Podcasts about escrt

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.28.551012v1?rss=1 Authors: Santiago, J. V., Natu, A., Ramelow, C. C., Rayaprolu, S., Xiao, H., Kumar, V., Seyfried, N., Rangaraju, S. Abstract: Microglia are resident immune cells of the brain that play important roles in mediating inflammatory responses in several neurological diseases via direct and indirect mechanisms. One indirect mechanism may involve extracellular vesicle (EV) release, so that the molecular cargo transported by microglia-derived EVs can have functional effects by facilitating intercellular communication. The molecular composition of microglia-derived EVs, and how microglial activation states impacts EV composition and EV-mediated effects in neuroinflammation, remain poorly understood. We hypothesize that microglia-derived EVs have unique molecular profiles that are determined by microglial activation state. Using size-exclusion chromatography to purify EVs from BV2 microglia, combined with proteomic (label-free quantitative mass spectrometry or LFQ-MS) and transcriptomic (mRNA and non-coding RNA seq) methods, we obtained comprehensive molecular profiles of microglia-derived EVs. LFQ-MS identified several classic EV proteins (tetraspanins, ESCRT machinery, and heat shock proteins), in addition to over 200 proteins not previously reported in the literature. Unique mRNA and microRNA signatures of microglia-derived EVs were also identified. After treating BV2 microglia with lipopolysaccharide (LPS), interleukin-10, or transforming growth factor beta, to mimic pro-inflammatory, anti-inflammatory, or homeostatic states, respectively, LFQ-MS and RNA seq revealed novel state-specific proteomic and transcriptomic signatures of microglia-derived EVs. Particularly, LPS treatment had the most profound impact on proteomic and transcriptomic compositions of microglia-derived EVs. Furthermore, we found that EVs derived from LPS-activated microglia were able to induce pro-inflammatory transcriptomic changes in resting responder microglia, confirming the ability of microglia-derived EVs to relay functionally-relevant inflammatory signals. These comprehensive microglia-EV molecular datasets represent important resources for the neuroscience and glial communities, and provide novel insights into the role of microglia-derived EVs in neuroinflammation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.06.547997v1?rss=1 Authors: Suzuki, S., West, M., Zhang, Y., Fan, J. S., Roberts, R. T., Odorizzi, G., Emr, S. D. Abstract: Endosomes are specialized organelles that function in the secretory and endocytic protein sorting pathways. Endocytosed cell surface receptors and transporters destined for lysosomal degradation are sorted into intralumenal vesicles (ILVs) at endosomes by Endosomal Sorting Complex Required for Transport (ESCRT) proteins. The endosomes (multivesicular bodies, MVBs) then fuse with the lysosome. During endosomal maturation, the number of ILVs increases, but the size of endosomes does not decrease despite consumption of the limiting membrane during ILV formation. Vesicle-mediated trafficking is thought to provide lipids to support MVB biogenesis. However, we have uncovered an unexpected contribution of a large bridge-like lipid transfer protein, Vps13, in this process. Here, we reveal that Vps13-mediated lipid transfer at ER-endosome contact sites is required for the ESCRT pathway. We propose that Vps13 may play a critical role in supplying lipids to the endosome, ensuring continuous ESCRT-mediated sorting during MVB formation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.21.545954v1?rss=1 Authors: Thompson, J., Boisvert, F.-M., Salsman, J., Dellaire, G., Ridgway, N. D. Abstract: When exposed to excess fatty acids, specific cell types produce nuclear lipid droplets (nLDs) that associate with promyelocytic leukemia (PML) protein to form Lipid Associated PML Structures (LAPS) that are enriched in lipid biosynthetic enzymes but deficient in canonical proteins associated with PML nuclear bodies (PML NBs). To identify the PML interactome during lipid stress, we employed proximity-dependent biotin identification (BioID) in U2OS cells expressing PMLI and PMLII fused to the ascorbate peroxidase APEX2 and cultured in the absence or presence of oleate to enhance lipid droplet formation. The resulting interactome included proteins enriched under oleate-treated conditions, such mitogen activated protein kinase-activated protein kinase 2 (MK2), ESCRT proteins and the COPII vesicle transport proteins Sec23B, Sec24A and USO1. COPII proteins co-localized with both PML-NBs and LAPS but were selectively enriched in PML-NBs following oleate treatment. The nuclear localization of USO1 was uniquely dependent on PML expression. Thus, the APEX2-PML proximity interactome implicates PML domains in the nuclear function of a non-canonical network of COPII vesicle trafficking proteins. