-induced BiOtin LIgase diminiSHing). We used the endoplasmic reticulum insertase complex (EMC) to demonstrate our approaches and uncover new substrates. To make these tools available for systematic probing of both stable and transient interactions, we generated five full-genome collections of strains in which every yeast protein is tagged with each of the tested biotinylation machineries; some on the background of the ABOLISH system. This comprehensive toolkit enables functional interactomics of the entire yeast proteome.
Sept. 6, 9:00
Understanding the tumor microenvironment through high-sensitivity MS-based proteomics
Presenting author:
Weizmann Institute of Science, , Herzel 234, 7610001 Rehovot [IL], tami.geiger@weizmann.ac.il
Author(s):
Tami Geiger, Mariya Mardamshina, Shiri Karagach, Vishnu Mohan
Cancer heterogeneity presents a significant challenge to effective treatment strategies. Genetic variations and cellular interactions within the tumor microenvironment (TME) contribute to the diverse molecular characteristics observed among different tumor clones. Understanding the functional proteomic layer of tumor subpopulations and their interactions with the microenvironment is crucial. In this study, we integrated mass spectrometry-based proteomics with spatial multiplexed imaging of cells from the TME to unravel the functional proteomic layer of breast cancer heterogeneity. Our approach combined clinical sample analysis, multilayer tissue imaging, and deep learning-based image processing to identify novel regulators of cancer phenotypes. Analyzing hundreds of breast cancer tumor regions, we discovered associations between clinical parameters, protein networks, and intra-tumor heterogeneity. Proteins related to cell adhesion and interactions with the immune system exhibited the highest variability, while proteins related to cell proliferation remained constant. Furthermore, our analyses highlighted the proteomic impact of distance from blood vessels, tumor center, and immune cells, including T-cells and macrophages. By integrating mass spectrometry-based proteomics and spatial multiplexed imaging, we provide valuable insights into the functional proteomic layer of breast cancer heterogeneity, offering new avenues for targeted therapies and personalized medicine.
M 09
Combining Data Independent Acquisition with Spike-in SILAC (DIA-SiS)
Presenting author:
MDC Berlin, , Robert-Rössle-Straße 10, 13125 Berlin [DE], maximilian.gerwien@mdc-berlin.de
Author(s):
Maximilian Gerwien, Anna Sophie Welter
SILAC-based quantification has been extensively applied in DDA proteomics for many years due to its superb quantitative performance. However, SILAC involves the metabolic labelling of cultured cells. Since this is not always possible or convenient (e.g., clinical samples), a previously prepared SILAC spike-in can be employed. Recently, DIA proteomics became more popular. It offers unbiased and reproducible profiling of peptides over a broad dynamic range. To combine the merits of spike-in SILAC with DIA proteomics, we devised DIA spike-in SILAC (DIA-SiS). As a stable isotope labelling method, it is precise and accurate. As a spike-in method, it is almost as easy and fast to use as a label-free approach. And, as a DIA method, it benefits from the unbiased and comprehensive profiling of precursor ions. To assess the quantitative performance (number of identifications, accuracy and precision) of DIA-SiS compared to label-free DIA, we created a benchmark dataset with known quantities. Here, we show that DIA-SiS improves the identification and quantification of precursors and proteins of low-input samples. From 10 ng human proteome digest, we quantify >2000 proteins with spike-in compared to ca. 1000 proteins without spike-in. Overall, coefficients of variation of proteins are lower with the spike-in. In summary, DIA-SiS offers improved identification and quantification of low-abundant samples compared to label-free DIA.
G 12
Nuclear localization of non-imported mitochondrial proteins modulates epigenetic landscape
Presenting author:
RPTU Kaiserslautern-Landau, DEPARTMENT OF CELL BIOLOGY, kohlenhofstrasse 3, 67663 kaiserslautern [DE], nikita.gupta@rhrk.uni-kl.de
Author(s):
Nikita Gupta, Johannes Herrmann
Most of the mitochondrial proteins are synthesized in the cytosol and are translocated to mitochondria via the mitochondrial import machinery. However, under import failure, the non-imported mitochondrial precursor proteins get accumulated in many regions of the cell, with the nucleus being one of the key locations for quality control. Still, it remains unclear what drives these non-imported mitochondrial precursor proteins to the nucleus and whether these mitoproteins exhibit any metabolic or regulatory function in the nucleus. To elucidate the consequences of mitochondrial import failure in the epigenetic landscape of the cell, we then investigated histone synthesis under the expression of a clogger protein. To our surprise, we observed that the synthesis of the histone gene is strongly repressed under import failure hinting towards a potential role of non-imported mitochondrial proteins which accumulate in the nucleus in regulating the epigenetic landscape of the cell. The aim of my study is to characterize the functional role of non-imported mitochondrial proteins which localize to the nucleus upon mitochondrial dysfunction.
C 01
Loss of nuclear pore complex function and cellular compartmentalization in the steroid resistant nephrotic syndrome
Presenting author:
Uniklinik Aachen, Biochemie (AG Antonin), Pauwelsstraße 30, 52074 Aachen [DE], mhamed@ukaachen.de
Author(s):
Mohamed Ismail Hamed
Focal segmental glomerulosclerosis (FSGS) is a progressive pathology with gradual loss of kidney function and end-stage kidney failure. FSGS is characterized by loss of podocyte function, a cell type that forms the kidneys’ filtration barrier. Podocytes are post-mitotic cells with no proliferative capacity which accordingly reside within the kidney for life time. As a result, proteins and protein complexes with long residual times are prone to insults such as mutations if dedicated repair mechanisms are lacking. Nuclear pore complexes (NPCs) are a multi-protein complexes integrated within the nuclear envelope (NE). The NE separates the cytoplasmic and nuclear compartments and NPCs act as the transport gates. Mutations in a number of NPC proteins cause the steroid resistant nephrotic syndrome, a FSGS with childhood-onset, characterized by podocyte and kidney function loss otherwise typically seen in older patients. We hypothesize that these mutations weaken the NPCs and lead to a progressive loss of cellular compartmentalization, which in healthy persons is only observed much later in life. Using mass-spectroscopy and immunofluorescence microscopy, we characterize compartmentalization loss in podocytes of aging mice and define specific markers to follow disease progression. Furthermore, we have established cellular assays where loss of compartmentalization by NPC defects can be recapitulated and which are currently used for compound screening to retain proper NPC function.
S 04 & Short talk 1
SNARE complex regulation by Complexin-1 - a structural mass spectrometry study
Presenting author:
Johannes Gutenberg University Mainz, Chemistry - Biochemistry, Hanns-Dieter-Hüsch-Weg 17, 55128 Mainz [DE], julia.hesselbarth@uni-mainz.de
Author(s):
Julia Hesselbarth, Carla Schmidt
Signal transmission between neurons is mediated by the SNARE complex that is responsible for fusion of synaptic vesicles with the presynaptic membrane. This ternary complex assembles from vesicular Synaptobrevin-2 as well as SNAP25 and Syntaxin-1A, which are both anchored to the presynaptic membrane. SNAP25 contributes two alpha-helices and Syntaxin-1A and Synaptobrevin-2 both contribute one alpha-helix forming a stable four-helix bundle. The SNARE assembly is a highly regulated process involving Complexin-1, which is known to bind a groove formed by Synaptobrevin-2 and Syntaxin-1A, however, the regulatory mechanism is largely unknown. Using native mass spectrometry, we first investigated interactions of Complexin-1 with individual SNAREs and binary SNARE complexes to elucidate a regulatory function in early states of SNARE assembly. While Complexin-1 does not interact with individual SNAREs or binary complexes similar to the SNAP25:Syntaxin-1A complex, Complexin-1 binding stabilizes the Syntaxin-1A:Synaptobrevin-2 interface leading to formation of a complex that imitates SNARE complex stoichiometry. Following incubation of all three SNAREs, formation of the SNARE complex and oligomers thereof was observed. Addition of Complexin-1 disassembled these oligomers indicating an inhibitory role for SNARE oligomerisation. Specific interaction sites of Complexin-1 within this assembly were further explored by chemical cross-linking providing a model of the SNARE:Complexin-1 complex.
M 12
De novo Protein Interactome Profiling of Small Molecule and Antisense Oligonucleotide Drugs
Presenting author:
Eberhard Karls Universität Tübingen, Interfaculty Institute of Biochemistry, Auf der Morgenstelle 15, 72076 Tübingen [DE], daniel.hofacker@uni-tuebingen.de
Author(s):
Daniel Hofacker, Alfred Hanswillemenke, Thorsten Stafforst
Protein interactions determine the pharmacological properties of a drug including toxicity, immunogenicity, efficacy, metabolism, and adverse effects. In this yet unpublished work, we present a toolbox to comprehensively identify the protein interactome for several drug types de novo in living cells. This includes small molecule drugs as well as antisense oligonucleotide (ASO) drugs, which have gained notable clinical relevance in the past few years. The interactome is identified by covalent recruitment of a biotin ligase to the drug of interest followed by targeted protein biotinylation, and biotin enrichment coupled to MS². Compared to pulldown-based approaches that identify RNA drug interactions in vitro, our novel approach can distinguish between different ASO chemistries at pharmacologically effective intracellular concentrations, and furthermore, mesoscale changes in the RNA/protein interactome in response to stress were identified. We further extended the method to discover the interactome of endogenous lncRNAs that recruit proteins to specific nuclear condensates. In contrast to established protocols, our assay does not need any genetic modification of the cells, uses simple probes, and requires up to 100-fold less input material than in vitro pulldown-based methods, making it suitable for hard-to-transfect and non-dividing cells. Overall, this powerful tool helps to unravel the intricate RNA/protein interplay and offers valuable insights into RNA drugs and RNA biology.
