Exocyst dynamics during vesicle tethering and fusion

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.

Maintaining order: COG complex controls Golgi trafficking, processing, and sorting

“…Additionally, isolated COG subcomplexes show lobe‐specific pattern of interaction with different protein partners including β‐COP, p115, and STX5 . The COG complex assembly/disassembly model is in good agreement with several models proposed for the mammalian exocyst .…”

The Exocyst: Dynamic Machine or Static Tethering Complex?

“…Recent structural studies show that exocyst consists of two subcomplexes with equal stoichiometry, and the connectivity among the subunits is conserved between budding yeast and mouse. Subcomplex I (SC I) consists of Sec3, Sec5, Sec6, and Sec8; whereas Sec10, Sec15, Exo70, and Exo84 are assembled into SC II (Figure , in box) . Furthermore, live cell imaging of CRISPR‐mediated exocyst subunit‐superfolder green fluorescent protein (sfGFP) knock‐in cells showed that each subcomplex can arrive at the PM without its partner subcomplex, even though ≈65% of SC I are associated in the cytoplasm .…”

“…Subcomplex I (SC I) consists of Sec3, Sec5, Sec6, and Sec8; whereas Sec10, Sec15, Exo70, and Exo84 are assembled into SC II (Figure , in box) . Furthermore, live cell imaging of CRISPR‐mediated exocyst subunit‐superfolder green fluorescent protein (sfGFP) knock‐in cells showed that each subcomplex can arrive at the PM without its partner subcomplex, even though ≈65% of SC I are associated in the cytoplasm . These observations suggest that distinct regulators might control each subcomplex and their association with one another.…”

“…However, the molecular mechanism by which exocyst tethers vesicles to the PM is still largely unknown, especially in mammals because knockout of exocyst subunits in mice is lethal and manipulating the genome of cultured cell lines has until recently been very difficult. But with the development of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9‐mediated genome editing in 2012, we can now explore protein function by tagging the endogenous loci with fluorescent proteins or small peptides, without the artifacts caused by overexpression . Furthermore, microscopy technologies have improved sufficiently to detect single molecules within living cells at high temporal resolution.…”

“…Furthermore, microscopy technologies have improved sufficiently to detect single molecules within living cells at high temporal resolution. The combination of genome‐edited cells and high‐quality microscopy allows us to understand the dynamics of endogenous proteins in living cells . In this review, we discuss our changing view of exocyst dynamics and regulation, with a focus on mammalian cells.…”