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.22.537920v1?rss=1 Authors: Dresselhaus, E. C., Harris, K. P., Koles, K., Pescosolido, M. F., Ermanoska, B., Rozencwaig, M., Soslowsky, R. C., Stewart, B. A., Rodal, A. A. Abstract: Exosomes are membrane-bound vesicles released by many cells including neurons, carrying cargoes involved in signaling and disease. It has been unclear whether exosomes promote intercellular signaling in vivo or serve primarily to dispose of unwanted cargo. This is because manipulations of exosome cargo expression or traffic often result in their depletion from the donor cell, making it difficult to distinguish whether these cargoes act cell-autonomously or through transcellular transfer. Exosomes arise when multivesicular endosomes fuse with the plasma membrane, releasing their intralumenal vesicles outside the cell. We show that loss of multivesicular endosome-generating ESCRT (endosomal sorting complex required for transport) machinery disrupts release of exosome cargoes from Drosophila motor neurons, without depleting them from the donor presynaptic terminal. Cargoes and autophagic vacuoles accumulate in presynaptic terminals, suggesting that compensatory autophagy follows endosome dysfunction. Surprisingly, exosome cargoes Synaptotagmin-4 (Syt4) and Evenness Interrupted (Evi) retain many of their signaling activities upon ESCRT depletion, despite being trapped in presynaptic terminals. Thus, these cargoes may not require intercellular transfer, and instead are likely to function cell autonomously in the motor neuron. Our results indicate that synaptic exosome release depends on ESCRT, and serves primarily as a proteostatic mechanism for at least some cargoes. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.27.534444v1?rss=1 Authors: Chou, C.-C., Vest, R., Prado, M. A., Wilson-Grady, J., Paulo, J. A., Shibuya, Y., Moran-Losada, P., Lee, T.-T., Luo, J., Gygi, S. P., Kelly, J. W., Finley, D., Wernig, M., Wyss-Coray, T., Frydman, J. Abstract: The role of proteostasis and organelle homeostasis dysfunction in human aging and Alzheimers disease (AD) remains unclear. Analyzing proteome-wide changes in human donor fibroblasts and their corresponding transdifferentiated neurons (tNeurons), we find aging and AD synergistically impair multiple proteostasis pathways, most notably lysosomal quality control (LQC). In particular, we show that ESCRT-mediated lysosomal repair defects are associated with both sporadic and PSEN1 familial AD. Aging- and AD-linked defects are detected in fibroblasts but highly exacerbated in tNeurons, leading to enhanced neuronal vulnerability, unrepaired lysosomal damage, inflammatory factor secretion and cytotoxicity. Surprisingly, tNeurons from aged and AD donors spontaneously develop amyloid-{beta} inclusions co-localizing with LQC markers, LAMP1/2-positive lysosomes and proteostasis factors; we observe similar inclusions in brain tissue from AD patients and APP-transgenic mice. Importantly, compounds enhancing lysosomal function broadly ameliorate these AD-associated pathologies. Our findings establish cell-autonomous LQC dysfunction in neurons as a central vulnerability in aging and AD pathogenesis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.12.520126v1?rss=1 Authors: Ader, N. R., Chen, L., Surovtsev, I. V., Chadwick, W. L., King, M. C., Lusk, C. P. Abstract: The reformation of the nuclear envelope (NE) at the end of metazoan mitosis requires the sealing of numerous fenestrations that recruit the endosomal sorting complexes required for transport (ESCRT) machinery. The molecular mechanisms by which ESCRT proteins drive NE sealing and their necessity to the reestablishment of the nuclear compartment have yet to be fully defined. Here, we leveraged the single NE hole generated by mitotic extrusion of the Schizosaccharomyces pombe spindle pole body to reveal two modes of ESCRT function carried out by unique complements of ESCRT-III proteins, both of which depend on the NE-specific CHMP7/Cmp7. The first is a grommet-like function, where we tie the presence of specific ESCRTs to a constriction of the NE hole from ~150 to ~50 nm in anaphase B. Consistent with a direct role for ESCRTs in restricting this NE hole diameter, the hole dilates a remarkable five-fold in cells lacking Cmp7. Second, in the subsequent G1/S-phases, a sealing module of ESCRT proteins replaces Cmp7 and is required to drive closure of the NE. Surprisingly, in the absence of Cmp7, nucleocytoplasmic compartmentalization remains intact despite discontinuities in the NE as large as 400 nm, suggesting a mechanism to establish a diffusion barrier(s) over persistent NE holes. Indeed, we demonstrate that the ortholog of the pericentriolar material protein, pericentrin (Pcp1), establishes such a barrier, likely through its ability to form a biomolecular condensate. NE remodeling mechanisms therefore cooperate with proteinaceous diffusion barriers beyond nuclear pore complexes to protect the nuclear compartment. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.05.519172v1?rss=1 Authors: Byrd, A. T., Marmorale, L., Addison, V., Marcinowski, S., Buchan, J. R. Abstract: A key pathological hallmark in greater than 97% of all Amyotrophic Lateral Sclerosis (ALS) cases is the cytoplasmic mislocalization and aggregation of a nuclear RNA binding protein, TDP-43. Driving clearance of cytoplasmic TDP-43 reduces toxicity in various ALS models, though how TDP-43 clearance is regulated remains controversial. To address this, we conducted an unbiased yeast genome-wide screen using high-throughput dot blots to identify genes that affect TDP-43 levels. Our screen identified ESCRT complex factors, which induce membrane invagination (particularly at multi-vesicular bodies; MVBs) and K63-linked ubiquitination as key facilitators of TDP-43 endolysosomal clearance. TDP-43 co-localized and bound Rsp5/NEDD4 and ESCRT proteins, and perturbations to either increased TDP-43 aggregation and accumulation. NEDD4 also ubiquitinates TDP-43. Lastly, TDP-43 accumulation caused formation of "giant" MVBs which could reflect a pathological consequence of TDP-43 pertinent to ALS. Our studies shed light on endolysosomal-mediated cytoplasmic protein degradation, which likely impacts multiple substrates, and may be a target for novel ALS therapeutic strategies. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.22.517322v1?rss=1 Authors: Pust, S., Brech, A., Wegner, C. S., Stenmark, H., Haglund, K. Abstract: Cellular abscission is the final step of cytokinesis that leads to the physical separation of the two daughter cells. The scaffold protein ALIX and the ESCRT-I protein TSG101 contribute to recruiting ESCRT-III to the midbody, which orchestrates the final membrane scission of the intercellular bridge. Here, we addressed by which mechanisms ALIX and the ESCRT-III subunit CHMP4B are transported to the midbody. Structured illumination microscopy revealed gradual accumulation of ALIX at the midbody, resulting in the formation of spiral-like structures extending from the midbody to the abscission site, which strongly co-localized with CHMP4B. Live-cell microscopy uncovered that ALIX appeared together with CHMP4B in vesicular structures, whose motility was microtubule-dependent. Depletion of ALIX led to structural alterations of the midbody and delayed recruitment of CHMP4B, resulting in delayed abscission. Likewise, depletion of the kinesin-1 motor KIF5B reduced the motility of ALIX-positive vesicles and caused delayed recruitment of ALIX, TSG101 and CHMP4B to the midbody, accompanied by impeded abscission. We propose that ALIX, TSG101 and CHMP4B are associated with endosomal vesicles transported along microtubules by kinesin-1, leading to their directional transport to the cytokinetic bridge and midbody, thereby contributing to their function in abscission. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.20.211854v1?rss=1 Authors: Laporte, M. H., Chi, K. I., Rolland, M., Caudal, L. C., Martinez-Hernandez, J., Martineau, M., Chatellard, C., Denarier, E., Mercier, V., Lemaitre, F., Blot, B., Moutaux, E., Cazorla, M., Buisson, A., Perrais, D., Fraboulet, S., Lante, F., Kirchhoff, F., Hemming, F. J., Sadoul, R. Abstract: In chemical synapses undergoing high frequency stimulation, vesicle components can be retrieved from the plasma membrane via a clathrin-independent process called activity dependent bulk endocytosis (ADBE). Alix (ALG-2 interacting protein X)/ PDCD6IP) is an adaptor protein binding to ESCRT and endophilin-A proteins and thereby driving deformation and fission of endosomal and cell surface membranes. In fibroblasts, Alix is required for clathrin-independent endocytosis. Here, using electron microscopy, we show that synapses from mice lacking Alix have subtle defects in presynaptic compartments, translating into flawed synaptic plasticity. Using cultured neurons, we demonstrate that Alix is required for ADBE. We further demonstrate that in order to perform ADBE, Alix must be recruited to synapses by the calcium-binding protein ALG-2 and interact with endophilin-A. Finally, we show that mutant mice lacking Alix in the forebrain undergo less seizures during kainate-induced status epilepticus. Furthermore, propagation of the epileptiform activity to the contralateral side of kainate injection is reduced. These results thus highlight Alix ko mice as an invaluable model to study the exact role of ADBE at synapses undergoing physiological or pathological stimulations. