Endosomal Protein Research Articles

Manganese Exposure Enhances the Release of Misfolded α-Synuclein via Exosomes by Impairing Endosomal Trafficking and Protein Degradation Mechanisms

Manganese Exposure Enhances the Release of Misfolded α-Synuclein via Exosomes by Impairing Endosomal Trafficking and Protein Degradation Mechanisms

Defects in exosome biogenesis are associated with sensorimotor defects in zebrafish vps4a mutants.

Mutations in human VPS4A are associated with neurodevelopmental defects, including motor delays and defective muscle tone. VPS4A encodes a AAA-ATPase required for membrane scission, but how mutations in VPS4A lead to impaired control of motor function is not known. Here we identified a mutation in zebrafish vps4a, T248I, that affects sensorimotor transformation. Biochemical analyses indicate that the T248I mutation reduces the ATPase activity of Vps4a and disassembly of ESCRT filaments, which mediate membrane scission. Consistent with the role for Vps4a in exosome biogenesis, vps4aT248I larvae have enlarged endosomal compartments in the CNS and decreased numbers of circulating exosomes in brain ventricles. Resembling the central form of hypotonia in VPS4A patients, motor neurons and muscle cells are functional in mutant zebrafish. Both somatosensory and vestibular inputs robustly evoke tail and eye movements, respectively. In contrast, optomotor responses, vestibulospinal, and acoustic startle reflexes are absent or strongly impaired in vps4aT248I larvae, indicating a greater sensitivity of these circuits to the T248I mutation. ERG recordings revealed intensity-dependent deficits in the retina, and in vivo calcium imaging of the auditory pathway identified a moderate reduction in afferent neuron activity, partially accounting for the severe motor impairments in mutant larvae. Further investigation of central pathways in vps4aT248I mutants showed that activation of descending vestibulospinal and midbrain motor command neurons by sensory cues is strongly reduced. Our results suggest that defects in sensorimotor transformation underly the profound yet selective effects on motor reflexes resulting from the loss of membrane scission mediated by Vps4a.Significance Statement Here we present a T248I mutation in vps4a, which causes sensorimotor defects in zebrafish larvae. Vps4a plays a key role in membrane scission. Spanning biochemical to systems level analyses, our study indicates that a reduction in Vps4a enzymatic activity leads to abnormalities in membrane-scission dependent processes such as endosomal protein trafficking and exosome biogenesis, resulting in pronounced deficits in sensorimotor transformation of visual, auditory, and vestibular cues. We suggest that the mechanisms underlying this type of dysfunction in zebrafish may also contribute to the condition seen in human patients with de novo mutations in the human VPS4A orthologue.

Divergent and Convergent TMEM106B Pathology in Murine Models of Neurodegeneration and Human Disease.

TMEM106B is a lysosomal/late endosome protein that is a potent genetic modifier of multiple neurodegenerative diseases as well as general aging. Recently, TMEM106B was shown to form insoluble aggregates in postmortem human brain tissue, drawing attention to TMEM106B pathology and the potential role of TMEM106B aggregation in disease. In the context of neurodegenerative diseases, TMEM106B has been studied in vivo using animal models of neurodegeneration, but these studies rely on overexpression or knockdown approaches. To date, endogenous TMEM106B pathology and its relationship to known canonical pathology in animal models has not been reported. Here, we analyze histological patterns of TMEM106B in murine models of C9ORF72 -related amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD), SOD1-related ALS, and tauopathy and compare these to postmortem human tissue from patients with C9-ALS/FTD, Alzheimer's disease (AD), and AD with limbic-predominant age-related TDP-43 encephalopathy (AD/LATE). We show that there are significant differences between TMEM106B pathology in mouse models and human patient tissue. Importantly, we also identified convergent evidence from both murine models and human patients that links TMEM106B pathology to TDP-43 nuclear clearance specifically in C9-ALS. Similarly, we find a relationship at the cellular level between TMEM106B pathology and phosphorylated Tau burden in Alzheimer's disease. By characterizing endogenous TMEM106B pathology in both mice and human postmortem tissue, our work reveals considerations that must be taken into account when analyzing data from in vivo mouse studies and elucidates new insights supporting the involvement of TMEM106B in the pathogenesis and progression of multiple neurodegenerative diseases.

