Regulated exocytosis, which underlies many intercellular signaling events, is a tightly controlled process often triggered by calcium ion(s) (Ca2+). Despite considerable insight into the central components involved, namely, the core fusion machinery [soluble N-ethylmaleimide?sensitive factor attachment protein receptor (SNARE)] and the principal Ca2+ sensor [C2-domain proteins like synaptotagmin (Syt)], the molecular mechanism of Ca2+-dependent release has been unclear. Here, we report that the Ca2+-sensitive oligomers of Syt1, a conserved structural feature among several C2-domain proteins, play a critical role in orchestrating Ca2+-coupled vesicular release. This follows from pHluorin-based imaging of single-vesicle exocytosis in pheochromocytoma (PC12) cells showing that selective disruption of Syt1 oligomerization using a structure-directed mutation (F349A) dramatically increases the normally low levels of constitutive exocytosis to effectively occlude Ca2+-stimulated release. We propose a parsimonious model whereby Ca2+-sensitive oligomers of Syt (or a similar C2-domain protein) assembled at the site of docking physically block spontaneous fusion until disrupted by Ca2+. Our data further suggest Ca2+-coupled vesicular release is triggered by removal of the inhibition, rather than by direct activation of the fusion machinery.Fil: Bello, Oscar Daniel. University of Yale. School of Medicine; Estados Unidos. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Mendoza. Instituto de Histologia y Embriologia de Mendoza Dr. Mario H. Burgos. Grupo Vinculado de Investigacion y Desarrollo Biotecnologico Aplicado Al Diagnostico Al Ihem | Universidad Nacional de Cuyo. Facultad de Ciencias Medicas. Instituto de Histologia y Embriologia de Mendoza Dr. Mario H. Burgos. Grupo Vinculado de Investigacion y Desarrollo Biotecnologico Aplicado Al Diagnostico Al Ihem.; Argentina. University College London; Estados UnidosFil: Jouannot, Ouardane. University of Yale. School of Medicine; Estados UnidosFil: Chaudhuri, Arunima. University of Yale. School of Medicine; Estados UnidosFil: Stroeva, Ekaterina. University of Yale. School of Medicine; Estados UnidosFil: Coleman, Jeff. University of Yale. School of Medicine; Estados UnidosFil: Volynski, Kirill E.. University College London; Reino UnidoFil: Rothman, James E.. University of Yale. School of Medicine; Estados Unidos. University College London; Estados UnidosFil: Krishnakumar, Shyam S.. University of Yale. School of Medicine; Estados Unidos. University College London; Estados Unido