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.04.133546v1?rss=1 Authors: Sun, X., Deng, X., Hu, R., Duan, Y., Zhang, K., Cui, J., Ni, J., Wang, Q., Chen, Y., Li, A., Fang, Y. Abstract: Protein inclusions containing phosphorylated TDP-43 are a shared pathology in several neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, most ALS/FTD patients do not have a mutation in TDP-43 or the enzymes directly regulating its phosphorylation. It is intriguing how TDP-43 becomes hyperphosphorylated in each disease condition. In a genetic screen for novel TDP-43 modifiers, we found that knockdown (KD) of CHMP2B, a key component of the endosomal sorting complex required for transport (ESCRT) machinery, suppressed TDP-43-mediated neurodegeneration in Drosophila. Further investigation using mammalian cells indicated that CHMP2B KD decreased whereas its overexpression (OE) increased TDP-43 phosphorylation levels. Moreover, a known FTD-causing mutation CHMP2Bintron5 promoted hyperphosphorylation, insolubility and cytoplasmic accumulation of TDP-43. Interestingly, CHMP2B did not manifest these effects by its well-known function in the autophagy-lysosomal pathway. Instead, the kinase CK1 tightly regulated TDP-43 phosphorylation level in cells, and CHMP2B OE or CHMP2BIntron5 significantly decreased ubiquitination levels and the turnover of CK1 via the ubiquitin-proteasome (UPS) pathway. Finally, we showed that CHMP2B protein levels increased in the cerebral cortices of aged mice, which might underlie the age-associated TDP-43 pathology and disease onset. Together, our findings reveal a molecular link between the two ALS/FTD-pathogenic proteins CHMP2B and TDP-43, and provide an autophagy-independent mechanism for CHMP2B in pathogenesis. Copy rights belong to original authors. Visit the link for more info

The TWiV team covers outbreaks of eastern equine encephalitis virus in the US and poliovirus in the Philippines, and explain how a chemokine induced by HIV-1 infection helps release more virus particles from cells. Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, Rich Condit, Kathy Spindler, and Brianne Barker Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode Poliomyelitis returns to the Philippines (Mashable, virology blog) EEE in the USA (Am J Trop Med Hyg) Chemokine helps HIV-1 release (eLIFE) Image credit Letters read on TWiV 567 Timestamps by Jolene. Thanks! Weekly Science Picks Brianne - History of infectious disease before/after vaccines Alan - NIH Ham Radio Club Rich - Renewable jet fuel from air Dickson - Newly Discovered Comet Is Likely Interstellar Visitor Kathy - How professors spend their time Vincent - Non-polio enterovirus surveillance network Listener Picks Alexey - Periodic Table Podcasts and Videos Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

The TWiV team covers outbreaks of eastern equine encephalitis virus in the US and poliovirus in the Philippines, and explain how a chemokine induced by HIV-1 infection helps release more virus particles from cells. Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, Rich Condit, Kathy Spindler, and Brianne Barker Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode Poliomyelitis returns to the Philippines (Mashable, virology blog) EEE in the USA (Am J Trop Med Hyg) Chemokine helps HIV-1 release (eLIFE) Image credit Letters read on TWiV 567 Timestamps by Jolene. Thanks! Weekly Science Picks Brianne - History of infectious disease before/after vaccines Alan - NIH Ham Radio Club Rich - Renewable jet fuel from air Dickson - Newly Discovered Comet Is Likely Interstellar Visitor Kathy - How professors spend their time Vincent - Non-polio enterovirus surveillance network Listener Picks Alexey - Periodic Table Podcasts and Videos Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

The TWiVers present mitoviruses, which infect mitochondria, and how quasi-enveloped hepatitis A virus gets naked again. Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, and Kathy Spindler Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode Evidence for plant mitoviruses (Virology) Uncoating of quasi-enveloped HAV (eLife) Image credit Letters read on TWiV 541 Timestamps by Jolene. Thanks! Weekly Science Picks Alan- Where and when to see cherry blossoms Dickson- Nebraska floods Kathy- Peeps fungal infection experiment Vincent - Emergency declared in NY over measles, unvaccinated barred from public spaces Listener Pick Sam- STLR conversations Josh- Tree of Life Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

The TWiVers present mitoviruses, which infect mitochondria, and how quasi-enveloped hepatitis A virus gets naked again. Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, and Kathy Spindler Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode Evidence for plant mitoviruses (Virology) Uncoating of quasi-enveloped HAV (eLife) Image credit Letters read on TWiV 541 Timestamps by Jolene. Thanks! Weekly Science Picks Alan- Where and when to see cherry blossoms Dickson- Nebraska floods Kathy- Peeps fungal infection experiment Vincent - Emergency declared in NY over measles, unvaccinated barred from public spaces Listener Pick Sam- STLR conversations Josh- Tree of Life Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