Endosomal actin branching, fission, and receptor recycling require FCHSD2 recruitment by MICAL-L1.

Endosome fission is required for the release of carrier vesicles and the recycling of receptors to the plasma membrane. Early events in endosome budding and fission rely on actin branching to constrict the endosomal membrane, ultimately leading to nucleotide hydrolysis and enzymatic fission. However, our current understanding of this process is limited, particularly regarding the coordination between the early and late steps of endosomal fission. Here we have identified a novel interaction between the endosomal scaffolding protein, MICAL-L1, and the human homologue of the Drosophila Nervous Wreck (Nwk) protein, FCH and double SH3 domains protein 2 (FCHSD2). We demonstrate that MICAL-L1 recruits FCHSD2 to the endosomal membrane, where it is required for ARP2/3-mediated generation of branched actin, endosome fission and receptor recycling to the plasma membrane. Because MICAL-L1 first recruits FCHSD2 to the endosomal membrane, and is subsequently responsible for recruitment of the ATPase and fission protein EHD1 to endosomes, our findings support a model in which MICAL-L1 orchestrates endosomal fission by connecting between the early actin-driven and subsequent nucleotide hydrolysis steps of the process.

The lysosomal lipid transporter LIMP-2/SCARB2 is part of lysosome-endoplasmic reticulum STARD3-VAPB-dependent contact sites.

SCARB2/LIMP-2 is an abundant lysosomal membrane protein. Previous studies have shown LIMP-2 functions as a virus receptor, a chaperone for lysosomal enzyme targeting, and a lipid transporter. The large luminal domain of LIMP-2 contains a hydrophobic tunnel that enables transport of phospholipids, sphingosine and cholesterol from the lysosomal lumen to the membrane. The question about the fate of the lipids after LIMP-2-mediated transport is largely unexplored. To elucidate whether LIMP-2 is part of contact sites between lysosomes and the endoplasmic reticulum (ER), we performed a proximity-based interaction screen. This revealed that LIMP-2 interacts with the endosomal protein STARD3 and the ER-resident protein VAPB. Using imaging and co-immunoprecipitation, we demonstrated colocalization and physical interaction between LIMP-2 and these proteins. Moreover, we found that interaction of LIMP-2 with VAPB required the presence of STARD3. Our findings suggest that LIMP-2 is part of ER-lysosome contact sites, possibly facilitating cholesterol transport from the lysosomal to the ER membrane. This suggests a novel mechanism for inter-organelle communication and lipid trafficking mediated by LIMP-2.

The schizophrenia risk gene C4 induces pathological synaptic loss by impairing AMPAR trafficking.

Neuroimmune interactions play a significant role in regulating synaptic plasticity in both the healthy and diseased brain. The complement pathway, an extracellular proteolytic cascade, exemplifies these interactions. Its activation triggers microglia-dependent synaptic elimination via the complement receptor 3 (CR3). Current models of pathological complement activity in the brain propose that accelerated synaptic loss resulting from overexpression of C4 (C4-OE), a gene associated with schizophrenia, follows this pathway. Here, we report that C4-mediated cortical hypoconnectivity is CR3-independent. Instead, C4-OE triggers impaired GluR1 trafficking through an intracellular mechanism involving the endosomal protein SNX27, resulting in pathological synaptic loss. Moreover, C4 circuit alterations in the prefrontal cortex, a brain region associated with neuropsychiatric disorders, were rescued by increasing neuronal levels of SNX27, which we identify as an interacting partner of this neuroimmune protein. Our results link excessive complement activity to an intracellular endo-lysosomal trafficking pathway altering synaptic plasticity.

Genome-wide CRISPR/Cas9 library screen identifies C16orf62 as a host dependency factor for porcine deltacoronavirus infection

