Posterabstracts (sorted by presenting author)

I02 - Annesha, Fariha
O13 - Bader, Verian
O15 - Barzideh, Jaleh
O01 - Bertgen, Lea
E05 - Broutzakis, George
L14 - Bülow, Margret
L15 - Bykov, Yury
I01 - Chatzimpinou, Anthoula
L01 - Chmyrov, Andriy
S01 - Crenner, Andrijana
O02 - Dempsey, Bianca
L02 - Dengler, Lisa
I06 - Durso, William
E01 - Fleckenstein, Hannah
O03 - Flohr, Tamara
O04 - Großerüschkamp, Frederik
O05 - Gupta, Nikita
I07 - Hamed, Mohamed Ismail

E06 - Haupt, Caroline
O06 - Heußer, Anna-Lena
O07 - Ho, Napyin
E03 - Hoffmann, Patrick
L16 - Holtmannspötter, Michael
L17 - Jekabson, Rafaella
S02 - Kessler, Laurell
I03 - Knapp, Sarah
S03 - Kock, Cilian
L18 - Kondadi, Arun Kumar
S04 - Kunz, Lea
O08 - Kurpa, Olha
L03 - Lehnart, Stephan E.
L19 - Lehners, Moritz
O09 - Lenhard, Svenja
L04 - Liebing, Aenne-Dorothea

L05 - López Sierra, Antonio
L20 - Lukas, Fabian
L06 - Maritzen, Tanja
I04 - Moreno-Andrés, Daniel
E04 - Nazari Banyarani, Delnia
L07 - Neumann-Staubitz, Petra
O10 - Nutz, Annika
L08 - Ocket, Esther
L09 - Peselj, Carlotta
O11 - Peter, Robert
L10 - Petropoulou, Anastasia
L11 - Plug, Martijn
E07 - Puchkov, Dmytro
L12 - Simakin, Pavel
O14 - Tamanna, Sadia
O12 - Verheyden, Nikita
E08 - Weismehl, Marius
I05 - Westenberger, Anke
L13 - Wiesmann, Sabine

Posterabstracts (sorted by poster number)

E01 - Fleckenstein, Hannah
E02 - withdrawn
E03 - Hoffmann, Patrick
E04 - Nazari Banyarani, Delnia
E05 - Broutzakis, George
E06 - Haupt, Caroline
E07 - Puchkov, Dmytro
E08 - Weismehl, Marius
I01 - Chatzimpinou, Anthoula
I02 - Annesha, Fariha
I03 - Knapp, Sarah
I04 - Moreno-Andrés, Daniel
I05 - Westenberger, Anke
I06 - Durso, William
I07 - Hamed, Mohamed Ismail
L01 - Chmyrov, Andriy
L02 - Dengler, Lisa
L03 - Lehnart, Stephan E.
L04 - Liebing, Aenne-Dorothea

L05 - López Sierra, Antonio
L06 - Maritzen, Tanja
L07 - Neumann-Staubitz, Petra
L08 - Ocket, Esther
L09 - Peselj, Carlotta
L10 - Petropoulou, Anastasia
L11 - Plug, Martijn
L12 - Simakin, Pavel
L13 - Wiesmann, Sabine
L14 - Bülow, Margret
L15 - Bykov, Yury
L16 - Holtmannspötter, Michael
L17 - Jekabson, Rafaella
L18 - Kondadi, Arun Kumar
L19 - Lehners, Moritz
L20 - Lukas, Fabian

O01 - Bertgen, Lea
O02 - Dempsey, Bianca
O03 - Flohr, Tamara
O04 - Großerüschkamp, Frederik
O05 - Gupta, Nikita
O06 - Heußer, Anna-Lena
O07 - Ho, Napyin
O08 - Kurpa, Olha
O09 - Lenhard, Svenja
O10 - Nutz, Annika
O11 - Peter, Robert
O12 - Verheyden, Nikita
O13 - Bader, Verian
O14 - Tamanna, Sadia
O15 - Barzideh, Jaleh
S01 - Crenner, Andrijana
S02 - Kessler, Laurell
S03 - Kock, Cilian
S04 - Kunz, Lea

I02
Holo vs. Soft X-ray Tomography: A comparison of advanced, label-free 3D cellular imaging techniques

Presenting author:
Fariha Annesha

Heidelberg University, Heidelberg [DE], fariha.annesha@cos.uni-heidelberg.de

Author(s):
Fariha Annesha, Dr. Venera Weinhardt

Accurately visualizing cellular structures is critical for biological research and medical diagnostics. This study compares holotomography and soft X-ray tomography in terms of quantitative analysis of cellular organelles in living or ‘near-native’ state without labelling. A key difference between these techniques lies in the strength of interactions between light and X-rays as measured by refractive index (RI and linear attenuation coefficients (LAC), respectively. Both parameters have been shown to be correlated with, cellular mass, density, and distribution of molecules within cells In our work, we quantitatively compare these two methods and their respective RI and LAC on phantom objects and different cells in order to understand their sensitivity, reproducibility and therefore value in quantitative analysis. By examining the strengths of each technique, we probe the potential applications for each method and potential of correlation between RI and LAC to bridge live imaging light microscopy with static but high resolution soft X-ray tomography.