Vincent travels to Penn State College of Medicine in Hershey, PA to speak with Nick and Leslie about their careers and their work on human cytomegalovirus and retroviruses. Host: Vincent Racaniello Guests: Nicholas Buchkovich and Leslie Parent Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode HIV-1 gag membrane binding domain (J Virol) Nuclear experience for gag (PNAS) Gag in nucleolus (J Virol) YB-1 for retrovirus assembly (J Virol) HCMV controls BiP levels (J Virol) HCMV regulates La (J Virol) Inhibition of HCMV by modulating syntaxin 5 (J Virol) Non-envelopment role for ESCRT-III during HCMV infection (J Virol) Late retroviral budding function (J Virol) Grit by Angela Duckworth The Scientists by John Gribbin Timestamps by Jolene. Thanks! Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

Hosts: Vincent Racaniello, Dickson Despommier, and Daniel Griffin The TWiPniks solve the case of the Man With AIDS, and explore how secretion of extracellular vesicles influences the social motility of Trypanosoma brucei. Become a patron of TWiP. Links for this episode: T. brucei exosomes and social motility (PLoS Path) Image credit Parasites Without Borders Letters read on TWiP 130 This episode is brought to you by Blue Apron. Blue Apron is the #1 fresh ingredient and recipe delivery service in the country. See what’s on the menu this week and get your first 3 meals free with your first purchase - WITH FREE SHIPPING - by going to blueapron.com/twip. Case Study for TWiP 130 Back in Peru. 24 yo female seen in ER, lives in rural area outside of big city, adobe house in highlands, thatched roof, dirt floor, 3000 meters. Quite ill with skin lesion for 48 h. 2 days before was pulling on pjs, felt sharp sudden pain in right upper leg. Next day found small living creature in pajamas, inguinal region. Developed red lesion, enlarged, developed black central dot. Then begins vomiting, comes to hospital. No fever, breathing fast 20, hr 70, bp 160/10, on exam see in right inguinal region an enlarging, necrotic area 1-2 cm, starting to look sick. Whites at 26000, left shift, 200 platelets, eosinophils 4%, bilirubin 3.5, creatinine 4.9 (going into renal failure, not making much urine). Hematocrit 14, BUN 59.7, CPK 227, RBCs and leukocytes in urine. No health problems, no surgeries, first interaction with health system. No toxic habit. Brings in the small creature! Send your case diagnosis, questions and comments to twip@microbe.tv Music by Ronald Jenkees

Fluorescence microscopy is a widely used technique for imaging of biological structures due to its noninvasiveness although resolution of conventional fluorescence microscopes is limited to about 200-300 nm due to the diffraction limit of light. Super-resolution fluorescence microscopy offers an extension of the original method that allows optical imaging below the diffraction limit. In this thesis, a microscope for localization-based super-resolution fluorescence microscopy techniques such as Stochastic Optical Reconstruction Microscopy (STORM) or Photoactivated Localization Microscopy (PALM) was established. An epifluorescence microscope was built for this purpose that provides both widefield and Total Internal Reflection Fluorescence (TIRF) excitation modalities and focus was put on the special requirements of localization-based super-resolution methods. This included a high mechanical and optical stability realized by a compact design and implementation of a home-built perfect focus system. The setup was further designed to allow both two- and three-dimensional imaging. The work also included both the development of a setup control software and a software for the analysis of the required data. Different analysis methods and parameters were tested on simulated data before the performance of the microscope was demonstrated in two and three dimensions at appropriate test samples such as the cellular microtubule network. These experiments showed the capability of super-resolution microscopy to reveal underlying structures that cannot be resolved by conventional fluorescence microscopy. Resolutions could be achieved down to approximately 30 nm in the lateral and 115 nm in the axial dimension. Subsequently, the established method was applied to two biological systems. The first is a study of the budding of the human immunodeficiency virus type 1 (HIV-1) from the host cell. In this step of the viral reproduction cycle, the virus hijacks the cellular endosomal sorting complex required for transport (ESCRT) machinery to achieve membrane fission. ESCRT consists of the subcomplexes ESCRT-0, -I, -II and -III and additional related proteins, from which HIV-1 recruits certain components. The fission process is initiated by the HIV-1 Gag protein, which recruits the ESCRT-I protein Tsg101 and the ESCRT-related protein ALIX to the virus assembly site. Subsequently, ESCRT-III proteins CHMP4 and CHMP2 form transient lattices at the membrane, which are actively involved in membrane fission. However, the actual geometry of the ESCRT machinery assembling at the HIV-1 budding site that is driving the