Biology subjects

Elbaz-Alon, Y.

Publications and source records attributed to Elbaz-Alon, Y..

4 recordsLinked to original sources

bioRxiv2026

Vesicular pseudopodia define the fusion site on large secretory vesicles of the Drosophila salivary glands

Large secretory vesicles (LSVs) pose a scaling problem for regulated exocytosis. Their micron-scale dimensions greatly increase the vesicular membrane surface area, making productive engagement between the vesicular and target membrane fusion machinery unlikely. Here, we show that vesicular pseudopodia define the fusion sites of LSVs in Drosophila larval salivary glands. Focused ion beam scanning electron microscopy revealed that most LSVs project polarized pseudopodia that interconnect neighboring vesicles and orient toward the apical membrane. Exposed pseudopodia were frequently observed at the apical surface and associated with narrow fusion pores, indicating that fusion occurs at these structures. Three-dimensional correlative light and electron microscopy showed that the I-BAR protein Missing in Metastasis (MIM) selectively localizes to exposed pseudopodia. Proteomic analysis based on a MIM pull-down assay identified exocyst components, including Sec15, which localizes to pseudopodia and persists at fusion sites throughout secretion. Finally, the tetraspanin Tsp42Ee marked complementary apical fusion domains and was required for efficient exocytosis. Our findings support a model in which prepatterned vesicular and apical membrane domains coordinate efficient exocytosis. SummaryRegulated exocytosis of large secretory vesicles is facilitated by vesicular pseudopodia and an apical fusion domain that spatially organizes membrane tethering and fusion during secretion.

cell biology↗

bioRxiv2023

High throughput screening identifies broad-spectrum Coronavirus entry inhibitors

The Covid-19 pandemic highlighted the pressing need for antiviral therapeutics capable of mitigating infection and spread of emerging coronaviruses (CoVs). A promising therapeutic strategy lies in inhibiting viral entry mediated by the Spike (S) glycoprotein. To identify small molecule inhibitors that block entry downstream of receptor binding, we established a high-throughput screening (HTS) platform based on pseudoviruses. We employed a three-step process to screen nearly 200,000 small molecules. First, we identified potential inhibitors by assessing their ability to inhibit pseudoviruses bearing the SARS-CoV-2 S glycoprotein. Subsequent counter-screening against pseudoviruses with the Vesicular Stomatitis Virus glycoprotein (VSV-G), yielding sixty-five SARS-CoV-2 S-specific inhibitors. These were further tested against pseudoviruses bearing the MERS-CoV S glycoprotein, which uses a different receptor. Out of these, five compounds including the known broad-spectrum inhibitor Nafamostat, were subjected to further validation and tested them against pseudoviruses bearing the S glycoprotein of the alpha, delta, and omicron variants as well as against bona fide SARS-CoV-2 in vitro. This rigorous approach revealed a novel inhibitor and its derivative as a potential broad-spectrum antiviral. These results validate the HTS platform and set the stage for lead optimization and future pre-clinical, in vivo studies.

microbiology↗

bioRxiv2023

Fusion pore dynamics of large secretory vesicles define a distinct mechanism of exocytosis

Exocrine cells utilize large secretory vesicles (LSVs) up to 10 m in diameter. LSVs fuse with the apical surface, often recruiting actomyosin to extrude their content through dynamic fusion pores. The molecular mechanism regulating pore dynamics remains largely uncharacterized. We observe that the fusion pore of LSVs in the Drosophila larval salivary glands expand, stabilize, and constrict. Arp2/3 is essential for pore expansion and stabilization, while myosin II is essential for pore constriction. We identify several Bin-Amphiphysin-Rvs (BAR) homology domain proteins that regulate fusion pore expansion and stabilization. We show that the I-BAR protein Missing-in-Metastasis (MIM) localizes to the fusion site and is essential for pore expansion and stabilization. The MIM I-BAR domain is essential but not sufficient for localization and function. We conclude that MIM acts in concert with actin, myosin II, and additional BAR-domain proteins to control fusion pore dynamics, mediating a distinct mode of exocytosis that facilitates actomyosin-dependent content release that maintains apical membrane homeostasis during secretion.

cell biology↗

bioRxiv2022

Extracellular vesicle fusion visualized by cryo-EM

Extracellular vesicles (EVs) transfer bioactive molecules between cells in a process reminiscent of enveloped viruses. EV cargo delivery is thought to occur by protein-mediated and pH-dependent membrane fusion of the EV and the cellular membrane. However, there is a lack of methods to identify the fusion proteins and resolve their mechanism. We developed and benchmarked an in vitro biophysical assay to investigate EV membrane fusion. The assay was standardized by directly comparing EV- and viral-fusion with liposomes. We show that EVs and retroviruses fuse with liposomes mimicking the membrane composition of the late endosome in a pH and protein-dependent manner. Moreover, we directly visualize the stages of membrane fusion using cryo-electron tomography. We find that, unlike most retroviruses, EVs remain fusogenic after acidification and re-neutralization. These results provide novel insights into the EV cargo delivery mechanism and an experimental approach to identify the EV fusion machinery.

biophysics↗