Transient pores in vesicles

International audienceWe present our observations of transient pores in giant unilamellar vesicles, placed under tension, by optical illumination. When the membrane tension reached a certain level, transient pores appeared. Pore opening is driven by the membrane tension, s, and its closure by the pore's line tension, T. By use of viscous mixtures of glycerol and water, we slowed down the leak out of the inner liquid in the presence of a pore. This allowed pores to reach large sizes (a few micrometres) and last at least a few seconds so that they could be visualized by fluorescence videomicroscopy. Line tension was inferred from the measurements of the closure velocity of the pores. By addition of cholesterol, which increased T (reducing pore lifetimes), or of surfactants, which decreased T (increasing pore lifetimes), we demonstrate how T , and consequently pore lifetimes, can be controlled over nearly two orders of magnitude. Addition of surfactants also has a dramatic effect on vesicle fusion. We discuss how our results can be extended to less viscous aqueous solutions which are more relevant for liposomal drug delivery formulations

Nanodisc-cell fusion: Control of fusion pore nucleation and lifetimes by SNARE protein transmembrane domains

The initial, nanometer-sized connection between the plasma membrane and a hormone- or neurotransmitter-filled vesicle-the fusion pore- can flicker open and closed repeatedly before dilating or resealing irreversibly. Pore dynamics determine release and vesicle recycling kinetics, but pore properties are poorly known because biochemically defined single-pore assays are lacking. We isolated single flickering pores connecting v-SNARE-reconstituted nanodiscs to cells ectopically expressing cognate, "flipped" t-SNAREs. Conductance through single, voltage-clamped fusion pores directly reported sub-millisecond pore dynamics. Pore currents fluctuated, transiently returned to baseline multiple times, and disappeared ∼6 s after initial opening, as if the fusion pore fluctuated in size, flickered, and resealed. We found that interactions between v- and t-SNARE transmembrane domains (TMDs) promote, but are not essential for pore nucleation. Surprisingly, TMD modifications designed to disrupt v- and t-SNARE TMD zippering prolonged pore lifetimes dramatically. We propose that the post-fusion geometry of the proteins contribute to pore stability.Fil: Wu, Zhenyong. University of Yale; Estados Unidos. University of Yale. School of Medicine; Estados UnidosFil: Auclair, Sarah M.. University of Yale. School of Medicine; Estados Unidos. University of Yale; Estados UnidosFil: Bello, Oscar Daniel. University of Yale. School of Medicine; Estados Unidos. University of Yale; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Vennekate, Wensi. University of Yale. School of Medicine; Estados Unidos. University of Yale; Estados UnidosFil: Dudzinski, Natasha R.. University of Yale; Estados Unidos. University of Yale. School of Medicine; Estados UnidosFil: Krishnakumar, Shyam S.. University of Yale. School of Medicine; Estados Unidos. University of Yale; Estados UnidosFil: Karatekin, Erdem. University of Yale; Estados Unidos. University of Yale. School of Medicine; Estados Unidos. Universite Paris Descartes; Francia. Centre National de la Recherche Scientifique; Franci

Dilation of fusion pores by crowding of SNARE proteins

Hormones and neurotransmitters are released through fluctuating exocytotic fusion pores that can flicker open and shut multiple times. Cargo release and vesicle recycling depend on the fate of the pore, which may reseal or dilate irreversibly. Pore nucleation requires zippering between vesicle-associated v-SNAREs and target membrane t-SNAREs, but the mechanisms governing the subsequent pore dilation are not understood. Here, we probed the dilation of single fusion pores using v-SNARE-reconstituted ~23-nm-diameter discoidal nanolipoprotein particles (vNLPs) as fusion partners with cells ectopically expressing cognate, ’flipped’ t-SNAREs. Pore nucleation required a minimum of two v-SNAREs per NLP face, and further increases in v-SNARE copy numbers did not affect nucleation rate. By contrast, the probability of pore dilation increased with increasing v-SNARE copies and was far from saturating at 15 v-SNARE copies per face, the NLP capacity. Our experimental and computational results suggest that SNARE availability may be pivotal in determining whether neurotransmitters or hormones are released through a transient (’kiss and run’) or an irreversibly dilating pore (full fusion).Fil: Wu, Zhenyong. University of Yale; Estados Unidos. University of Yale. School of Medicine; Estados UnidosFil: Bello, Oscar Daniel. University of Yale. School of Medicine; Estados Unidos. University of Yale; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Thiyagarajan, Sathish. Columbia University; Estados UnidosFil: Auclair, Sarah Marie. University of Yale. School of Medicine; Estados Unidos. University of Yale; Estados UnidosFil: Vennekate, Wensi. University of Yale; Estados Unidos. University of Yale. School of Medicine; Estados UnidosFil: Krishnakumar, Shyam S. University of Yale; Estados Unidos. University of Yale. School of Medicine; Estados UnidosFil: O'Shaughnessy, Ben. Columbia University; Estados UnidosFil: Karatekin, Erdem. University of Yale; Estados Unidos. Universite Paris Descartes; Francia. University of Yale. School of Medicine; Estados Unido

