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.
Highlights
COG consists of two subcomplexes, each containing four subunits, which function together within the Golgi[4,5,6]
The advent of CRISPR/Cas9-mediated gene editing coupled with the development of high-efficiency scientific CMOS cameras has the potential to revolutionize our understanding of protein dynamics in the living cell
In mammary epithelial cells, exocyst connectivity is different from previous models of the mammalian exocyst but is consistent with the proposed connectivity in budding yeast[19], with two tetrameric subcomplexes, SC1 and SC2, that associate to form the complete octamer
Summary
COG consists of two subcomplexes, each containing four subunits, which function together within the Golgi[4,5,6]. The advent of CRISPR/Cas9-mediated gene editing coupled with the development of high-efficiency scientific CMOS (sCMOS) cameras has the potential to revolutionize our understanding of protein dynamics in the living cell We have exploited these technologies to generate multiple tagged alleles of exocyst subunits by gene editing, and coupled proteomics with highspeed TIRFM and fluorescence cross-correlation spectroscopy (FCCS) to quantify exocyst dynamics in unprecedented detail. Cross-correlation of SEC3 to other subunits is significantly reduced Taken together, these data are inconsistent with prior exocyst models and suggest that, in mammalian cells, exocyst subunits are in dynamic equilibrium with assembled complexes and the PM, that intact subcomplexes assemble on secretory vesicles as they dock, and that SEC3 is preferentially released prior to fusion
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