G 03
Gliflozin drug class and its effect on the proteome of cardiomyocytes
Presenting author:
, , Albrechtsbergergasse 19/2, 1120 Wien [AT], julia.hoehlschen@tuwien.ac.at
Author(s):
Julia Höhlschen, Tamara Tomin, Ruth Birner-Grünberger
Death from cardiovascular (CV) diseases is the most relevant macrovascular complication in type 2 diabetes. A new drug class that offers cardioprotective properties are sodium-glucose co-transporter-2 (SGLT-2) inhibitors, initially used for the treatment of type 2 diabetes. Meanwhile trials have shown that these properties are beyond the effect of lowering glucose concentrations in the blood. Therefore, their application in non-diabetic patients suffering from heart diseases has recently been approved. This project aims to identify the not yet understood mechanisms leading to the observed cardioprotective effects. In a first experiment I applied redox-proteomics to check if the drugs show antioxidative effects on a differentiated, human cardiomyocyte cell line (AC16) by mimicking disruption of oxygen supply (similar to heart failure): 1% oxygen (hypoxia), 21% oxygen (Control) and reperfusion injury (1% followed by re-oxygenation at 21% oxygen), as oxidative stress is one potential cause of heart failure.
O 04
Akt isoforms differentially affect Rho signaling pathways in H23 non-small cell lung carcinoma cells
Presenting author:
Justus Liebig University Giessen, Signal Transduction of Cellular Motility, Aulweg 128, 35392 Giessen [DE], Bahareh.Jooyeh@innere.med.uni-giessen.de
Author(s):
Bahareh Jooyeh, Stefanie Wirth, Manfred Jücker, Andre Menke, Klaudia Giehl
Studies in our group showed that oncogenic K-Ras regulates cell migration of carcinoma cells by modulating the PI3-K/Akt pathway and expression of the three Akt isoforms. In this study, H23 lung carcinoma cells harboring K-Ras(G12C), were used to elucidate a possible cross-talk between Akt and Rho signaling. Stable lentiviral transduced Akt isoform-specific knockdown (kd) H23 cell clones were generated to investigate the impact of each Akt isoform. Additionally, different pharmacological inhibitors for Akt, PI3-K, RhoA, and ROCK were used. MK-2206, a pan-Akt inhibitor, downregulated the phosphorylation of all Akt isoforms, but it did not affect the phosphorylation of the ribosomal protein S6. LY-294002 inhibited Akt and S6 phosphorylation. Inhibition of RhoA by Rhosin resulted in decreased phosphorylation of Akt in all analyzed cell clones, without affecting S6. However, we observed no changes on Akt and S6 by Y-27632, ROCK inhibitor. Western blot analyses revealed that RhoA protein expression was upregulated in Akt2-kd H23 cells, whereas Akt1-kd cells displayed an increased expression of Rac1b. The upregulated RhoA expression resulted in a higher content of active RhoA in Akt2-kd cells. Furthermore, knockdown of Akt1 and Akt2 led to an upregulation of cofilin and an increase in the phosphorylated form. With our findings, we aim to elucidate the intracellular communication between K-Ras/Akt and Rho signaling pathways and their impact on cell migration and metastasis.
M 04
Improved dia-PASEF isolation window schemes for proteomics measurements
Presenting author:
Bruker Daltonics GmbH & Co KG, , Fahrenheitstraße 4, 28359 Bremen [DE], stephanie.kaspar-schoenefeld@bruker.com
Author(s):
Markus Lubeck, Stephanie Kaspar-Schoenefeld, Christoph Krisp, Andreas Schmidt, Gary Kruppa
DIA is widely used for proteomics as it promises reproducible and accurate protein identification and quantitation. dia-PASEF is both more sensitive and selective than traditional DIA approaches as it combines the advantages of DIA with the inherent ion-usage efficiency of PASEF. Making use of the correlation of molecular weight and CCS coded information, dia-PASEF enables highly confident identification. The two-dimensional mass and mobility space enables method creation with extensively different window schemes. Here, a variety of fixed-width as well as more advanced window schemes were evaluated. Dilution series of tryptic digests from human cell lines were separated using different nanoLC gradients. Different isolation windows widths were compared to more sophisticated approaches like schemes with variable window widths based on precursor density (py_diAID). Data were processed using Spectronaut 18 (Biognosys). In the presented study, we limited the dia-PASEF windows to the mass and mobility range of highest precursor density. For sample amounts in the 10-50 ng range identifications were remarkable similar among the different tested acquisition schemes. Lower sample amounts benefit from a lower number of broader windows. For higher sample loads, acquisition schemes of more narrow isolation windows resulted in improved identifications due to their higher specificity. Optimal methods for a broad range of sample amounts and gradient lengths could be determined.
G 15
Using proteomics to characterize RNF213- a unique AAA+ ATPase and E3 ligase
Presenting author:
Institute of Biochemistry II, , Theodor-Stern-Kai 7, 60590 Frankfurt am Main [DE], kohli@med.uni-frankfurt.de
Author(s):
Aneesha Kohli, Christian Münch
Moyamoya disease associated protein RNF213 is a large AAA+ ATPase and an E3 ligase. Recently, it was reported to play a role in xenophagy, cancer and lipid metabolism apart from its previously briefly described role in immune response, inflammation and angiogenesis. Despite the rising interest, its role in basal and inflammatory states remains largely unknown. We aim to use proteomics as a way of characterizing the functional role of RNF213 in basal and stressed states wherein for the latter we employ IFNγ, a known inducer of RNF213. In this regard, we have thus far, applied global proteomics approach to identify the key proteins and the associated pathways that are modulated in the absence of RNF213 using knockdown assays. We have used interactomics to identify its direct or complex associated interaction partners as well as E3 targets. Our proteomic analyses provide an insight into the role of RNF213 in immune response as reported earlier but also in other pathways such as mitochondrial biogenesis and ribosomal machinery, protein transport and rather crucially, also in heme biogenesis and ferroptosis. Next, we aim to further our findings by complementing our work with ubiquitinomics to correctly identify the E3 targets and sites of its associated activity on the target proteins in the future and dive further into its mechanistic role in ferroptosis.
G 17
Single-cell multiomics on brain organoid models of autism spectrum disorder
Presenting author:
Max Delbrück Center (MDC), , Robert-Rössle Str. 10, 13125 Berlin [DE], marianna.kokoli@mdc-berlin.de
Author(s):
Marianna Kokoli, Matthias Selbach
Autism Spectrum Disorder (ASD) is one of the most complex neurodevelopmental disorders, characterized by atypical social, behavioral and cognitive function. ASD exhibits remarkable heterogeneity in terms of genotypes and phenotypes, yet currently lacks early diagnostic methods and effective treatments. In recent years, dysregulated protein synthesis has emerged as a prominent feature of ASD. This project aims to shed light on the molecular mechanisms underlying ASD with respect to clinically-relevant mutations in FMR1, PTEN and TSC2 genes which affect protein synthesis. In order to better recapitulate and model neurodevelopment during ASD, we plan to generate brain cerebral and micropatterned organoids from wildtype induced pluripotent stem cells (iPSCs) and iPSCs harboring the aforementioned ASD-related mutations. A multiomics approach of transcriptomics and proteomics both at the bulk and the single-cell level will be employed so as to investigate the correlation between mRNA and protein levels, as well as to identify alterations in protein translation over time. Overall, with this study we anticipate to explore proteome dynamics in the context of ASD and capture both differences in cellular composition of organoids and cell autonomous changes. Through our findings we hope to deepen our understanding of the molecular underpinnings of this intricate disorder and pave the way for the development of more precise diagnostic methods and targeted therapeutic interventions.
G 09
Investigating the role of Arabidopsis HISTONE DEACETYLASE 14 in chloroplasts
Presenting author:
University Münster, IBBP AG Finkemeier, Schlossplatz 7, 48149 Münster [DE], florian.kotnik@uni-muenster.de
Author(s):
Florian Kotnik, Claudia Markiton, Jürgen Eirich, Iris Finkemeier
Lysine acetylation is an important post-translational protein modification that plays a vital role in plant development and in responses to different environmental stimuli. Histone deacetylases (HDACs) are responsible for removing lysine acetylation on various proteins. While most work has focussed on the role of Arabidopsis HDACs on histone acetylation, their role in the deacetylation of non-histone proteins is much less known, although proteins of many different organelles have been found to be lysine-acetylated. From the 18 HDACs found in Arabidopsis, only HDA14 has been found to be dual-localized in plastids and mitochondria. Here we performed a quantitative mass spectrometry-based approach, using isobaric TMT labelling, to profile the lysine acetylome of an Arabidopsis hda14 mutant compared to WT. We identified 1509 acetylation sites on 881 Arabidopsis protein groups, of which 56 sites were de-regulated in the hda14 mutant. Most of these sites were derived from chloroplast proteins. In addition, we used different co-immunoprecipitation approaches to identify possible interaction partners of HDA14 and to identify its function in the regulation of organellar metabolism.