ABSTRACT Porcine deltacoronavirus (PDCoV) is an emerging pathogen that can cause severe diarrhoea and high mortality in suckling piglets. Moreover, evidence of PDCoV infection in humans has raised concerns regarding potential public health risks. To identify potential therapeutic targets for PDCoV, we performed a genome-wide CRISPR/Cas9 library screening to find key host factors important to PDCoV infection. Several host genes in this screen were enriched, including ANPEP, which encodes the PDCoV receptor aminopeptidase N (APN). Furthermore, we discovered C16orf62, also known as the VPS35 endosomal protein sorting factor like (VPS35L), as an important host factor required for PDCoV infection. C16orf62 is an important component of the multiprotein retriever complex involved in protein recycling in the endosomal compartment and its gene knockout led to a remarkable decrease in the binding and internalization of PDCoV into host cells. While we did not find evidence for direct interaction between C16orf62 and the viral s (spike) protein, C16orf62 gene knockout was shown to downregulate APN expression at the cell surface. This study marks the first instance of a genome-wide CRISPR/Cas9-based screen tailored for PDCoV, revealing C16orf62 as a host factor required for PDCoV replication. These insights may provide promising avenues for the development of antiviral drugs against PDCoV infection.

Regulation mechanism of microRNAs in cardiac cells-derived exosomes in cell crosstalk.

The heart is a multicellular system, and the intercellular crosstalk mechanism is very important for the growth and development of the heart and even the organs, tissues, and cells at a distance. As a kind of extracellular vesicle, exosomes are released by different types of cells and can carry specific genetic material, endosomal proteins, cytokines, etc., which are the main material basis for mediating cell crosstalk mechanism. Among them, microRNA carried by cardiac cells-derived exosomes have highly conserved sequences and play a key role in regulating the function of organs, tissues, and cells related to cardiovascular diseases and their complications and comorbidities, which have attracted extensive attention in the medical community in recent years. Following up on the latest research progress at home and abroad, this review systematically summarized the regulatory role of microRNA from cardiac cells-derived exosomes in various cell crosstalk, including not only cardiac cells (including cardiomyocytes, fibroblasts, myofibroblast, cardiac progenitor cells, cardiac microvascular endothelial cells, cardiosphere-derived cells, etc.) but also tumor cells, bone marrow progenitor cells, and other tissue cells, in order to provide a reference for the prevention and treatment of cardiovascular diseases and their complications and comorbidities.

Truncated tau interferes with the autophagy and endolysosomal pathway and results in lipid accumulation

The autophagy-lysosomal pathway plays a critical role in the clearance of tau protein aggregates that deposit in the brain in tauopathies, and defects in this system are associated with disease pathogenesis. Here, we report that expression of Tau35, a tauopathy-associated carboxy-terminal fragment of tau, leads to lipid accumulation in cell lines and primary cortical neurons. Our findings suggest that this is likely due to a deleterious block of autophagic clearance and lysosomal degradative capacity by Tau35. Notably, upon induction of autophagy by Torin 1, Tau35 inhibited nuclear translocation of transcription factor EB (TFEB), a key regulator of lysosomal biogenesis. Both cell lines and primary cortical neurons expressing Tau35 also exhibited changes in endosomal protein expression. These findings implicate autophagic and endolysosomal dysfunction as key pathological mechanisms through which disease-associated tau fragments could lead to the development and progression of tauopathy.

Endosomal protein DENND10/FAM45A integrates extracellular vesicle release with cancer cell migration

BackgroundMounting evidence shows that tumor-derived extracellular vesicles (EVs) are critical constituents in the tumor microenvironment. The composition and function of EVs often change during cancer progression. However, it remains less clear how cancer cells modulate their own EV biogenesis to promote tumor development. The release of EVs is closely linked to the endolysosome system residing within the cell. The current study aims to decipher the role of endosomal protein DENND10 in cancer development.ResultsBioinformatics data mining showed that DENND10 expression is significantly associated with poor prognosis in multiple cancer types and up-regulated in metastatic breast cancer cell lines. DENND10 knockout (DENND10-KO) in breast cancer cells led to defective EV biogenesis due to impaired endolysosomal trafficking. Intriguingly, DENND10-KO cells exhibited reductions in cell spreading, migration, invasion, and metastatic potential in vivo. These deficiencies in cell motility were associated with compromised cytoskeleton organization. Importantly, wild-type conditioned medium or EVs restored the migratory ability and cytoskeletal organization of DENND10-KO cells. Global proteomic profiling revealed that DENND10 depletion led to a distinct EV compositional landscape with remodeled profiles of extracellular matrix (ECM) and adhesion molecules. Consistently, exogenous application of ECM molecules rescued the spreading and migration of DENND10-KO cells.ConclusionsIn summary, our study unveiled DENND10 as an intrinsic regulator of cell migration that modifies the tumor microenvironment through autocrine EV release, which could be exploited for developing targeted therapies for tumor metastasis.

