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

Untersuchung der neuronalen Membranfusion mit der Fluoreszenz Korrelations Spektroskopie

Die Neurotransmission erfordert die schnelle Fusion zwischen den synaptischen Vesikeln und der presynaptischen Membran. Die Membranfusion wird durch den Calciumeinstrom ausgelöst und benötigt die SNARE-Proteine. Das Vesikelprotein, Synaptotagmin-1, dient als ein Calciumsensor, und die Bindung des Synaptotagmin-1 an die Plasmamembran ist ein essentieller Schritt für die Membranfusion. In dieser Arbeit wurden einige Details der Membranfusion untersucht und diskutiert. Als Hauptergebnis wird die cis- und trans-Membraninteraktion von Synaptotagmin-1 präsentiert