M 03
Edman degradation relaunched for unequivocal analysis of disulfide-rich peptides
Presenting author:
University of Bonn , Pharmaceutical Biochemistry and Bioanalytics, An der Immenburg 4, 53121 Bonn [DE], tkuhl@uni-bonn.de
Author(s):
Yomnah Y. Elsayed, Karl G. Wagner, Toni Kühl, Diana Imhof
N-terminal sequencing introduced by Peer Edman in 1949, was the gold standard for protein analysis for many years til the 1990s, when the rise of mass spectrometry superseded the stepwise chemical degradation of proteins in this field til today [1,2]. However, several scientific problems cannot be easily solved without the application of Edman degradation, e.g., the differentiation of isoleucin and leucin in protein sequences, the analysis of immobilized compounds or the identification of posttranslational modifications [3,4]. The analysis of disulfide bonds in cysteine-rich peptides is another tempting application for N-terminal sequencing. However, this requires the (re)establishment of suitable standard compounds and the development of a protocol for the rapid analysis of the disulfide connectivities in peptide and protein sequences. We present the application of such standard compounds in an optimized workflow in which partial reduction, alkylation, fractionation and Edman degradation are applied in a sequential manner. Peptides of different complexity (different length, 1-3 disulfide bridges), such as the conotoxins CCAP-vil and µ-KIIIA, were analyzed. With this study, we aim to relaunch N-terminal sequencing by applying and developing favorable protocols for rapid analysis exploiting this method. [1]Edman (1949) Arch Biochem 22,475 [2]Stehen, Mann (2004) Nat Rev Mol Cell Biol 5,699-711 [3]Lukas et al. (2022) Biol Chem 403,1099-1105 [4]Fitzner et al. (2023) Food Chem 136698
G 05
Establishing a cytosolic version of the mitochondrial processing peptidase to study mitochondrial protein import
Presenting author:
RPTU Kaiserslautern-Landau, Standort Kaiserslautern, AG Zellbiologie, Erwin-Schrödinger-Straße 13, 67663 Kaiserslautern [DE], lenhard@rhrk.uni-kl.de
Author(s):
Svenja Lenhard
Mitochondria consist of many hundreds of different proteins that are synthesized on cytosolic ribosomes. Mitochondrial protein import mechanisms have been extensively studied in the past. Aminoterminal presequences ensure the reliable targeting of client proteins into mitochondria. Subsequently to the import of these proteins, the presequences are proteolytically removed in the mitochondrial matrix by the mitochondrial processing peptidase, MPP. Strikingly, the processes occurring right before the translocation of a polypeptide remain unclear. In order to better understand the timing of the synthesis and import of precursor proteins, we engineered a yeast strain which expresses MPP in the cytosol. Expression of this cytosolic MPP (cytoMPP) is highly toxic as MPP cleavage in the cytosol obviously competes with mitochondrial import of precursor proteins. Establishment of this tool is expected to provide novel insights into (1) how different precursors are sequestered to the mitochondrial surface, (2) the determinants of post- or co-translational protein import, (3) which proteins are particularly sensitive to cleavage by cytoMPP and (4) which factors determine the import efficiency into mitochondria. Furthermore, we aim to characterize the conserved C-terminus of the MPP α-subunit concerning thus far unknown structure-function relationships. Therefore, this study aims to investigate both, endogenous as well as cytosolic MPP.
G 29
The natural small molecule compound prodigiosin targets the Golgi stacking protein GRASP55/GORASP2
Presenting author:
Heinrich Heine Universität, MPL / BMFZ, Universitätsstraße 1, 40225 Düsseldorf [DE], thomas.lenz@hhu.de
Author(s):
Thomas Lenz, Lena Berning, Ann Kathrin Bergmann, Björn Stork, Kai Stühler
Background Prodigiosin is a bacterial secondary metabolite that has been shown to have anticancer, antimalarial, antibacterial and immunomodulatory properties. It has been reported to affect cancer cells but not non-malignant cells, making it a promising lead compound for anticancer drug discovery. A direct protein target has not yet been experimentally identified. Methods In order to identify target proteins of prodigiosin, mass spectrometry-based thermal proteome profiling was used in its temperature range (TPP-TR) and compound concentration range (TPP-CCR) variants. TPP-TR was performed such that effects of prodigiosin treatment on both protein thermal stability and protein abundance could be determined simultaneously using the ratio-based thermal shift assay analysis (RTSA). Target validation was performed by a genetic knockout approach and electron microscopy. Results The Golgi stacking protein GRASP55/GORASP2 was identified as a target protein of prodigiosin. Among the prodigiosin-affected proteins (TPP-TR/RTSA), GRASP55 was the statistically most significant thermally stabilized protein with the lowest EC₅₀ (2.6 nM, TPP-CCR). Prodigiosin treatment severely affects Golgi morphology and functionality, and prodigiosin-dependent cytotoxicity is partially reduced in GRASP55 knockout cells. Furthermore, prodigiosin treatment results in decreased cathepsin activity and overall blocks autophagic flux probably involving also other mechanisms such as organelle alkalization.
S 01
Bridging top-down proteomics and native mass spectrometry: A consortium-based study
Presenting author:
Technical University of Darmstadt, , Peter-Grünberg-Strasse 4, 64287 Darmstadt [DE], frederik.lermyte@tu-darmstadt.de
Author(s):
Frederik Lermyte
Native mass spectrometry allows the study of the quaternary structure of protein complexes, while top-down protein analysis provides proteoform-specific insights into the structure of individual protein chains. The combination of both methods – i.e., top-down fragmentation after native ionisation – allows the study of how specific proteoforms interact to form complexes. This powerful combination has led to important biological insights in recent years; however, due to a lack of standardisation, only a handful of labs regularly carry out this type of work. Here, we have brought together an international consortium of users with different experience levels, and have developed and tested standard protocols for native MS combined with top-down fragmentation. QTOF, Orbitrap, and FTICR instruments were all represented. The set of samples contained monomeric proteins as well as complexes, and water-soluble as well as membrane proteins. All participants successfully ionised and activated at least part of the set of native-like proteins, resulting in monomer ejection and backbone fragmentation. Both native precursor spectra and fragmentation patterns were remarkably consistent between labs. Overall, this work provides an entry point for newcomers to combine native with top-down MS. It serves as a robust benchmark for the expected results of such an experiment, and shows that these results are more dependent on inherent properties of the protein than on precise experimental conditions.
M 02
Nano-flow HILIC-MS-based site-specific assessment of RNA modifications
Presenting author:
Goethe University, Faculty of Biochemistry, Chemistry, Pharmacy, Max-von-Laue-Str. 9, 60439 Frankfurt am Main [DE], li@pharmchem.uni-frankfurt.de
Author(s):
Chengkang Li, Stefanie Kaiser
RNAs might undergo multiple modifications (epitranscriptome) post-transcriptionally, affecting their structures and functions accordingly, some of which may involve in disease development, e.g. cancers. Therefore, a better understanding of the modification type, quantity, and location in RNA will be beneficial to the mechanism study of disease and the development of targeted therapeutic drugs. Accurate identification and quantification of multiple RNA modifications are recently achieved using advanced mass spectrometric approaches, e.g. the Nucleic Acid Isotope Labeling Mass Spectrometry (NAIL-MS). However, a robust approach for site-specific localization of RNA modifications is still an unsolved but promising challenge in epitranscriptomic study. In order to avoid potential practical limitations, e.g. impaired MS sensitivity and instrument contamination, brought by the most popular ion-pairing reagent assisted reverse phase chromatography in LC-MS-based epitranscriptomic study yet, we separate different lengths (up to 30 base pairs long) of oligonucleotides using a cleaner ion-pairing reagent free technique, i.e. hydrophilic interaction chromatography, particularly under nano-flow. In the following MS analyses, a nearly infinite signal-to-noise ratio is recorded in the corresponding (MS1) extracted ion chromatogram by only injecting samples in “ng” magnitude, along with great (≥ 73%) MS2 fragmentation coverage rates under data-dependent acquisition mode.
G 08
Secretome Analysis Revealing Effects of Kallikrein-related Peptidase 6 (KLK6) in Pancreatic Ductal Adenocarcinoma
Presenting author:
Uniklinik Freiburg, Institute of Clinical Pathology, Breisacher Strasse 115a, 79106 Freiburg im Breisgau [DE], mujia.li@uniklinik-freiburg.de
Author(s):
Mujia Li, Bettina Wehrle, Patrick Bernhard, Janina Werner, Oliver Schilling
Kallikrein-related peptidase 6 (KLK6) is a secreted serine protease involved in inflammatory pathways. Additionally, its overexpression was detected in several tumor entities including PDAC. However, distinct substrates of KLK6 are only sparsely identified and the extent of its biological effects remains to be fully understood. This study aims to elucidate biological mechanisms of KLK6 in a MiaPaCa2 knockdown model. Cell conditioned medium was used for explorative proteomic analyses, revealing over 1400 secreted proteins. By using an isobaric labeling approach (TMT 16-plex), a quantitative analysis and comparison between control and KLK6 knockdown condition was achieved. Differential abundances of KLK6-related proteins suggest that KLK6 is part of a homeostatic system with feedback controls to maintain its equilibrium. Furthermore, effectors of the extracellular matrix were significantly differentially regulated, proposing an impact of KLK6 on extracellular matrix remodeling. As expected, we detected several pro- and anti-inflammatory proteins (PTX3, TGFB2, CFH, PTGDS, CCL6, IL1R1, S100A16) differentially regulated in the knockdown condition compared to control. Altogether, this study represents first secretome analysis to unravel the biological effects of KLK6 in PDAC. Given that KLK6 is already considered as a therapeutic target, our findings promise to furnish crucial and valuable insights into the physiological mechanisms that could be influenced by KLK6 inhibition.
G 21
Proteomics-based evaluation of different cell culture models for the development of treatments for psoriasis
Presenting author:
PharmBioTec GmbH, Drug Delivery, Am Nusskopf 32, 66578 Schiffweiler [DE], s.lichtner@pharmbiotec.de
Author(s):
Simone Lichtner, Kathrin Schunck, Carina Groh, Marc Schneider, Marius Hittinger
As a replacement for animal testing, novel cell culture models offer a promising opportunity for the assessment of safety and efficacy of drugs. Of particular interest in this context are co-culture models, which consist of several cell types and thus can make a precise prediction for human-relevant data. However, these models have often not been fully characterized yet. Proteomic profiling of different cell culture models can help to elucidate the functional interaction and lead to an optimization of the models. This knowledge will then be used to test different drugs for psoriasis. Finally, the mode of action of the active ingredients within different cell culture models will be investigated.