Pathogenic variants in TMEM184B cause a neurodevelopmental syndrome via alteration of metabolic signaling.

Transmembrane protein 184B (TMEM184B) is an endosomal 7-pass transmembrane protein with evolutionarily conserved roles in synaptic structure and axon degeneration. We report six pediatric patients who have de novo heterozygous variants in TMEM184B. All individuals harbor rare missense or mRNA splicing changes and have neurodevelopmental deficits including intellectual disability, corpus callosum hypoplasia, seizures, and/or microcephaly. TMEM184B is predicted to contain a pore domain, wherein many human disease-associated variants cluster. Structural modeling suggests that all missense variants alter TMEM184B protein stability. To understand the contribution of TMEM184B to neural development in vivo, we suppressed the TMEM184B ortholog in zebrafish and observed microcephaly and reduced anterior commissural neurons, aligning with patient symptoms. Ectopic TMEM184B expression resulted in dominant effects for K184E and G162R. However, in vivo complementation studies demonstrate that all other variants tested result in diminished protein function and indicate a haploinsufficiency basis for disease. Expression of K184E and other variants increased apoptosis in cell lines and altered nuclear localization of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, suggesting disrupted nutrient signaling pathways. Together, our data indicate that TMEM184B variants cause cellular metabolic disruption likely through divergent molecular effects that all result in abnormal neural development.

Endosomal actin branching, fission and receptor recycling require FCHSD2 recruitment by MICAL-L1.

Endosome fission is required for the release of carrier vesicles and the recycling of receptors to the plasma membrane. Early events in endosome budding and fission rely on actin branching to constrict the endosomal membrane, ultimately leading to nucleotide hydrolysis and enzymatic fission. However, our current understanding of this process is limited, particularly regarding the coordination between the early and late steps of endosomal fission. Here we have identified a novel interaction between the endosomal scaffolding protein, MICAL-L1, and the human homolog of the Drosophila Nervous Wreck (Nwk) protein, FCH and double SH3 domains protein 2 (FCHSD2). We demonstrate that MICAL-L1 recruits FCHSD2 to the endosomal membrane, where it is required for ARP2/3-mediated generation of branched actin, endosome fission and receptor recycling to the plasma membrane. Since MICAL-L1 first recruits FCHSD2 to the endosomal membrane, and is subsequently responsible for recruitment of the ATPase and fission protein EHD1 to endosomes, our findings support a model in which MICAL-L1 orchestrates endosomal fission by connecting between the early actin-driven and subsequent nucleotide hydrolysis steps of the process.

Increasing the Survival of a Neuronal Model of Alzheimer's Disease Using Docosahexaenoic Acid, Restoring Endolysosomal Functioning by Modifying the Interactions between the Membrane Proteins C99 and Rab5.

Docosahexaenoic acid (DHA, C22:6 ω3) may be involved in various neuroprotective mechanisms that could prevent Alzheimer's disease (AD). Its influence has still been little explored regarding the dysfunction of the endolysosomal pathway, known as an early key event in the physiopathological continuum triggering AD. This dysfunction could result from the accumulation of degradation products of the precursor protein of AD, in particular the C99 fragment, capable of interacting with endosomal proteins and thus contributing to altering this pathway from the early stages of AD. This study aims to evaluate whether neuroprotection mediated by DHA can also preserve the endolysosomal function. AD-typical endolysosomal abnormalities were recorded in differentiated human SH-SY5Y neuroblastoma cells expressing the Swedish form of human amyloid precursor protein. This altered phenotype included endosome enlargement, the reduced secretion of exosomes, and a higher level of apoptosis, which confirmed the relevance of the cellular model chosen for studying the associated deleterious mechanisms. Second, neuroprotection mediated by DHA was associated with a reduced interaction of C99 with the Rab5 GTPase, lower endosome size, restored exosome production, and reduced neuronal apoptosis. Our data reveal that DHA may influence protein localization and interactions in the neuronal membrane environment, thereby correcting the dysfunction of endocytosis and vesicular trafficking associated with AD.