fission process is still not fully understood. Different models proposed either constriction of the budding neck by lattices surrounding the neck, by ESCRT structures within the neck or within the bud itself. A problematic aspect in previous studies was the usage of modified, tagged versions of the involved proteins for visualization. In this study, super-resolution microscopy was therefore applied to endogenous Tsg101, ALIX and CHMP2 isoform CHMP2A and to a version of CHMP4 isoform CHMP4B with a small HA-tag to elucidate the size and the distribution of the structure relative to the HIV-1 assembly sites. ESCRT structures colocalizing with HIV-1 exhibited closed, circular structures with an average size restricted to 45 and 60 nm in diameter. This size was significantly smaller than found for HIV-1 assembly sites and the constriction of the size, which was not observed for non-colocalizing ESCRT structures at the cell membrane, ruled out an external restriction model. Super-resolution imaging of ALIX often revealed an additional cloud-like structure of individual molecules surrounding the central clusters. This was attributed to ALIX molecules incorporated into the nascent HIV-1 Gag shell. Together with experiments that confirmed the non-physiological behavior of tagged Tsg101 and a relative orientation of ESCRT clusters towards the edge of the assembly site, the results strongly point toward a within-neck model. A second project focused on the influence of external constriction on cell migration. The latter plays an important role in various processes in the human body ranging from wound healing to metastasis formation by cancer cells. Migration is driven by the lamellipodium, which is a meshwork of fine actin filaments that drive membrane protrusion. Endothelial cells were grown on micropatterns that confined the freedom of movement of the cells. Three-dimensional super-resolution imaging revealed that the lamellipodia of these cells showed a much broader axial extension than was the case for control cells that grew without confinement of migration. The different organization of the actin filament network showed a clear effect of environmental conditions on cellular migration. Overall, it was possible to build a super-resolution fluorescence microscope over the course of this study and establish the required analysis methods to allow STORM and PALM imaging below the diffraction limit of light. Two applications further showed that these tools are capable of answering currently discussed questions in the biological sciences.

Deploying exosomes in a battle of the sexes The paired accessory glands of male Drosophila secrete multiple signaling factors into the seminal fluid that promote reproductive success by altering the recipient female's physiology and behavior. This biosights episode presents the paper by Corrigan et al. from the September 1, 2014, issue of The Journal of Cell Biology and includes an interview with corresponding author Alexander Bershadsky (Weizmann Institute, Israel, and Mechanobiology Institute, National University of Singapore). Produced by Caitlin Sedwick and Ben Short. See the associated paper in JCB for details on the funding provided to support this original research. Subscribe to biosights via iTunes or RSS View biosights archive The Rockefeller University Press biosights@rockefeller.edu

Actin is one of the most abundant proteins in eukaryotic cells and regulation of the microfilament system is crucial for a wide range of cellular functions including cell shape, cell motility, cell division and membrane dynamics. The aim of this thesis was (1) to gain a better understanding of the function of distinct actin binding domains in the regulation of the actin cytoskeleton and (2) to elucidate the role of actin variants. WH2 domains (WH2, Wiskott-Aldrich syndrome protein homology 2) are ubiquitous multifunctional regulators of actin dynamics. The protein Spire contains four central WH2 domains A-B-C-D with about 20 amino acids each and the cyclase-associated protein CAP2 contains only one WH2 domain. Under certain conditions, they can (1) nucleate actin polymerization, (2) disintegrate actin filaments and (3) sequester actin monomers. Here, the influence of selected Drosophila melanogaster Spire-WH2 and Mus musculus CAP2-WH2 domain constructs on actin dynamics was tested in vitro. To act as a filament nucleator, at least two WH2 domains are required, and nucleation of actin polymerization was only observed at substoichiometric concentrations of WH2 domains over actin. At higher concentrations, the sequestering activity of WH2 domains takes over. Preformed and purified SpireWH2-actin complexes act as extremely efficient nuclei for actin polymerization, even at superstoichiometric WH2 concentrations, under which free WH2 domains would sequester actin. All analyzed constructs, including these with only a single WH2 domain, sequester actin as well as they can disrupt