M 06
Assessment of cellular redox regulation via proteomics: Establishment of an appropriate sulfenic acid labeling procedure in human bronchial epithelial cells
Presenting author:
Karlsruhe Insitute of Technology (KIT), Food Chemistry and Toxicology, Adenauerring 20a, 76131 Karlsruhe [DE], martin.link@kit.edu
Author(s):
Martin Link, Jana Kuhn, Marlene Parsdorfer, Andrea Hartwig
Cysteine sulfenic acids occur as short-lived intermediates, and their detection appears to be a sensitive indicator of redox-regulated pathways. By using a sensitive labeling approach, we aim to identify proteins being redox-regulated by oxidative stress and to investigate the effects of toxic metal compounds on cellular redox regulation. Based on the approach published by Alcock et al. (Chembiochem 2020, 21, 1329–1334), we applied sulfenic acid labeling using the norbornene-biotin (norb-bio) probe in human bronchial epithelial cells. To define appropriate treatment conditions for reliable protein identification by LC-MS/MS, cells were first incubated with norb-bio, followed by oxidative stimulation with H₂O₂. With respect to sulfenic acid detection by streptavidin-HRP, BEAS-2B cells showed a dose-dependent increase in sulfenic acid formation after H₂O₂ stimulation. We found that 1.5 mM norb-bio for 2 h and an H₂O₂ stimulation with 150 to 200 µM for 1°h was sufficient for the labeling in intact BEAS-2B cells. However, since only very low levels of biotinylated protein were obtained, an efficient LC-MS/MS sample processing method needs to be selected. Up to now, we demonstrated that protein sulfenic acid is formed in BEAS-2B cells upon oxidative stimulation. Next, the LC-MS/MS measurement conditions will be adjusted in order to obtain an optimal readout to identify specific proteins involved in redox regulation, as well as the impact of toxic metal ions on this process.
G 26
Biogenesis of the presynaptic compartment
Presenting author:
Leibniz Forschungsinstitut für Molekulare Pharmakologie (FMP), Molecular Physiology & Cell Biology, Robert-Roessle-Strasse 10, 13125 Berlin [DE], lucht@fmp-berlin.de
Author(s):
Max Thomas Lucht, Filiz Sila Rizalar, Dmytro Puchkov, Volker Haucke
One of the most striking properties of a neuron is the great distance between its soma and the presynapses (up to one meter in humans). Since the majority of proteins are synthesized in the soma, the delivery of the machinery necessary to form functional synapses poses a unique challenge. To improve the understanding of this transport process, how these precursor vesicles (PV) might be assembled and their transport regulated, it is crucial to investigate their fundamental properties. To this end, uncovering their protein composition and their ultrastructure will help to integrate the transport of presynaptic proteins into the overarching neuronal processes. The main challenges arise from the transient nature of PVs as well as their large proteomic overlap with the vastly more abundant synaptic vesicles. In this project we investigate the protein composition of PVs as well as their structural identity. By redirecting PVs to mitochondria, an electron-dense organelle, and combining conventional live imaging with focused ion beam scanning electron microscopy (FIB-SEM), we are able to show their high morphological variability. To study their protein composition via mass spectrometry, especially in terms of sorting factors and regulators, it will be necessary to isolate a large number of vesicles with a sufficient purity. Therefore, we will pursue multiple strategies that target PV cargo proteins as well as the main motor protein responsible for PV transport.
Sept 5, 13:15
Advances in Mass spectrometry-based proteomics for body fluid and single cell type-resolved tissue proteomics
Presenting author:
Max Planck Institute of Biochemistry, Martinsried, Germany
Author(s):
Matthias Mann
Recent breakthroughs in high-content imaging, mass spectrometry-based proteomics and computational biology are transforming bioscience. In this talk, I will introduce our Python-based open-source AlphaPept software suite, designed for rapid and efficient processing of large MS datasets. Additionally, I will highlight our advancements in MS-based technologies, enable large scale interactomics studies as well as large plasma-cohort analysis to identify diagnostic and prognostic biomarkers of chronic diseases. Finally, I will describe our new workflow termed Deep Visual Proteomics that enables single cell analysis to describe cellular heterogeneity, such as those that arise in cancer. DVP combines high-content microscopy, AI-driven image recognition, and laser microdissection with ultrahigh sensitivity MS to connect visual, spatial, and molecular proteomics data. Applied to various diseases, such as borderline ovarian cancers, rare cutaneous drug reactions, and liver diseases, this provides a comprehensive understanding of cellular function at resolution specific to the cell type, identifying potential therapeutic targets. DVP has great potential in facilitating diagnosis and prognosis and moving us towards personalized cancer medicine, which is our ambition moving forward.
Short talk 6
µPhos: a scalable and sensitive platform for functional phosphoproteomics
Presenting author:
Universitätsklinikum Jena, Funktionelle Proteomanalyse, Am Klinikum 1, 07747 Jena [DE], florian.meier@med.uni-jena.de
Author(s):
Denys Oliinyk, Andreas Will, Felix Schneidmadel, Maximilian Böhme, Jenny Rinke, Andreas Hochhaus, Thomas Ernst, Markus Lubeck, Oliver Raether, Sean Humphrey, Florian Meier
Mass spectrometry has revolutionized cell signaling research by vastly simplifying the analysis of protein phosphorylation on a systems scale. However, disentangling the functionality of the phosphoproteome remains a particularly challenging task, considering that only few of the well over 100,000 reported phosphorylation sites have known cognate kinases, and even fewer are functionally characterized. There is therefore a growing need to further increase the throughput, sensitivity and robustness of MS-based phosphoproteomics workflows to study cellular responses to perturbations in space and time. Here we introduce µPhos (‘microPhos’), an accessible phosphoproteomics platform that permits phosphopeptide enrichment from 96-well cell culture experiments in 30,000 unique phosphopeptides in a human cancer cell line using 20 µg starting material, and confidently localize ~6,500 phosphosites from 1 µg. This depth covers key signaling pathways, rendering sample-limited applications and perturbation experiments with hundreds of samples viable as we demonstrate by profiling the time-resolved response of a chronic myeoloid leukemia model to tyrosine kinase inhibitors.
G 28
Detection of known and novel small proteins in Pseudomonas stutzeri using a combination of bottom-up and digest-free proteomics and proteogenomics
Presenting author:
MPI of Biophysics, , Max-von-Laue-Str. 3, 60438 Frankfurt [DE], jakob.meier-credo@biophys.mpg.de
Author(s):
Jakob Meier-Credo, Benjamin Heiniger, Christian Ahrens, Julian Langer
Small proteins of around 50 aa have been largely overlooked in biochemical assays due to the inherent challenges with detecting and characterizing them. Recent discoveries of their critical roles in many biological processes have led to an increased recognition of the importance of small proteins for basic research and as potential new drug targets. One example is CcoM, a 36 aa subunit, that plays an essential role in adaptation to oxygen-limited conditions in P.stutzeri, a model for the clinically relevant pathogen P. aeruginosa. However, as no comprehensive data were available in P. stutzeri, we devised an integrated, generic approach to study small proteins more systematically. Using the first complete genome as basis, we conducted proteomics analyses and established a digest-free, direct-sequencing approach to study cells grown under aerobic and oxygen-limiting conditions. Finally, we also applied a proteogenomics pipeline to identify missed protein-coding genes. We identified 2921 known and 29 novel proteins, many of which were differentially regulated. Among 176 small proteins 16 were novel. Direct sequencing exhibited advantages in the detection of small proteins with higher sequence coverage and more PSMs. Three novel small proteins, uniquely identified by direct sequencing and not conserved beyond P. stutzeri, were predicted to form an operon with a conserved protein and may represent de novo genes.
M 08
APEX-based proximity labeling for time-resolved, subcellular proteomics of primary cilia to study proteome dynamics during active signaling
Presenting author:
Saarland University Medical Center, Medical Biochemistry and Molecular Biology, Kirrberger Str. 100, 66421 Homburg [DE], david.mick@uks.eu
Author(s):
Tommy Sroka, Elena May, David Mick
The primary cilium is a fL-sized compartment of vertebrate cells that initiates signaling cascades in response to external stimuli. Effective cilia signaling depends on the dynamic transport of signaling components such as receptors and effectors into and out of the cilium. Yet, the precise content and the extent of the proteomic remodeling of primary cilia during active signaling remained largely unknown. We employ proximity labeling methods using cilia-localized ascorbate peroxidase (cilia-APEX) in combination with tandem-mass-tags for quantitation by synchronous precursor selection-MS³ approaches to profile the cilia proteome in a time-resolved manner after signal pathway stimulation. By inducing the hallmark primary cilium signaling pathway, Hedgehog signaling, we could reconcile known dynamics in the localization of known signaling components. We further revealed a fast removal of the cAMP-dependent protein kinase (PKA) holoenzyme, including its unconventional A-kinase anchoring protein GPR161 from primary cilia. Hierarchical clustering identified the putative phosphatase PALD1 that accumulates in cilia in response to active Hedgehog signaling to dampen signaling in a cell type-specific manner. Our unbiased analyses demonstrate that proximity labeling in combination with quantitative proteomics allows time-resolved proteomics of subcellular compartments and provide novel insights into how primary cilia orchestrate signaling processes.
G 16
Identification of mitotic DNA-protein complexes formed after replication stress by ChIP-MS analysis
Presenting author:
Kaiserslautern University, , Fruchthallstraße, 67655 Kaiserslautern [DE], fmohseni@rptu.de
Author(s):
Farbod Mohseni, Angela Wieland, Andrea Tirincsi, Markus Räschle
Replication stress challenges genome stability, leading to replication fork stalling or collapse. Incomplete replication gives rise to mitotic errors, including mis-segregation of chromosomes and formation of ultrafine bridges (UFBs) connecting segregating sister chromatids. Most UFBs disappear during late mitosis, however the mechanism of UFB resolution remains unknown. In this study, we apply Chromatin Immunoprecipitation Mass Spectrometry (ChIP-MS), a powerful technique widely used for the characterization of protein-DNA complexes formed during transcription or DNA repair. ChIP-MS involves crosslinking of transient protein assemblies with the bound DNA, isolation of chromatin and its fragmentation. Solubilized protein-DNA complexes are then subjected to immune precipitation and quantitative MS analysis. Here we conduct ChIP-MS experiments using antibodies recognizing known UFB-associated proteins, including the BLM and PICH helicase, as well as the DNA repair factor FancD2. By comparing the formation of protein assemblies induced by mild replication stress in S-phase and mitotic cells, we aim to identify novel UFB-associated proteins. Currently, we are in the process of validating various mitosis-specific interaction partners through reciprocal ChIP-MS experiments, immunofluorescence microscopy, and phenotypic assays. The identification of novel UFB-associated proteins will enhance our understanding of UFB resolution and their role in maintaining genomic stability.