Identification of residues in Lassa virus glycoprotein 1 involved in receptor switch

Lassa virus (LASV) is an enveloped, negative-sense RNA virus that causes Lassa hemorrhagic fever. Successful entry of LASV requires the viral glycoprotein 1 (GP1) to undergo a receptor switch from its primary receptor alpha-dystroglycan (α-DG) to its endosomal receptor lysosome-associated membrane protein 1 (LAMP1). A conserved histidine triad in LASV GP1 has been reported to be responsible for receptor switch. To test the hypothesis that other non-conserved residues also contribute to receptor switch, we constructed a series of mutant LASV GP1 proteins and tested them for binding to LAMP1. Four residues, L84, K88, L107, and H170, were identified as critical for receptor switch. Substituting any of the four residues with the corresponding lymphocytic choriomeningitis virus (LCMV) residue (L84 ​N, K88E, L10F, and H170S) reduced the binding affinity of LASV GP1 for LAMP1. Moreover, all mutations caused decreases in glycoprotein precursor (GPC)-mediated membrane fusion at both pH 4.5 and 5.2. The infectivity of pseudotyped viruses bearing either GPCL84N or GPCK88E decreased sharply in multiple cell types, while L107F and H170S had only mild effects on infectivity. Using biolayer light interferometry assay, we found that all four mutants had decreased binding affinity to LAMP1, in the order of binding affinity being L84 ​N ​> ​L107F ​> ​K88E ​> ​H170S. The four amino acid loci identified for the first time in this study have important reference significance for the in-depth investigation of the mechanism of receptor switching and immune escape of LASV occurrence and the development of reserve anti-LASV infection drugs.

Regulation of YAP and Wnt signaling by the endosomal protein MAMDC4.

Maintenance of the intestinal epithelium requires constant self-renewal and regeneration. Tight regulation of proliferation and differentiation of intestinal stem cells within the crypt region is critical to maintaining homeostasis. The transcriptional co-factors β-catenin and YAP are required for proliferation during normal homeostasis as well as intestinal regeneration after injury: aberrant signaling activity results in over proliferation and tumorigenesis. Although both YAP and β-catenin activity are controlled along canonical pathways, it is becoming increasingly clear that non-canonical regulation of these transcriptional regulators plays a role in fine tuning their activity. We have shown previously that MAMDC4 (Endotubin, AEGP), an integral membrane protein present in endosomes, regulates both YAP and β-catenin activity in kidney epithelial cells and in the developing intestinal epithelium. Here we show that MAMDC4 interacts with members of the signalosome and mediates cross-talk between YAP and β-catenin. Interestingly, this cross-talk occurs through a non-canonical pathway involving interactions between AMOT:YAP and AMOT:β-catenin.

Coxiella burnetii-containing vacuoles interact with host recycling endosomal proteins Rab11a and Rab35 for vacuolar expansion and bacterial growth.

Coxiella burnetii is a gram-negative obligate intracellular bacterium and a zoonotic pathogen that causes human Q fever. The lack of effective antibiotics and a licensed vaccine for Coxiella in the U.S. warrants further research into Coxiella pathogenesis. Within the host cells, Coxiella replicates in an acidic phagolysosome-like vacuole termed Coxiella-containing vacuole (CCV). Previously, we have shown that the CCV pH is critical for Coxiella survival and that the Coxiella Type 4B secretion system regulates CCV pH by inhibiting the host endosomal maturation pathway. However, the trafficking pattern of the 'immature' endosomes in Coxiella- infected cells remained unclear. We transfected HeLa cells with GFP-tagged Rab proteins and subsequently infected them with mCherry-Coxiella to visualize Rab protein localization. Infected cells were immunostained with anti-Rab antibodies to confirm the Rab localization to the CCV, to quantitate Rab11a and Rab35- positive CCVs, and to quantitate total recycling endosome content of infected cells. A dual-hit siRNA mediated knockdown combined with either immunofluorescent assay or an agarose-based colony-forming unit assay were used to measure the effects of Rab11a and Rab35 knockdown on CCV area and Coxiella intracellular growth. The CCV localization screen with host Rab proteins revealed that recycling endosome-associated proteins Rab11a and Rab35 localize to the CCV during infection, suggesting that CCV interacts with host recycling endosomes during maturation. Interestingly, only a subset of CCVs were Rab11a or Rab35-positive at any given time point. Quantitation of Rab11a/Rab35-positive CCVs revealed that while Rab11a interacts with the CCV more at 3 dpi, Rab35 is significantly more prevalent at CCVs at 6 dpi, suggesting that the CCV preferentially interacts with Rab11a and Rab35 depending on the stage of infection. Furthermore, we observed a significant increase in Rab11a and Rab35 fluorescent intensity in Coxiella-infected cells compared to mock, suggesting that Coxiella increases the recycling endosome content in infected cells. Finally, siRNA-mediated knockdown of Rab11a and Rab35 resulted in significantly smaller CCVs and reduced Coxiella intracellular growth, suggesting that recycling endosomal Rab proteins are essential for CCV expansion and bacterial multiplication. Our data, for the first time, show that the CCV dynamically interacts with host recycling endosomes for Coxiella intracellular survival and potentially uncovers novel host cell factors essential for Coxiella pathogenesis.

