Imaging The Rapid Recruitment Of Dynamins At The Exocytic Fusion Pore

The size and shape of the exocytic fusion pore directly modulates the release of vesicle cargoes like insulin, hormones, and neurotransmitters. Though a number of proteins have been implicated in fusion pore regulation, the molecular details of this process and how these proteins work together to shape the membrane are largely unknown. Using TIRF microscopy in living INS-1 cells we directly image the coincident and transient recruitment of dynamin proteins, the regulatory lipid PIP2, and BAR domain proteins amphiphysin, syndapin, and endophilin to exocytic sites. Recruitment occurs at the moment of membrane fusion. Evidence from imaging and siRNA depletion of endogenous dynamins suggests that dynamin-1 and dynamin-2 have distinct functional roles at sites of exocytosis. We show that perturbing dynamin-1 interaction with PIP2 or amphiphysin blocks its recruitment to exocytic sites and has functional effects on vesicle cargo release. We turn to stimulated emission depletion (STED) super-resolution fluorescence microscopy to directly visualize the organization of dynamins at sites of exocytosis in living cells. The spatiotemporal coincident recruitment of dynamins, PIP2, and BAR domain proteins, and their known interactions, strongly suggest these molecules form a complex at the nascent exocytic fusion pore. Such a protein and lipid assembly represents a novel paradigm for controlling fusion pore size and shape and directly modulating endocrine cargo release.