Sept. 5, 16:00
Native mass spectrometry: How to probe molecular principles of assembly and interactions of protein complexes
Presenting author:
Frankfurt University, Physical and Theoretical Chemistry, Max-von-Laue-Str. 9 , 60438 Frankfurt [DE], morgner@chemie.uni-frankfurt.de
Author(s):
Nina Morgner
Protein complex assembly as well as their interplay are controlled by the non-covalent interactions of all biomolecular partners. Native mass spectrometry and ion mobility are ideally suited to unravel the molecular principles which tightly control these interactions. Here I will present what we can learn about well-choreographed assembly strategies of a multi protein complex such as an ATPase or rather unwanted aggregation as seen for the Alzheimer related Amyloid b peptide. For the example of photoreceptors I will show how instrumental modifications can allow for time resolved studies of light dependent conformational rearrangements upon illumination.
G 10
Single cell proteome analysis with ultra-high sensitivity using a timsTOF mass spectrometer
Presenting author:
Bruker Daltonics GmbH + Co KG, , Fahrenheitstr 4, 28359 Bremen [DE], tor.mueller@bruker.com
Author(s):
Christoph Krisp, Anjali Seth, David Hartlmayr, Torsten Müller, Guilhem Tourniaire, Markus Lubeck, Gary Kruppa
For single cell proteome analysis, ultra-high sensitivity mass spectrometry is a key to reach proteome coverages necessary for understanding the cellular heterogeneity on a cell-by-cell level. Latest enhancements in ion transfer with a larger transfer capillary, an additional higher-pressure segment for more effective ion collection and two orthogonal deflections, to maintain robustness, and high-capacity trapped ion mobility spectrometry (TIMS) pushes the limits of detection to single cell level. Here, we assessed the sensitivity of a timsTOF Ultra mass spectrometer using a dilution series of K562 cell digest showing excellent identification rates, reproducibility, and quantification accuracy per concentration replicates. Processing of the dia-PASEF data identified >1,000 protein groups out of 15 pg, and >7,000 protein groups out of 16 ng K562 peptides loaded on column. The quantitative accuracy improved inversely with loaded peptide amounts with 19% at 15 pg to 4% at loads of 4, 8 and 16 ng. Analysis of the isolated HeLa cells resulted in good identification rates and good reproducibility per individual cell count group with expected increase in protein abundance from the single cells to 20 cells. The timsTOF Ultra combined with automated single cell isolation and sample preparation using the cellenONE® platform for protein-loss reduced preparation and transfer with the proteoCHIP format leads to deep proteome coverage and high reproducibility.
Sept. 5, 16:30
Narrow-window DIA for ultra-fast quantitative analysis of comprehensive proteomes with high sequencing depth
Presenting author:
University of Copenhagen, Novo Nordisk Foundation Center for Protein Research, Blegdamsvej 3b, 2200 Copenhagen [DK], jesper.olsen@cpr.ku.dk
Author(s):
Jesper Olsen
The goal of mass spectrometry (MS)-based proteomics is to efficiently and reliably characterize complete proteomes. We introduce a narrow window data-independent acquisition (DIA) method utilizing 2-Th precursor isolation windows. This method dissolve the distinctions between data-dependent acquisition (DDA) and DIA approaches. To achieve this, we make use of the new Quadrupole Orbitrap mass spectrometer coupled to an asymmetric track lossless (Astral) analyzer, which offers exceptional features such as >200 Hz MS/MS scanning speed, high resolving power, high sensitivity, and low ppm-mass accuracy. By using narrow-window DIA, we are able to profile >100 full yeast proteomes within a single day or approximately 10,000 human proteins in just half-an-hour. Additionally, by acquiring multiple shots of fractionated samples, we can comprehensively cover human proteomes in approximately 3 hours. This approach demonstrates a similar level of depth as next-generation RNA sequencing, but with 10 time’s higher throughput compared to the current leading MS techniques. We demonstrate high quantitative precision and accuracy based on 3-species mixture analysis. Overall, our ultra-fast scanning narrow window DIA strategy offers a significant advancement in proteomics research, enabling rapid and accurate protein characterization with impressive throughput and quantification capabilities.
M 01
Identification of novel cellular targets of α-13’-COOH and garcinoic acid using a compound-centric chemoproteomic method
Presenting author:
Friedrich Schiller Universität Jena, Institute of Nutrition, Dornburgerstrasse 25, 07743 Jena [DE], sylvia.omage@uni-jena.de
Author(s):
Sylvia Omage, Maria Wallert, Stefan Lorkowski
Identifying the cellular targets of novel natural products increases the understanding of their biological actions. Most proteomic approaches lead to numerous false-negative/false-positive hits. We present an optimised chemoproteomic method that leads to a streamlined list of targets of α-13’-COOH and garcinoic acid (GA). Our approach is particularly suitable for hydrophobic compounds, since the methacrylic resin used is resistant to organic solvents and extreme pH, unlike the more commonly used sepharose resin. With the optimized approach α-13’-COOH and GA were separately coupled to the insoluble methacrylate resin, Toyopearl AF amino 650M, using their carboxyl groups. The coupled resins were incubated with appropriately processed cell lysates. After extensive washing of the resin, the proteins bound to the resin-coupled compounds were eluted and identified using mass spectrometry. We found 17 proteins involved in lipid metabolism, antioxidant response, glucose metabolism as well as stress and immune response. We have validated one of the targets, 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1), using western blotting. Since HMGCS1 is involved in cholesterol synthesis, this target is in line with previous findings that α-13’-COOH regulates cellular lipid metabolism. Validation of the remaining proposed targets is ongoing. In conclusion, compound-centric chemoproteomics has enabled us to identify HMGCS1 as a potential molecular target of α-13’-COOH and GA.
O 03
Coordination of DNA damage and aging by ubiquitin signaling and the ubiquitin-proteasome system
Presenting author:
CECAD Research Center , , Joseph-Stelzmann-Straße, 26, 50931 Köln [DE], mpandrea@uni-koeln.de
Author(s):
Maria Pandrea, Thorsten Hoppe
Double strand breaks (DSBs) are severe types of DNA lesions that if left unrepaired can lead to genomic instability and premature ageing. We are using the C. elegans germline to study homologous recombination DSB repair and its regulation by ubiquitin signaling. It remains unclear whether the age-associated decline in ubiquitin proteasome system (UPS) efficiency contributes to age-related genomic instability and disease progression. The E3/E4 ubiquitin ligase UFD-2 accumulates in the nuclei of irradiated germ cells within ubiquitination hubs to regulate DSB repair and DNA damage-induced apoptosis. Therefore, we are investigating the tissue-specific function of UFD-2 to characterize the coordination between DNA damage and aging by ubiquitin signaling and the UPS. Using an integrated proteomics approach consisting of large-scale proteomic studies and biotinylation-based protein-protein interaction assays we will generate a list of potential co-factors and substrates of UFD-2. So far, we have identified a putative UBQL4 ortholog F49C12.9 as an interactor of UFD-2 capable of regulating DSB responses. In addition, we observe that upon UFD-2 loss worms fail to inhibit RNA processing, ribosomal assembly and translation-associated processes following genotoxic insults. We expect to obtain an extensive picture of how genotoxic stress controls localization and activities of repair factors and how this is coordinated by ubiquitin signaling dynamics during development and aging.
C 04
Proteome analysis of precursor lesions from pancreatobiliary cancer to improve early cancer diagnostic
Presenting author:
Molecular Proteomics Laboratory, BMFZ, Heinrich Heine Universität, Düsseldorf, 22.07., Universitätsstr. 1 , 40225 Düsseldorf [DE], stella.pauls@hhu.de
Author(s):
Stella Pauls, Anja Stefanski, Christin Hafermann, Friederike Opitz, Sandra Biskup, Irene Esposito, Kai Stühler
Objective: Cell‑subpopulation analysis for the detection of proteins that are involved in tumor progression, enables the potential to find new disease-specific biomarker to improve diagnostics. Methods for morphomolecular characterization of pancreatobiliary (PB) precursors as well as molecular subtyping of different precursor stages applied on FFPE tissues has been combined with quantitative proteomics. Methods: For label-free analysis of pancreatic FFPE tissue, a modified tissue lysis protocol is used to disrupt cells, reverse the formalin fixation and to extract proteins. For protein purification and processing for MS analysis an optimized protocol for a single-pot solid-phase-enhanced sample preparation (SP3) method was applied. Furthermore, an optimized data independent acquisition method (DIA) was applied for LC-MS/MS analysis and data processing was performed using DIA-NN. Results: Using these optimized methods, we are able to analyze 2.5 mm² FFPE pancreatic tissues (approx. 8,600 cells) and to identify around 2,700 proteins per slice. We apply these optimized methods on precise morphological characterized and microdissected areas of intraductal papillary mucinous neoplasm (IPMN) from pancreatic FFPE tissues and identify over 5,000 proteins in 53 tissue slices of different tissue types. By using ANOVA and soft clustering methods, it is possible to find interesting biomarker candidates and obtain deeper insights of biological processes involved in tumor progression.