TLR7 in channel catfish (Ictalurus punctatus) is expressed in the endolysosome and is stimulated by synthetic ssRNA analogs, imiquimod, and resiquimod.

Toll-like receptors (TLRs) are pivotal pattern recognition receptors (PRRs) and key mediators of innate immunity. Despite the significance of channel catfish (Ictalurus punctatus) in comparative immunology and aquaculture, its 20 TLR genes remain largely functionally uncharacterized. In this study, our aim was to determine the catfish TLR7 agonists, signaling potential, and cellular localization. Using a mammalian reporter system, we identified imiquimod and resiquimod, typical ssRNA analogs, as potent catfish TLR7 agonists. Notably, unlike grass carp TLR7, catfish TLR7 lacks the ability to respond to poly (I:C). Confocal microscopy revealed predominant catfish TLR7 expression in lysosomes, co-localizing with the endosomal chaperone protein, UNC93B1. Furthermore, imiquimod stimulation elicited robust IFNb transcription in peripheral blood leukocytes isolated from adult catfish. These findings underscore the conservation of TLR7 signaling in catfish, reminiscent of mammalian TLR7 responses. Our study sheds light on the functional aspects of catfish TLR7 and contributes to a better understanding of its role in immune defense mechanisms.

Illuminating the Cryptococcus neoformans species complex: unveiling intracellular structures with fluorescent-protein-based markers.

Cryptococcus neoformans is a fungal pathogen of the top critical priority recognized by the World Health Organization. This clinically important fungus also serves as a eukaryotic model organism. A variety of resources have been generated to facilitate investigation of the C. neoformans species complex, including congenic pairs, well-annotated genomes, genetic editing tools, and gene deletion sets. Here, we generated a set of strains with all major organelles fluorescently marked. We tested short organelle-specific targeting sequences and successfully labeled the following organelles by fusing the targeting sequences with a fluorescence protein: the plasma membrane, the nucleus, the peroxisome, and the mitochondrion. We used native cryptococcal Golgi and late endosomal proteins fused with a fluorescent protein to label these two organelles. These fluorescence markers were verified via colocalization using organelle-specific dyes. All the constructs for the fluorescent protein tags were integrated in an intergenic safe haven region. These organelle-marked strains were examined for growth and various phenotypes. We demonstrated that these tagged strains could be employed to track cryptococcal interaction with the host in phagocytosis assays. These strains also allowed us to discover remarkable differences in the dynamics of proteins targeted to different organelles during sexual reproduction. Additionally, we revealed that "dormant" spores transcribed and synthesized their own proteins and trafficked the proteins to the appropriate subcellular compartments, demonstrating that spores are metabolically active. We anticipate that these newly generated fluorescent markers will greatly facilitate further investigation of cryptococcal biology and pathogenesis.

