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Visualization of Membrane Pore in Live Cells Reveals a Dynamic-Pore Theory Governing Fusion and Endocytosis.

Cell (2018-04-03)
Wonchul Shin, Lihao Ge, Gianvito Arpino, Seth A Villarreal, Edaeni Hamid, Huisheng Liu, Wei-Dong Zhao, Peter J Wen, Hsueh-Cheng Chiang, Ling-Gang Wu
ABSTRACT

Fusion is thought to open a pore to release vesicular cargoes vital for many biological processes, including exocytosis, intracellular trafficking, fertilization, and viral entry. However, fusion pores have not been observed and thus proved in live cells. Its regulatory mechanisms and functions remain poorly understood. With super-resolution STED microscopy, we observed dynamic fusion pore behaviors in live (neuroendocrine) cells, including opening, expansion, constriction, and closure, where pore size may vary between 0 and 490 nm within 26 milliseconds to seconds (vesicle size: 180-720 nm). These pore dynamics crucially determine the efficiency of vesicular cargo release and vesicle retrieval. They are generated by competition between pore expansion and constriction. Pharmacology and mutation experiments suggest that expansion and constriction are mediated by F-actin-dependent membrane tension and calcium/dynamin, respectively. These findings provide the missing live-cell evidence, proving the fusion-pore hypothesis, and establish a live-cell dynamic-pore theory accounting for fusion, fission, and their regulation.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Latrunculin A, from sea sponge, ≥85% (HPLC), waxy solid
Sigma-Aldrich
Atto 532 NHS ester, BioReagent, suitable for fluorescence, ≥80% (coupling to amines)

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