B 01
Exploring Pathogenic Mutations on Phosphorylation Sites: Unraveling Disease Mechanisms via Interactome Studies
Presenting author:
Max Delbrück Center for Molecular Medicine, Proteome Dynamics, Robert Rossle str. 10, 13125 Berlin [DE], trendelina.rrustemi@mdc-berlin.de
Author(s):
Trendelina Rrustemi
With the advancement of sequencing technologies, identification of single nucleotide mutations surged, exceeding functional characterization capacity. Many of these mutations occur within structure-lacking intrinsically disordered regions (IDRs) of proteins. IDRs often contain short linear motifs (SLiMs) that are crucial for protein-protein interactions (PPIs) and are often subject to phosphorylation. Our approach involved immobilizing synthetic peptides representing mutated IDR regions onto cellulose membranes to capture interacting proteins from cellular extracts. This enabled simultaneous comparison of interaction partners between wild-type, phosphorylated, and mutated peptide forms, allowing functional assessment of individual mutations. We screened 36 disease-causing phosphorylation site mutations within IDRs, sourced from PTMVar database. The results revealed substantial differences between phosphorylated and mutated peptide interactomes, often due to disrupted phosphorylated SLiMs. We later focused on S102P mutation in GATAD1 that is linked to dilated cardiomyopathy. We found that the mutation disrupted a phosphorylation site crucial for interaction with 14-3-3 proteins. Further studies suggest that the GATAD1 peptide is important for nuclear localization and 14-3-3 binding prevents importin-GATAD1 interaction, highlighting its importance in proper nucleocytoplasmic transport.
G 06
In-depth exploration of the cyanobacterial secretome with trapped ion mobility spectrometry coupled to dia-PASEF
Presenting author:
Friedlich Schiller University Jena, Institute for Inorganic and Analytical Chemistry, Lessingstr. 8, 07743 Jena [DE], david.russo@uni-jena.de
Author(s):
David A. Russo, Denys Oliinyk, Florian Meier, Julie A. Z. Zedler
Extracellular proteins are involved in a remarkable number of fundamental processes in cyanobacteria. Yet, there is limited knowledge regarding the identity and function of these secreted proteins. Here, we develop an approach which combines single-pot, solid-phase-enhanced sample preparation (SP3) with trapped ion mobility spectrometry (TIMS), coupled to parallel accumulation-serial fragmentation with DIA (dia-PASEF) to enable description of the cyanobacterial secretome with unprecedented depth. Application to cyanobacteria from three distinct habitats, Synechocystis sp. PCC 6803, Synechcoccus sp. PCC 11901 and Nostoc punctiforme PCC 73102, allowed the identification of up to 62% of all predicted secreted proteins. The approach was then extended to compare the Synechocystis sp. PCC 6803 wild-type secretome with that of a bloom-like aggregated state and a secretion-impaired mutant. We also demonstrate that the method can be miniaturized and adapted to a 96-well format for high-throughput secretome analysis. These findings challenge the general belief that cyanobacteria lack secretory proteins and point to a functional conservation of the secretome across species from different environments. Our approach should be broadly applicable to bacterio- and phytoplankton, with the potential to open new avenues of investigation in microbial exoproteomics.
M 10
ENRICH-iST technology provides deeper coverage of the plasma proteome
Presenting author:
Bruker Daltonik GmbH & Co. KG, Bremen, Germany, AppDev - BLSMS, Fahrenheitstrsse 4, 28359 Bremen [DE], Andreas.Schmidt@bruker.com
Author(s):
Andreas Schmidt, Katrin Hartinger, Claudia Martelli, Zehan Hu, Katharina Limm, Sebastian Mueller, Xaver Wurzenberger, Nils A. Kulak
As a liquid biopsy, blood is easily available and very rich in information on personal health and wellness. Moreover, its steady contact with all tissues and quick turnover time allows for precise determination of disease progression or the effect of a treatment. As a derivative after removing the cellular fraction, plasma retains the valuable information in form of antibodies, protein released from tissue or cytokines. Due to its high dynamic range, it presents a challenging sample for proteomics analysis. We introduce a fast and robust method to reduce the dynamic range in protein abundance by binding proteins to the micro-particles and follow up proteomics analysis of the bound fraction. In comparison to selective technologies, high abundant plasma proteins are still present in the sample. The ENRICH-iST technology, preserves quantitative differences in plasma samples and is therefore suitable to study disease cohorts or treatment progression with high reproducibility. A model cohort of plasma samples derived from lung cancer patients and matched healthy donors was prepared with the ENRICH-iST kit and analyzed by dia-PASEF technology on the TimsTOF HT mass spectrometer. Applying a sample derived spectral library, we were able to cover more than 1500 proteins in both cohorts with only 30 min acquisition time/sample. Using the ENRICH technology, tripled the number the number of significantly enriched proteins, thus allowing for a more precise description of the disease state.
G 23
Characterizing mitochondrial protein import in senescence
Presenting author:
Institute of Biochemistry II, , Theodor-Stern-Kai 7, 60590 Frankfurt am Main [DE], jo.schmidt@med.uni-frankfurt.de
Author(s):
Jonas Schmidt, Christian Münch
During aging, senescent cell accumulation occurs, characterized by irreversible cell-cycle arrest, pro-inflammatory phenotype, and decline in proteostasis. Senescence also leads to changes in mitochondria, including enlargement, increased mass, reduced ATP production, and decreased membrane potential. These alterations likely affect mitochondrial protein import (MPI) that relies on membrane potential and ATP. Consequently, MPI impairment is highly plausible in aged mitochondria. Restoring membrane potential has been shown to extend the lifespan in C. elegans, and genetic variations in the mitochondrial protein import system are linked to lifespan differences in humans. Despite the significance of MPI, the impact of senescence on this process is largely unknown. This project aims to address this knowledge gap using mePRODmt, a SILAC-based proteomics approach for quantifying protein uptake into mitochondria in IMR90 cells. We will investigate alterations in MPI during senescence and examine affected pathways in the senescent phenotype. Understanding the impact of senescence on mitochondrial protein import is crucial for unraveling the complex interplay between aging, cellular homeostasis, and neurodegenerative diseases.
M 13
TurboID reveals the proxiomes of VIPP1 and VIPP2 in Chlamydomonas reinhardtii and confirms VPL2 and PGRL1 in the VIPP1 proxiome
Presenting author:
RPTU Kaiserslautern-Landau, Molecular Biotechnology & Systems Biology, Paul-Ehrlich-Str 23, 67663 Kaiserslautern [DE], m.schroda@rptu.de
Author(s):
Elena Kreis, Katharina König, Melissa Misir, Justus Niemeyer, Frederik Sommer, Michael Schroda
In Chlamydomonas reinhardtii, VIPP1 and VIPP2 play roles in the sensing, signaling and coping with membrane stress, triggering a chloroplast unfolded protein response (cpUPR), and in the biogenesis of thylakoid membranes. To gain more insight into these processes, we aimed to identify proteins interacting with VIPP1/2 in the chloroplast and chose proximity labeling (PL) for this purpose. TurboID-mediated PL with VIPP1/2 as baits under ambient and H₂O₂ stress conditions confirmed known interactions of VIPP1 with VIPP2, HSP70B and CDJ2. Novel proteins in the VIPP1/2 proxiome can be grouped into proteins involved in the biogenesis of thylakoid membrane complexes and the regulation of photosynthetic electron transport. A third group comprises 11 proteins of unknown function whose genes are upregulated under chloroplast stress conditions. We named them VIPP PROXIMITY LABELING (VPL1-11). We confirmed VIPP1 in the proxiomes of VPL2 and PGRL1 in reciprocal experiments and aim to (co)-localize them in the chloroplast. Our results demonstrate the robustness of TurboID-mediated PL for studying protein interaction networks in the chloroplast of Chlamydomonas and pave the way for analyzing functions of VIPPs and their proximal proteins in thylakoid biogenesis and stress responses.
C 03
Proteomic Characterization of Colorectal Cancer Patients for Precision Oncology
Presenting author:
German Cancer Research Center (DKFZ), Molecular Genome Analysis, Im Neuenheimer Feld 280, 69120 Heidelberg [DE], luisa.schwarzmueller@dkfz-heidelberg.de
Author(s):
Luisa Schwarzmüller, Efstathios Vlachavas, Katja Beck, Katrin Pfütze, Theresa Mullholland, Johannes Betge, Stefan Fröhling, Dominic Helm, Stefan Wiemann
Although research has made major advances in the discovery of cancer biomarkers and the development of new therapeutic options, the majority of patients receive the standard treatment for their respective cancer type. Molecular tumor boards, such as within the NCT MASTER program, try to leverage recent technological developments for in-depth molecular tumor characterization to infer personalized therapy recommendations. This genome-driven precision oncology project considers each patient’s mutational status and mRNA expression for treatment guidance. However, including protein abundance and phosphorylation status would offer an additional layer of tumor characterization regarding cancer pathway activities. To advance the integration of high-throughput, unbiased proteomics into precision oncology, we established a mass spectrometry-based full and phospho proteome screening of tissue samples and applied it to a retrospective NCT MASTER cohort of 31 colorectal cancer patients. Adding the informational layers of protein expression and activity to the previously acquired genomic and transcriptomic information, offered new insights into oncogenic mechanisms and identified possible tumor vulnerabilities. The protein and pathway activity measurements could have a significant impact on improving the stratification of patients into more actionable treatment “baskets” and enhance personalized oncology.
Sept 6, 11:45
An Integrated Landscape of mRNA and Protein Isoforms
Presenting author:
Max Delbrück Center, Berlin, Germany
Author(s):
Matthias Selbach
Proteomic characterization of protein isoforms poses a significant challenge due to limitations in available methodologies. Current bottom-up proteomic approaches provide limited information on protein isoforms, while top-down proteomic workflows often fail to comprehensively capture them. In this study, we introduce peptide correlation profiling (PepCP) as a novel method for globally characterizing protein isoforms. PepCP involves protein fractionation via SDS-PAGE, followed by bottom-up proteomic analysis of individual fractions. By quantifying peptide abundances across protein fractions, we obtain peptide abundance profiles that enable identification of protein isoforms through a computational pipeline. Using PepCP, we identified approximately 20,000 protein isoforms for 10,000 genes in human RPE-1 cells. Our results demonstrate that PepCP can identify isoforms arising from diverse cellular mechanisms, such as alternative splicing, alternative translation, and proteolytic processing. Additionally, we complemented our proteomic data by conducting full-length mRNA sequencing. Our integrated landscape of mRNA and protein isoforms provides insights into how transcriptional, translational and post-translational processes contribute to proteome complexity.