O13
Visualization of Protein Quality Control Processes at Aggregate Interfaces

Presenting author:
Verian Bader

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.

O15
A computational dissection of microscopy files of human semen

Presenting author:
Jaleh Barzideh

Shiraz University of Medical Sciences, Shiraz [IR]
The University of Newcastle, Newcastle [AU], Jaleh.Barzideh@gmail.com

Author(s):
Jaleh Barzideh

Sperm dissection is a research very far from imagination from the historic time that it was discovered till now. This study explore that the semen is more than a medium for sperm conservation. Light sheet and confocal microscopy were applied for visualization of marker expression in semen. Photos exhibited sperm brain structure and its connection and synapses with the semen. Indeed, this review answers the main questions of philosophy and evolution and also clarifies the aim of this study of sperm structure. It also addresses puzzles such as sperm movement leading to the vague process of reproduction. Sperm dissection shows these organisms are capitulated by semen and in synapses with that.

O01
Distinct types of intramitochondrial protein aggregates protect mitochondria against proteotoxic stress

Presenting author:
Lea Bertgen

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.

E05
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:
George Broutzakis

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.

L15
A systematic bi-genomic split-GFP assay illuminates the mitochondrial matrix proteome and protein targeting routes

Presenting author:
Yury Bykov

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 (GFP_1-10) 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.

L14
Expansion microscopy and MALDI Imaging approaches to investigate inter-organelle communication

Presenting author:
Margret Bülow

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.

L01
Imaging Beyond The Visible: advantages of the Shortwave-Infrared spectral range for confocal microscopy and Raman scattering imaging.

Presenting author:
Andriy Chmyrov

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.

S01
Imaging of membrane-bound Y5 RNA using (bi)RhoBAST Fluorescent Light Up Aptamers

Presenting author:
Andrijana Crenner

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.

O02
TRC40 serves as a redox-regulated chaperone and interacts with protein quality control systems to cope with oxidative unfolding stress

Presenting author:
Bianca Dempsey

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.

L02
Why is mtDNA lost in cells lacking an active mitochondrial fusion machinery?

Presenting author:
Lisa Dengler

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.

I06
Restoring ER morphology and lysosome dynamics as therapeutic targets for axonopathies

Presenting author:
William Durso

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.

E01
Correlative X-Ray and Volume Electron Microscopy to Study Cerebral Malaria Pathogenesis in a 3D Blood Brain Barrier Model

Presenting author:
Hannah Fleckenstein

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.

O03
MitoTraP: Mitochondria triage precursor proteins into the intermembrane space to protect the cellular proteome from orphaned ribosomal proteins

Presenting author:
Tamara Flohr

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.

E02
withdrawn

O04
Label-free Infrared Imaging for Cancer Diagnostics in Computational Pathology

Presenting author:
Frederik Großerüschkamp

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

O05
NUCLEAR LOCALIZATION OF NON-IMPORTED MITOCHONDRIAL PROTEINS

Presenting author:
Nikita Gupta

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.

I07
Characterization of interphase nuclear pore complex assembly

Presenting author:
Mohamed Ismail Hamed

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.

E06
Fluorescence and electron microscopy study to elucidate the structure of Arf1-coated membrane tubules

Presenting author:
Caroline Haupt

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.

O06
Unraveling the consequences for cellular rescue and protein degradation responses upon allotopic expression

Presenting author:
Anna-Lena Heußer

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.

O07
Characterizing the role of TRIB3 in fine-tuning chronic mtUPR mediated cell death transition.

Presenting author:
Napyin Ho

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.

E03
Porous flow across nuclear pores and the in situ NPC structure of Dictyostelium in response to osmotic stress

Presenting author:
Patrick Hoffmann

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.

L16
The reHaloTag: Reversible labeling for live cell long-term high- and superresolution imaging

Presenting author:
Michael Holtmannspötter

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.

L17
Actin reorganization controls different phases of Plasma Membrane Repair

Presenting author:
Rafaella Jekabson

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.

S02
SELF-QUENCHED FLUOROPHORE LABELS FOR DNA-PAINT AND STED MICROSCOPY

Presenting author:
Laurell Kessler

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.

I03
Analysing nuclear foci and their subnuclear localisation using CellProfiler

Presenting author:
Sarah Knapp

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

S03
Elucidating the Roles of Glutathione and Reactive Oxygen Species in Basal Land Plant Development through Redox Sensor Microscopy

Presenting author:
Cilian Kock

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.

L18
Live-cell STED super-resolution nanoscopy reveals modulation of cristae dynamics in bioenergetically compromised mitochondria

Presenting author:
Arun Kumar Kondadi

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.

S04
Development of a ratiometric biosensor to measure ciliary cAMP levels and to elucidate Paladin1 accumulation in the primary cilia

Presenting author:
Lea Kunz

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.

