Exocyst dynamics during vesicle tethering and fusion

Syed Mukhtar Ahmed (), Hisayo Nishida-Fukuda, Yuchong Li, W. Hayes McDonald, Claudiu C. Gradinaru and Ian G. Macara ()
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Syed Mukhtar Ahmed: Vanderbilt University School of Medicine
Hisayo Nishida-Fukuda: Vanderbilt University School of Medicine
Yuchong Li: University of Toronto
W. Hayes McDonald: Vanderbilt University School of Medicine
Claudiu C. Gradinaru: University of Toronto
Ian G. Macara: Vanderbilt University School of Medicine

Nature Communications, 2018, vol. 9, issue 1, 1-17

Abstract: Abstract The exocyst is a conserved octameric complex that tethers exocytic vesicles to the plasma membrane prior to fusion. Exocyst assembly and delivery mechanisms remain unclear, especially in mammalian cells. Here we tagged multiple endogenous exocyst subunits with sfGFP or Halo using Cas9 gene-editing, to create single and double knock-in lines of mammary epithelial cells, and interrogated exocyst dynamics by high-speed imaging and correlation spectroscopy. We discovered that mammalian exocyst is comprised of tetrameric subcomplexes that can associate independently with vesicles and plasma membrane and are in dynamic equilibrium with octamer and monomers. Membrane arrival times are similar for subunits and vesicles, but with a small delay (~80msec) between subcomplexes. Departure of SEC3 occurs prior to fusion, whereas other subunits depart just after fusion. About 9 exocyst complexes are associated per vesicle. These data reveal the mammalian exocyst as a remarkably dynamic two-part complex and provide important insights into assembly/disassembly mechanisms.

Date: 2018
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DOI: 10.1038/s41467-018-07467-5

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