O 02
A modular cloning (MoClo) toolkit for reliable intracellular protein targeting in the yeast Saccharomyces cerevisiae
Presenting author:
RPTU Kaiserslautern-Landau, Standort Kaiserslautern, AG Zellbiologie, Erwin-Schrödinger-Straße 13, 67663 Kaiserslautern [DE], simakin@rhrk.uni-kl.de
Author(s):
Pavel Simakin, Christian Koch, Johannes M. Herrmann
Modular Cloning (MoClo) allows the combinatorial assembly of plasmids from standardized genetic parts without the need of error-prone PCR reactions. It is a very powerful strategy which enables highly flexible expression patterns without the need of repetitive cloning procedures. In this study, we describe an advanced MoClo toolkit that is designed for the baker’s yeast Saccharomyces cerevisiae and optimized for the targeting of proteins of interest to specific cellular compartments. Comparing different targeting sequences, we developed signals to direct proteins with high specificity to the different mitochondrial subcompartments, such as the matrix and the intermembrane space (IMS). Furthermore, we optimized the subcellular targeting by controlling expression levels using a collection of different promoter cassettes; the MoClo strategy allows it to generate arrays of expression plasmids in parallel to optimize gene expression levels and reliable targeting for each given protein and cellular compartment. Thus, the MoClo strategy enables the generation of protein-expressing yeast plasmids that accurately target proteins of interest to various cellular compartments.
M 07
Quantitative Translation and Import Proteomics using mePROD
Presenting author:
Institute of Biochemistry II, , Theodor-Stern-Kai 7, 60590 Frankfurt am Main [DE], tascher@med.uni-frankfurt.de
Author(s):
Georg Tascher, Jasmin Schäfer, Suleyman Bozkurt, Christian Münch
Measuring protein translation is an invaluable tool for understanding cellular stress-responses and protein homeostasis. Classic pulsed stable isotope labeling with amino acids in cell culture (pSILAC) requires relatively long pulse time for sufficient incorporation of heavy isotopes into the proteome. Hence, we developed multiplexed enhanced protein dynamics mass spectrometry (mePROD) combining pSILAC with Tandem mass tags (TMT), enabling robust quantification of translation on a proteome wide scale in experiments with short labeling times. This was achieved by incorporating a „booster-channel” containing only heavy-labeled peptides to increase acquisition of MS2-spectra and thus quantification of newly synthesized peptides as well as a “noise-channel“ containing only light peptides to determine background noise levels and co-isolation interference for each individual peptide. We recently expanded the method to study mitochondrial protein import by using a booster-channel comprised of enriched mitochondria. We show that mePROD provides an easy and cost-efficient method to profile proteome-wide translatome changes at a temporal resolution of minutes. The method already has brought valuable insight into different biomedical contexts, such as SARS-CoV2-Infection and acute myeloid leukemia. Notably, the noise-channel included in mePROD makes ratio compression, caused by co-isolation of non-targeted ions, as typically observed in TMT MS2-based methods, largely negligible.
G 19
Structural & Functional Analysis of MICOS & the Mitochondrial Intermembrane Space Bridging Complex (MIB)
Presenting author:
Universität des Saarlandes, Medical Biochemistry & Molecular Biology, Kirrberger Straße 100, Gebäude 45.2, 66421 Homburg [DE], martin.van-der-laan@uks.eu
Author(s):
Alexander von der Malsburg, Martin van der Laan
Mitochondria are surrounded by two distinct membrane systems. The outer membrane (OM) mediates communication with the cytosol and other organelles. The inner membrane (IM) is particularly protein-rich and harbors the machinery for ATP synthesis by oxidative phosphorylation. Intimate cooperation of both membranes is required for key functions of mitochondria, like lipid synthesis, channeling of metabolites an ions, like Calcium, and apoptosis. We and others have identified and initially described a direct OM-IM contact site in yeast mitochondria formed by the Mitochondrial Contact Site and Cristae Organizing System (MICOS) in the IM and the Sorting and Assembly Machinery (SAM) in the OM. Our recent proteomic and biochemical studies on this Mitochondrial Intermembrane Space Bridging (MIB) super-complex in human mitochondria have revealed a novel mechanism for the biogenesis of OM beta-barrel proteins, like VDACs, that requires the Hsp40 co-chaperone DNAJC11 at the MIB.
G 30 & Short talk 2
The proteomic landscape of synaptic diversity across brain regions and cell types
Presenting author:
MPI Brain Research, , Max-von-Laue Strasse 4, 60438 Frankfurt am Main [DE], marc.van-oostrum@brain.mpg.de
Author(s):
Marc van Oostrum, Thomas Blok, Stefano L. Giandomenico, Susanne tom Dieck, Georgi Tushev, Nicole Fürst, Julian Langer, Erin M. Schuman
Neurons diversify synaptic contacts using protein combinations that define the specificity and function of synapses. While there is ample evidence of diverse synaptic structures, states or functional properties, the diversity of the underlying individual synaptic proteomes remains largely unexplored. We used 7 different Cre-driver mouse lines crossed with a floxed mouse line in which the presynaptic terminals were fluorescently labeled (SypTOM) to identify the proteomes that underlie synaptic diversity. We used fluorescent-activated synaptosome sorting to isolate and analyze using quantitative mass spectrometry 18 types of synapses and their underlying synaptic proteomes. We discovered ~1’800 unique synapse type-enriched proteins and allocated thousands of proteins to different types of synapses. We identify commonly shared synaptic protein modules and highlight the hotspots for proteome specialization. A protein-protein correlation network classifies proteins into modules and their association with synaptic traits reveals synaptic protein communities that correlate with neurotransmitter identity. We reveal specializations and commonalities of the striatal dopaminergic proteome and highlighting proteome signatures that relate to the functional properties of interneuron synapse types. This study opens the door for molecular systems-biology analysis of synapses and provides a framework to integrate type-specific proteomic information with cellular or circuit-level experiments.
G 01
The electrophilic immunometabolite itaconate causes an acid stress response as well as S-bacillithiolation and S-itaconation in the thiol proteome of Staphylococcus aureus
Presenting author:
Freie Universitat Berlin, Institut für Biologie-Mikrobiologie, Königin-Luise-Straße 12-16, 14195 Berlin [DE], vu.v.loi@fu-berlin.de
Author(s):
Van Loi Vu, Tobias Busche, Susanne Eva Müller, Benno Kuropka, Karen Methling, Michael Lalk, Jörn Kalinowski, Haike Antelmann
Using RNA-seq transcriptomics and Northern blot transcriptional analyses, we analysed the specific stress responses caused by itaconate. Shotgun proteomics was applied to identify the targets of itaconation and S-bacillithiolation by itaconate in S. aureus. Phenotype analyses of mutants were used to analyse the role of specific defense mechanisms against itaconate stress. In the RNA-seq transcriptome, itaconate caused predominantly an acid stress response as revealed by the induction of the GlnR, KdpDE, CidR, SigB and GraRS regulons and the urease-encoding operon in S. aureus. The urease and urea supplementation were found to protect S. aureus from itaconate-induced acid stress. The generation of ROS and oxidative protein damage by itaconate was indicated by the up-regulation of the PerR, CtsR and HrcA regulons. Using shotgun proteomics, itaconate was shown to cause widespread S-bacillithiolation and S-itaconation of redox-sensitive antioxidant and metabolic enzymes, ribosomal proteins and translation factors in S. aureus, supporting the oxidative and electrophilic mode of action of itaconate in S. aureus. In phenotype analyses, the catalase KatA and the low molecular weight thiol bacillithiol (BSH) were found to provide protection against itaconate-induced ROS in S. aureus. Our results revealed that the antimicrobial mode of action of the itaconate in S. aureus is mediated by acid stress, oxidative and electrophilic stress, leading to S-bacillithiolation and itaconation
G 20
Unraveling the Link between Neuronal Activity Patterns and Proteome Remodeling through Optogenetic Stimulation and Mass Spectrometry Analysis
Presenting author:
Max Planck Institute for Brain Research, Synaptic Plasticity, Max von Laue Str. 4, 60438 Frankfurt [DE], quinn.waselenchuk@brain.mpg.de
Author(s):
Quinn Waselenchuk, Kristina Desch, Julian Langer, Erin Schuman
Understanding how neurons encode and process information is crucial for understanding synaptic transmission and plasticity. Indeed, neuronal activity patterns, represented by action potential firing, play a pivotal role in triggering downstream pathways and adaptive processes such as synaptic plasticity. Manipulating neuronal activity has been shown to induce changes in the transcriptome, proteome, and phosphoproteome, highlighting their interconnectedness. However, discrete temporal firing pattern-associated proteome dynamics remain unexplored. This project aims to fill this gap by tightly controlling and reading out neuronal activity using all-optical methods and determining neuronal proteomic and phosphoproteomic changes through mass spectrometry-based analysis. Primary cultured hippocampal neurons expressing light-gated ion channels will be subjected to defined firing patterns through light pulses, followed by collection of cells for (phospho)proteomic analysis. Proteomic changes occurring at synapses will be further assessed by comparing results from whole neurons with synaptosomal preparations. Additionally, ex vivo hippocampal slices will be isolated and stimulated, enabling assessment of response heterogeneity within the brain region. This comprehensive approach aims to uncover the relationship between neuronal activity patterns and their downstream proteomic and phosphoproteomic responses, shedding light on mechanisms underlying synaptic transmission and plasticity.
