PT  - JOURNAL ARTICLE
AU  - Syed Mukhtar Ahmed
AU  - Hisayo Nishida-Fukuda
AU  - Yuchong Li
AU  - W. Hayes McDonald
AU  - Claudiu Gradinaru
AU  - Ian G. Macara
TI  - Exocyst Dynamics During Vesicle Tethering and Fusion
AID  - 10.1101/354449
DP  - 2018 Jan 01
TA  - bioRxiv
PG  - 354449
4099  - http://biorxiv.org/content/early/2018/08/03/354449.short
4100  - http://biorxiv.org/content/early/2018/08/03/354449.full
AB  - 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, unexpectedly, can associate independently with vesicles and plasma membrane and are in dynamic equilibrium. 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. Single molecule counting indicates ~9 exocyst complexes associated per vesicle. These data reveal the mammalian exocyst as a remarkably dynamic two-part complex and provide important new insights into assembly/disassembly mechanisms.