Abstract
Vesicle fusion is executed via formation of an Ω-shaped structure (Ω-profile), followed by closure (kiss-and-run) or merging of the Ω-profile into the plasma membrane (full fusion). Although Ω-profile closure limits release but recycles vesicles economically, Ω-profile merging facilitates release but couples to classical endocytosis for recycling. Despite its crucial role in determining exocytosis/endocytosis modes, how Ω-profile merging is mediated is poorly understood in endocrine cells and neurons containing small ∼30–300 nm vesicles. Here, using confocal and super-resolution STED imaging, force measurements, pharmacology and gene knockout, we show that dynamic assembly of filamentous actin, involving ATP hydrolysis, N-WASP and formin, mediates Ω-profile merging by providing sufficient plasma membrane tension to shrink the Ω-profile in neuroendocrine chromaffin cells containing ∼300 nm vesicles. Actin-directed compounds also induce Ω-profile accumulation at lamprey synaptic active zones, suggesting that actin may mediate Ω-profile merging at synapses. These results uncover molecular and biophysical mechanisms underlying Ω-profile merging.
Highlights
Vesicle fusion is executed via formation of an O-shaped structure (O-profile), followed by closure or merging of the O-profile into the plasma membrane
We found that the filamentous actin (F-actin) assembly pathway including hydrolysis of the energy molecule ATP, neuronal Wiskott–Aldrich syndrome protein (NWASP) and formin that activate F-actin assembly participates in mediating O-profile merging
We used a recently developed technique to image O-profile merging in live, primary-cultured bovine adrenal chromaffin cells containing B300 nm dense-core vesicles in a bath solution containing membrane-impermeable Alexa Fluor 647 (A647) and Alexa Fluor 488 (A488) (Fig. 1a)[11]
Summary
Vesicle fusion is executed via formation of an O-shaped structure (O-profile), followed by closure (kiss-and-run) or merging of the O-profile into the plasma membrane (full fusion). W Present addresses: Department of Pharmacology, College of Medicine, National Cheng Kung University, No.[1], University Road, Tainan city, Taiwan 701 (H.-C.C.); Centre for Cancer Research and Cell Biology, Vesicle fusion releases vesicular contents such as hormones, peptides and transmitters, to mediate many biological processes crucial to an animal’s life, such as stress responses, mood changes, synaptic transmission, neuronal network activity, and immune responses[1,2,3,4] It is executed via formation of an O-shape intermediate structure, termed O-profile, at the plasma membrane for releasing contents, followed by closure (called kiss-and-run) or merging of the O-profile into the plasma membrane (called full fusion)[1,2,3,4]. These results uncover novel molecular and biophysical mechanisms underlying O-profile merging in neuroendocrine cells and neurons, which mediates full fusion and couples exocytosis to classical endocytosis
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