S 03 & Short talk 3
Insights into Meiosis: Elucidating DNA Repair Modulation via Mass Spectrometry
Presenting author:
Friedrich Miescher Laboratory, , Max-Planck-Ring 9, 72073 Tübingen [DE], john.weir@tuebingen.mpg.de
Author(s):
Veronika Altmannova, Petra Janning, Franziska Müller, Tanja Bange, John Weir
Exploring meiosis is key to understanding eukaryotic propagation and diversity. The pivotal process in meiosis I is accurate segregation of homologous chromosomes, facilitated by physical linkages - crossovers - derived from programmed double-strand DNA breaks. Crossovers are essential in the germline, yet deleterious in somatic cells, highlighting a unique DNA repair modulation in meiosis. Given the limited availability of mammalian germline tissue, our research utilizes budding yeast as a model system. Our work has been focused on the Mer3 helicase, known as HFM1 in mammals. Using immunoprecipitation coupled with mass spectrometry (IP-MS), we identified potential Mer3 interactors, including several DNA repair factors. We generated recombinant proteins and complexes and characterisded them using techniques including cross-linking mass spectrometry (XL-MS), which validated protein complex models produced by AlphaFold2. We hypothesised that phosphorylation might govern several protein complexes' formation. Hence, we studied the phosphorylation state of recombiant proteins, comparing them to the sites from meiotic cultures, and initiated work on phosphosite mutants. This study not only deepens our understanding of fundamental biology but also suggests mechanisms behind misexpression of meiotic proteins in cancers.
C 02
Proteomic subtypes of intrahepatic cholangiocarcinoma are linked to patient’s time-to-recurrence
Presenting author:
Freiburg University Hospital, Institute for Surgical Pathology, Breisacher Straße 115a, 79106 Freiburg [DE], tilman.werner@uniklinik-freiburg.de
Author(s):
Tilman Werner, Klara-Luisa Budau, Miguel Cosenza Contreras, Hause Frank, Kurowski Konrad, Pinter Niko, Schüler Julia, Martin Werner, Sigel Carlie, Laura Tang, Peter Bronsert, Oliver Schilling
Intrahepatic cholangiocarcinoma (ICC) is a rare and insufficiently described cancer whose pathological classification remains challenging. Recurrences are frequent, but occur in patient-individual and unpredictable timeframes. In this study, we characterized proteomic profiles of tumors and adjacent tissue from 80 ICC patients via liquid-chromatography mass-spectrometry (LC-MS/MS) in data independent acquisition (DIA) mode to identify predictive markers for the time-to-recurrence (TTR). We found two tumor subgroups: cluster 1 was enriched with extracellular matrix (ECM) components, and cluster 2 showed increased expression of RNA- and protein turnover machinery components. Patients from cluster 1, which also showed increased proteolytic activity in a semi-tryptic analysis, had significantly longer TTRs. An independent survival-statistics model then extracted proteins whose expression correlates with TTR distribution and uncovered similar biological motifs as in the clustering approach as determinants for the TTR. 9 patient-derived ICC xenografts highlighted the role of tumor-stroma interactions. In a principal component analysis based on this multi-species proteomic approach, we observed ECM proteins in association with infiltrating stroma, while tumor proteins were enriched for splicing, translation, and metabolization of RNA. Overall, ICC recurrence appears to shaped by differing protein expression profiles, likely as a result of varying tumor-stroma interactions.
S 02
Maintenance on mitochondrial complexes ensures bioenergetic function in differentiated cells
Presenting author:
Goethe University, Functional Proteomics, Institute for Cardiovascular Physiology, Theodor-Stern-Kai 7, 60590 Frankfurt [DE], wittig@med.uni-frankfurt.de
Author(s):
Ilka Wittig, Juliana Heidler, Heiko Giese, Ralf Brandes
The assembly sequence of mitochondrial complexes has been extensively studied in proliferating cells. These studies mostly reflect de-novo assembly and provide limited information on the dynamics of protein complexes in differentiated cells and tissues. The state of protein complexes in post-mitotic tissues may rather be a balance between biosynthesis and degradation. An important question is whether protein complexes are always assembled de novo or whether remodelling and repair mechanisms maintain mitochondrial function. Complexome profiling combines blue native electrophoresis with quantitative mass spectrometry to identify rare sub-complexes, assembly intermediates and complex remodelling. In this study, we combined complexome profiling and pulse stable isotope labelling of amino acids in cell culture (Pulsed-SILAC) to investigate the turnover and half-life of individual proteins within protein complexes in differentiated post-mitotic C2C12 myotubes. The results represent a comprehensive collection of data on the dynamics of all stable mitochondrial protein complexes. The complete turnover of all complexes of the oxidative phosphorylation system (OXPHOS) takes about one month. We identified subunits of complex I with higher turnover rates in parts of the electron transport modules and service factors involved in these quality control mechanisms to ensure full bioenergetic function in post-mitotic tissues.
G 27
Learning from errors: Deducing the action of aminoglycoside antibiotics from error landscapes
Presenting author:
Max-Planck Institut für Multidisziplinäre Naturwissenschaften, Department for Physical Biochemistry, Am Fassberg 11, 37077 Göttingen [DE], Ingo.Wohlgemuth@mpinat.mpg.de
Author(s):
Ingo Wohlgemuth, Nilanjan Ghosh Dastidar, Nicola Freyer, Christof Lenz, Henning Urlaub, Marina V Rodnina
The accuracy of protein synthesis determines the quality of the proteome and the fitness of the cell. Errors in translation have been associated with aging, cancer and neurological diseases. On the other hand, many antibiotics compromise the fidelity of translation and kill pathogens by disturbing their proteostasis. We use different mass spectrometric workflows to quantify missense errors in cellular proteins and study their impact on protein stability and the fitness of the cell. Recently, our analysis helped to understand the mechanism and exceptional proteotoxicity of aminoglycoside antibiotics (AGAs). AGAs target the bacterial ribosome and induce mistranslation, yet which translation errors induce bacterial cell death was unclear. We found that AGAs stay bound to the translating ribosome and thereby induce strings of consecutive errors, with up to four incorrect amino acids incorporated along a stretch of seven amino acids in a protein. Proteins with such error clusters are enriched in aggregates, indicating stronger protein misfolding. Consistent with the notion that error clusters drive the bactericidal effect of AGA we show that resistance mechanisms towards aminoglycosides can be associated with a dramatic reduction of error cluster formation. Overall, our work shows how the analysis of the microheterogenity of the proteome can help to deduce the cellular action of drugs and to probe the fitness of the cell.
G 07
N-terminomics identifies substrates of the secreted Staphylococcus aureus protease Jep previously missed by classical label-free proteomics
Presenting author:
University Medicine Greifswald, Department of Functional Genomics, Felix-Hausdorff-Straße 8, 17475 Greifswald [DE], hannes.wolfgramm@uni-greifswald.de
Author(s):
Hannes Wolfgramm, Christopher Saade, Leif Steil, Alexander Reder, Stephan Michalik, Christian Hentschker, Manuela Gesell Salazar, Liliane M. Fernandes Hartzig, Patricia Trübe, Barbara M. Bröker, Keenan Lacey, Victor J. Torres, Kristin Surmann, Silva Holtfreter, Uwe Völker
Virulence of Staphylococcus aureus is shaped by a wide range of tightly regulated virulence factors, including several proteases. These proteases act on host factors, contributing to immune evasion and spreading. In addition, there is evidence that secreted S. aureus proteases regulate virulence by processing the pathogen's own virulence factors extracellularly. Protease deletion mutants show altered levels of secreted virulence factors and exhibit hypervirulence in many cases (e.g., Gimza et al., 2021). In our study, we focused on the novel serine protease Jep, which is found almost exclusively in mouse-associated S. aureus strains. We have shown that the deletion of jep in the mouse-associated S. aureus strain JSNZ led to hypervirulence in a murine bacteraemia model. However, using a classical label-free proteomic approach, no differences were found in the secretome pattern of the mutant strain compared to the wild-type strain. This unexpected contradiction was resolved by using N-terminomics, which revealed alterations in the N-termini of a number of secreted proteins, including known virulence factors such as the subunits of LukAB. Our results suggest that the protease Jep influences virulence rather by targeted proteolytic processing of secreted virulence factors than by protein degradation. This example illustrates the power of N-terminomics in the investigation of proteases, to reveal effects that cannot be covered by classical label-free proteomic approaches.
O 05
Analysis of 3CL Protease inhibitors: an automated assay for rapid screening of compounds
Presenting author:
Max von Laue Straße 3, 60438 Frankfurt am Main [DE], jonathan.zoeller@biophys.mpg.de
Author(s):
Jonathan Zöller, Frederic Farges, Barbara Rathmann, Kristina Desch, Joshua Vollrath, Nadide Altincekic, Harald Schwalbe, Julian Langer
This work outlines an innovative approach to investigate potential drugs for the treatment of SARS-CoV-2, the virus responsible for the 2019 novel virus pandemic. Specifically, we developed a MALDI-MS based activity assay, which can be used to rapidly screen for potential inhibitors of the 3CL protease, a key enzyme in the replication of the virus. Our data show that compounds such as a newly identified potential drug named Tamol and Nirmatrelvir, a known inhibitor, strongly inhibit 3CL protease activity. To further investigate the effects of these inhibitors, we utilized HDX-MS and showed that Tamol is likely to cover the surface of domain I of the 3CL protease and that Nirmatrelvir binds strongly to its active site. We also investigated binding of Tamol to the Coronavirus receptor binding domain, and observed only weak interactions. We further acquired preliminary NMR data on the 3CL protease bound to Tamol and observed similar unfolding effects. With effective inhibitors in high demand, further investigation into potential compounds is essential. The developed assays have the potential to significantly expedite the process of finding new compounds that can be used to treat SARS-CoV-2.
Abstract not submitted yet