filaments. This latter and most peculiar behavior of WH2 domains was observed in fluorometric, viscometric and TIRF assays. The WH2 domains seem to have such a high affinity for actin that they can forcefully sequester monomers even from filaments and filament bundles, thus breaking the whole structures. Taken together, the data clearly show that SpireWH2-actin complexes are the intermediates that account for the observed nucleating activity, whereas free WH2 domains can disrupt filaments and filament bundles within seconds, again underlining the intrinsic versatility of this regulator of actin dynamics. These data have been confirmed by crystallography in collaboration with the groups of Prof. Dr. Tad Holak and Prof. Dr. Robert Huber (Martinsried, Germany). Besides the well-studied conventional actins many organisms harbor actin variants with unknown function. The model organism Dictyostelium discoideum comprises an actinome of a total of 41 actins, actin isoforms and actin-related proteins. Among them is filactin, a highly conserved actin with an elongated N-terminus. The 105 kDa protein has a distinct domain organization and homologs of this protein are present in other Dictyosteliidae and in some pathogenic Entamoebae. Here, the functions of filactin were studied in vivo and in vitro. Immunofluorescence studies in D. discoideum localize endogenous and GFP-filactin in the cytoplasm at vesicle-like structures and in cortical regions of the cell. A most peculiar behavior is the stress-induced appearance of full length filactin in nuclear actin rods. To perform in vitro analyses recombinant filactin was expressed in Sf9 cells. Fluorescence studies with the filactin actin domain suggest that it interferes with actin polymerization by sequestering G-actin or even capping filaments. Gel filtration assays propose a tetrameric structure of full length filactin. Protein interaction studies suggest that filactin is involved in the ESCRT (endosomal sorting complexes required for transport) pathway which is responsible for multivesicular body formation. The data on filactin suggest that only the conventional actins are the backbone for the microfilamentous system whereas less related actin isoforms have highly specific and perhaps cytoskeleton-independent subcellular functions.

The processing of APP occurs in two alternative ways: upon release of the ectodomain by α-secretase, the neuroprotective APPsα-fragment is produced. But if APP is cleaved by the β-secretase the Aβ-peptide can be produced. To be able to influence the production of Aβ-peptides, it is essential to understand how it is decided if cleavage occurs by α- or β-secretase. At present little is known about the control of the alternate processing. Until now, the molecular mechanisms and especially the responsible cellular modulators are not understood in detail or not yet identified. To get a better understanding of cellular regulatory processes and to identify novel cellular modulators of APP ectodomain shedding, the present work chose two approaches: on the one hand cellular mechanisms of TMEM59-mediated inhibition ectodomain shedding of APP were investigated. On the other hand a genome-wide RNAi screening in Drosophila cells was performed in order to identify novel cellular modulators of APP ectodomain shedding in human cells. TMEM59 was identified as a novel modulator of APP ectodomain shedding in a cDNA expression screening in the lab (Neumann et al., 2006; Schobel et al., 2008; Schobel et al., 2006). TMEM59 is a Golgi protein that inhibits on the one hand processing and maturation of APP and on the other hand Golgi glycosylation reactions (Fischer, 2008). My own work could verify these effects of TMEM59 and its homolog TMEM59L on processing and maturation of APP. In particular, it was shown that these effects are not only true for transiently expressed APP but also for endogenous levels of APP. In detailed immunofluorescence studies it was shown that TMEM59 colocalizes with different markers of the Golgi subcompartments and that therefore TMEM59 is present throughout the whole Golgi apparatus. This finding points to a more general modulation of Golgi glycosylation reactions by TMEM59. To test if TMEM59-dependet modulation of Golgi glycosylation reactions also affects APP secretases ADAM10 and BACE1, which are also glycosylated proteins, the activities of these proteases were investigated. It was shown that proteolytic activities were not changed, ruling out that impairment of secretase activities by TMEM59 could cause the observed inhibition of APP processing. But interestingly, studies of intracellular APP transport could show that TMEM59 caused retention of APP in the Golgi apparatus and blockage of transport towards the cell surface and into endosomal compartments. Since APP is cleaved by α-secretase at the plasma membrane and by β-secretase in endosomes it is likely that a TMEM59-dependent APP transport block causes the