Methylation of ESCRT-III components regulates the timing of cytokinetic abscission

Abscission is the final stage of cytokinesis, which cleaves the intercellular bridge (ICB) connecting two daughter cells. Abscission requires tight control of the recruitment and polymerization of the Endosomal Protein Complex Required for Transport-III (ESCRT-III) components. We explore the role of post-translational modifications in regulating ESCRT dynamics. We discover that SMYD2 methylates the lysine 6 residue of human CHMP2B, a key ESCRT-III component, at the ICB, impacting the dynamic relocation of CHMP2B to sites of abscission. SMYD2 loss-of-function (genetically or pharmacologically) causes CHMP2B hypomethylation, delayed CHMP2B polymerization and delayed abscission. This is phenocopied by CHMP2B lysine 6 mutants that cannot be methylated. Conversely, SMYD2 gain-of-function causes CHMP2B hypermethylation and accelerated abscission, specifically in cells undergoing cytokinetic challenges, thereby bypassing the abscission checkpoint. Additional experiments highlight the importance of CHMP2B methylation beyond cytokinesis, namely during ESCRT-III-mediated HIV-1 budding. We propose that lysine methylation signaling fine-tunes the ESCRT-III machinery to regulate the timing of cytokinetic abscission and other ESCRT-III dependent functions.

Predicting Cell Type-Specific Extracellular Vesicle Biology Using an Organism-Wide Single Cell Transcriptomic Atlas - Insights from the Tabula Muris

Background: Extracellular vesicles (EVs) like exosomes are functional nanoparticles traffcked between cells and found in every biofluid. An incomplete understanding of which cells, from which tissues, are traffcking EVs in vivo has limited our ability to use EVs as biomarkers and therapeutics. However, recent discoveries have linked EV secretion to expression of genes and proteins responsible for EV biogenesis and found as cargo, which suggests that emerging “cell atlas” datasets could be used to begin understanding EV biology at the level of the organism and possibly in rare cell populations. To explore this possibility, here we analyzed 67 genes that are directly implicated in EV biogenesis and secretion, or carried as cargo, in ~44,000 cells obtained from 117 cell populations of the Tabula Muris. Methods: We obtained normalized count (FPKM) data from 44,779 FACS-sorted cells described in the Tabula Muris. These data were freely obtained from the Tabula Muris web portal operated by the Chan-Zuckerberg Biohub. We selected 67 EV genes based on their direct role in the regulation of endosome biogenesis, protein sorting, secretion or cargo. These genes include the endosome sorting complexes required for transport (ESCRT), multiple Rab GTPases, tetraspanins and syndecans. To identify the most abundant EV genes, we plotted mean expression (FPKM) across all cells against the fraction of cells with gene expression > 50 FPKM. To measure gene expression variance, we calculated %CV for each gene within each cell population and across all cell populations. For gene co-expression analysis, we performed linear regressions comparing selected EV genes (Cd9, Cd63, Cd81, Sdc4 and Sdcbp) to all other genes, within each cell population. Results: We found that the most abundant EV cargo proteins (tetraspanins (Cd9, Cd63 and Cd81) and syndecans (Sdc4 and Sdcbp)) are also the most abundant EV genes expressed across all cell populations. However, the expression of these genes varied greatly among cell populations. For example, Cd81 is most abundant in brain glial populations and multiple endothelial cell populations. By contrast, Cd9 expression is highest in "barrier" cells that support innate immunity while Cd63, a classic marker of exosomes, is most abundantly expressed by stem cells. Expression variance analysis was used to compare the transcriptional behavior of these abundant EV genes (Cd9, Cd63, Cd81, Sdc4 and Sdcbp). We found that Cd63 is the most dynamic EV gene while others are more constitutively expressed. This finding may help explain why Cd63 is typically found either within the cytoplasm or outside of the cell on EVs, whereas Cd9 and Cd81 are abundant both on the cell surface and on EVs. Finally, we used EV gene co-expression analysis to define cell population-specific transcriptional networks. We discovered that EV gene co-expression networks are heavily dependent on the EV cargo gene examined with different cell populations driving clustering. Conclusions: Our analysis has provided at least four independent observations which advance our understanding of EV biology. These are that: 1) abundant EV cargo genes are differentially expressed among cell populations, 2) skeletal muscle stem cells display the highest expression of both Cd63 and Sdc4, 3) Cd81 may be a primary EV cargo traffcked in the brain whiand 4) that rare cell populations can secrete EVs and thereby influence tissue biology. This analysis is the first, to our knowledge, to predict tissue- and cell type-specific EV biology at the level of the organism. As such, we expect this resource to be the first of many valuable tools for predicting the endogenous impact of specific cell populations on EV function in health and disease. National Institutes on Aging (R01AG078859). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.