O08
Aneuploidy-induced protein aggregation impairs mitochondria

Presenting author:
Olha Kurpa

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.

L03
Dysferlin recruits Vesicles to Calcium Release Units at Endomembrane Tubules in Cardiomyocytes

Presenting author:
Stephan E. Lehnart

, 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.

L19
Single-cell imaging identifies cGMP signalling heterogeneity in living VSMCs

Presenting author:
Moritz Lehners

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.

O09
Establishing a cytosolic version of the mitochondrial processing peptidase to study mitochondrial protein import

Presenting author:
Svenja Lenhard

, , , 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.

L04
The N terminus of the Oxoeicosanoid receptor 1–does its length affect the receptor’s localisation?

Presenting author:
Aenne-Dorothea Liebing

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.

L20
Canonical and non-canonical integrin-based adhesions dynamically interconvert

Presenting author:
Fabian Lukas

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.

L05
Imaging conformational rearrangements on the extracellular region of the neuropeptide Y Y5 receptor

Presenting author:
Antonio López Sierra

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.

L06
Canonical and non-canonical integrin-based adhesions dynamically interconvert

Presenting author:
Tanja Maritzen

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.

I04
LiveCellMiner: A new tool to analyze mitotic progression

Presenting author:
Daniel Moreno-Andrés

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.

E04
Investigating the Cytoskeletal Interactions with the Golgi Apparatus: A Quantitative Ultrastructural Study

Presenting author:
Delnia NAZARI BANYARANI

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

L07
Developping new concepts for drug discovery using high-content imaging

Presenting author:
Petra Neumann-Staubitz

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.

O10
Discovering the early stages of mitochondrial carrier biogenesis: a proximity-based labeling approach

Presenting author:
Annika Nutz

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.

L08
Insights into caveolae-mediated lipid transport

Presenting author:
Esther Ocket

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.

L09
Misfolded ER membrane proteins partition into the Reticulated Quality control compartment

Presenting author:
Carlotta Peselj

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.

O11
Structural and functional interaction of IRAG1 with IP₃R1

Presenting author:
Robert Peter

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.

L10
Investigation of the S. cerevisiae Start checkpoint and its regulation under acute carbon starvation

Presenting author:
Anastasia Petropoulou

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.

L11
Characterization of spindle ER determinants

Presenting author:
Martijn Plug

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.

E07
Optimized ultrathin embedding protocol to trace rare trafficking organelles in neuronal and other cells.

Presenting author:
Dmytro Puchkov

, , , [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

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

L12
A modular cloning (MoClo) toolkit for reliable intracellular protein targeting in Saccharomyces cerevisiae

Presenting author:
Pavel Simakin

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.

O14
Connected Plastid Networks: Incomplete Division or Fusion?

Presenting author:
Sadia S. Tamanna

Molecular Botany, Department of Biology, Rheinland-Pfälzische Technische Universität Kaiserslautern-Landau [DE], stamanna@rptu.de

Author(s):
Sadia S. Tamanna, Martin H. Schattat, Stefanie J. Müller-Schüssele

Plant cells contain various organelles that facilitate specialized reactions with distinct protein content and redox environment. Among these organelles, mitochondria and plastids are endosymbiont-derived and reproduce only through division. Mitochondria exchange their content through re-fusion and fission, while plastid bodies presumably remain discrete after division. However, there may be connections between different plastid bodies, either via incomplete fission or via secondary fusion facilitated by stromules. Stromules are thin, stroma-filled tubular structures that extend from plastids. Although stromule formation has been characterized in flowering plants, little is currently known about plastid connectivity in non-vascular plants, such as moss. In this study, we investigated the effects of stress on plastid connectivity in non-vascular plants, using the moss Physcomitrium patens. We used a stroma-targeted photoconvertible fluorescent protein (mMaple) to monitor and quantify the possibility of stroma content exchange with adjacent plastids under different abiotic stress conditions. Despite the appearance of stromules presumably connecting different plastid bodies, our experiments revealed no bulk transfer of photoconverted stroma. However, we show that in caulonema cell, plastid stroma exchange was possible, and hypothesize that plastid connectivity results from incomplete plastid division. These findings show that plastid connectivity in non-vascular plants is complex. The physiological significance of connected plastid bodies through incomplete fission requires further investigation.

O12
Identification of candidates from high-throughput proteomic profiling of PROTAC-induced degradation

Presenting author:
Nikita Verheyden

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.

I01
Microglia signs to distinct intracellular events upon acute inflammation

Presenting author:
Anthoula Chatzimpinou

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.

E08
Structural insights into the activation mechanism of antimicrobial GBP1

Presenting author:
Marius Weismehl

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.

I05
The cellular fate of RNA-nanoparticles - uptake, release & translation

Presenting author:
Anke Westenberger

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.

L13
A new task for eIF4A1: A RNA helicase in chromatin decondensation

Presenting author:
Sabine Wiesmann

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.