observed inhibition of APP ectodomain shedding. For further validation of TMEM59 and its homolog TMEM59L as modulators of APP ectodomain shedding, a double knockdown study was performed. In this approach effects on APP ectodomain shedding could also be established, affirming TMEM59 and its homolog TMEM59L as modulators of APP ectodomain shedding with novel cellular mechanisms. In order to identify novel cellular modulators of APP ectodomain shedding a genome wide RNAi screening in Drosophila cells was performed and candidate genes were investigated in human cells in present work. Initially a suitable Drosophila reporter cell line expressing a reporter construct of APP ectodomain shedding (HRP-APP) was established. Other constructs were used to monitor general secretion (GLuc) and transfection efficiency (FLuc). Using Kuzbanian, the α-secretase in Drosophila (Sapir et al., 2005), as a positive control guaranteed that transfection of cDNAs into Drosophila cells did not interfere with uptake of dsRNAs or efficiency of RNAi and that the reporter construct HRP-APP is normally produced and processed in reporter cells. After successful establishment of the reporter cell line the genome wide RNAi was performed in two steps: a primary screening revealed approx. 300 candidate genes out of which 43 could be confirmed in a secondary screening to be modulators of APP ectodomain shedding. The RNAi screening was verified by the several-fold appearance of Kuzbanian among the top modulators. For further investigation of the top candidates human ortholog genes were identified. The 30 human candidate genes were investigated in RNAi studies in human SH-SY5Y cells. In these cells, APP is processed by α-secretase ADAM10 as well as by β-secretase BACE1. Therefore effects on both shedding products (APPsα and APPsβ) were investigated upon depletion of candidate genes using siRNAs. It is known that siRNAs produce a high rate of off target effects, to this end a robust validation strategy was developed. Candidate genes were first depleted with two different siRNA pools and their effects on APP shedding were compared. Afterwards the remaining 12 candidate genes were depleted using single siRNA sequences and the effects were compared to those of the siRNA pool. Only when a reproduction of effects was obtained in a next step correlation of knockdown and phenotype were assessed. Using these steps of validation 5 candidate genes could be verified as modulators of APP shedding in human cells: next to genes coding for a histone protein (HIST1H4C), a ribosomal protein (RPL36AL), a protein of the minor spliceosom (ZMAT5), an unknown gene (METTL16) and the gene VPS24 („vacuolar protein sorting-associated protein 24“), coding for a protein of intracellular protein transport, were identified. VPS24 was chosen for further validation by a pathway analysis. VPS24 belongs to the ESCRT machinery („endosomal sorting complex required for transport“) and therefore participates in endosomal-lysosomal protein transport. In further RNAi studies other members of the ESCRT machinery were depleted in human cells and effects on APP shedding were compared to VPS24 depletion. For most of the ESCRT members a consistent reduction in APPsβ production could be observed. To engross these results VPS24 was depleted by using an alternative RNAi system. With this stable knockdown approach, the knockdown phenotype could be confirmed. This stepwise validation strategy for candidate genes of the initial Drosophila RNAi screening verified VPS24 as a modulator of APP ectodomain shedding in human cells.

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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.04.16.044818v1?rss=1 Authors: Birdsall, V., Imoto, Y., Waites, C., Watanabe, S. Abstract: Turnover of synaptic vesicle (SV) proteins is vital for the maintenance of healthy, functional synapses in neurons. Our previous work showed that the degradation of SV proteins is mediated by the endosomal sorting complex required for transport (ESCRT) pathway in an activity-dependent manner. Here, we characterize the axonal transport dynamics of ESCRT-0 proteins Hrs and STAM1, the first components of the ESCRT pathway critical for initiating SV protein degradation. Hrs- and STAM1-positive transport vesicles exhibit increased anterograde and bidirectional motility in response to neuronal firing, and frequent colocalization with SV pools. These ESCRT-0 vesicles are a subset of Rab5-positive structures in axons, likely representing pro-degradative early endosomes. Further, we identify kinesin motor protein KIF13A as essential for the activity-dependent transport of ESCRT-0 vesicles as well as the degradation of SV membrane proteins. Together, these data demonstrate a novel KIF13A-dependent mechanism for mobilizing axonal transport of ESCRT machinery to facilitate the degradation of SV proteins. Copy rights belong to original authors. Visit the link for more info