Model of SNARE-Mediated Membrane Adhesion Kinetics

SNARE proteins are conserved components of the core fusion machinery driving diverse membrane adhesion and fusion processes in the cell. In many cases micron-sized membranes adhere over large areas before fusion. Reconstituted in vitro assays have helped isolate SNARE mechanisms in small membrane adhesion-fusion and are emerging as powerful tools to study large membrane systems by use of giant unilamellar vesicles (GUVs). Here we model SNARE-mediated adhesion kinetics in SNARE-reconstituted GUV-GUV or GUV-supported bilayer experiments. Adhesion involves many SNAREs whose complexation pulls apposing membranes into contact. The contact region is a tightly bound rapidly expanding patch whose growth velocity increases with SNARE density . We find three patch expansion regimes: slow, intermediate, fast. Typical experiments belong to the fast regime where depends on SNARE diffusivities and complexation binding constant. The model predicts growth velocities s. The patch may provide a close contact region where SNAREs can trigger fusion. Extending the model to a simple description of fusion, a broad distribution of fusion times is predicted. Increasing SNARE density accelerates fusion by boosting the patch growth velocity, thereby providing more complexes to participate in fusion. This quantifies the notion of SNAREs as dual adhesion-fusion agents

PLSCR1 is a cell-autonomous defence factor against SARS-CoV-2 infection

Understanding protective immunity to COVID-19 facilitates preparedness for future pandemics and combats new SARS-CoV-2 variants emerging in the human population. Neutralizing antibodies have been widely studied; however, on the basis of large-scale exome sequencing of protected versus severely ill patients with COVID-19, local cell-autonomous defence is also crucial1,2,3,4. Here we identify phospholipid scramblase 1 (PLSCR1) as a potent cell-autonomous restriction factor against live SARS-CoV-2 infection in parallel genome-wide CRISPR–Cas9 screens of human lung epithelia and hepatocytes before and after stimulation with interferon-γ (IFNγ). IFNγ-induced PLSCR1 not only restricted SARS-CoV-2 USA-WA1/2020, but was also effective against the Delta B.1.617.2 and Omicron BA.1 lineages. Its robust activity extended to other highly pathogenic coronaviruses, was functionally conserved in bats and mice, and interfered with the uptake of SARS-CoV-2 in both the endocytic and the TMPRSS2-dependent fusion routes. Whole-cell 4Pi single-molecule switching nanoscopy together with bipartite nano-reporter assays found that PLSCR1 directly targeted SARS-CoV-2-containing vesicles to prevent spike-mediated fusion and viral escape. A PLSCR1 C-terminal β-barrel domain—but not lipid scramblase activity—was essential for this fusogenic blockade. Our mechanistic studies, together with reports that COVID-associated PLSCR1 mutations are found in some susceptible people3,4, identify an anti-coronavirus protein that interferes at a late entry step before viral RNA is released into the host-cell cytosol

Regulation of Exocytotic Fusion Pores by SNARE Protein Transmembrane Domains

Calcium-triggered exocytotic release of neurotransmitters and hormones from neurons and neuroendocrine cells underlies neuronal communication, motor activity and endocrine functions. The core of the neuronal exocytotic machinery is composed of soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs). Formation of complexes between vesicle-attached v- and plasma-membrane anchored t-SNAREs in a highly regulated fashion brings the membranes into close apposition. Small, soluble proteins called Complexins (Cpx) and calcium-sensing Synaptotagmins cooperate to block fusion at low resting calcium concentrations, but trigger release upon calcium increase. A growing body of evidence suggests that the transmembrane domains (TMDs) of SNARE proteins play important roles in regulating the processes of fusion and release, but the mechanisms involved are only starting to be uncovered. Here we review recent evidence that SNARE TMDs exert influence by regulating the dynamics of the fusion pore, the initial aqueous connection between the vesicular lumen and the extracellular space. Even after the fusion pore is established, hormone release by neuroendocrine cells is tightly controlled, and the same may be true of neurotransmitter release by neurons. The dynamics of the fusion pore can regulate the kinetics of cargo release and the net amount released, and can determine the mode of vesicle recycling. Manipulations of SNARE TMDs were found to affect fusion pore properties profoundly, both during exocytosis and in biochemical reconstitutions. To explain these effects, TMD flexibility, and interactions among TMDs or between TMDs and lipids have been invoked. Exocytosis has provided the best setting in which to unravel the underlying mechanisms, being unique among membrane fusion reactions in that single fusion pores can be probed using high-resolution methods. An important role will likely be played by methods that can probe single fusion pores in a biochemically defined setting which have recently become available. Finally, computer simulations are valuable mechanistic tools because they have the power to access small length scales and very short times that are experimentally inaccessible