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, 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.Key words: vesicles, membrane fusion, dynamics, TIRFM, CRISPR, gene-editing, protein complex.. CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/354449 doi: bioRxiv preprint first posted online Jun. 22, 2018; 3 Traffic between membrane-bound compartments requires the docking of cargo vesicles at target membranes, and their subsequent fusion through the interactions of SNARE proteins. The capture and fusion of vesicles are both promoted by molecular tethers known as multisubunit tethering complexes 1 . One group of such tethers, sometimes called CATCHR (complexes associate with tethering containing helical rods) comprises multisubunit complexes required for fusion in the secretory pathway, and includes COG, Dsl1p, GARP and the exocyst 2 . The endolysosomal pathway contains two different tethering complexes, CORVET and HOPS, with similar overall structures to the CATCHR group 3 .COG consists of two subcomplexes, each containing four subunits, which function together within the Golgi 4-6 . The exocyst is also octameric, and is necessary for exocytic vesicle fusion to the plasma membrane (PM), but the organization of the complex has been controversial [7][8][9][10] . Several studies in yeast suggest that one (Sec3) or two (Sec3 and Exo70)subunits associate with the PM and recruit a vesicle-bound subcomplex of the other subunits, but other work argues that the exocyst consists of 2 subcomplexes of 4 subunits each that form a stable octamer or, in mammalian cells, that 5 subunits at the PM recruit 3 other subunits on the vesicle [11][12][13][14][15][16][17][18][19][20][21][22] . Rab GTPases promote exocyst binding to the vesicle, and SNARES, Rho family GTPases, the PAR3 polarity protein, and phospho-inositide binding domains are all involved ...

Structure of membrane tethers and their role in fusion

“…On the other hand, the multilegged appearance of the complexes could be induced by the sample treatment in those studies. The hierarchical organization of exocyst and dynamic behavior of exocyst subunit in cells strengthen the concept that tethering could be driven by assembly. It is interesting to note that also the coiled‐coil protein EEA1 undergoes a conformational switch in tethering, albeit from higher to lower order.…”

“…Exocyst was proposed to assemble from subcomplexes that are located on the opposing membranes of the secretory vesicle and the plasma membrane . This concept was recently supported in a study of genome edited mammalian cell line, where endogenous exocyst subunits carried fluorescent tags and could be monitored by high‐speed TIRF microscopy and fluorescence cross‐correlation spectroscopy . The subunits showed a dynamic behavior.…”

“…Another open question is how disassembly of exocyst after tethering could be triggered. The more loosely associated subunit Sec3 could serve as a trigger, if its association with the remaining subunits was regulated . At this time, we can only speculate, but it will be exciting to define a mechanism for exocyst tethering that combines the dynamic behavior of exocyst in vivo and hierarchical architecture observed by structural analysis.…”

Phosphorylation of the exocyst subunit Exo70B2 contributes to the regulation of its function

“…The phosphonull variant may therefore represent a constitutively active (lipid-bound) form, in which PM dissociation is impaired. In human cells, SEC3 departure from the PM occurs prior to fusion, whereas Exo70 departs just after fusion (Ahmed et al, 2018). However, it remains unclear how the departure, i.e.…”

“…At early stages of post-Golgi trafficking STOMATAL CYTOKINESIS DEFECTIVE1 (SCD1) and SCD2 proteins are though to recruit RabE1 and the exocyst to vesicles (Mayers et al, 2017). During the delivery of secretory vesicles, Sec3 and Exo70 subunits of the exocyst mediate binding to the PM (Ahmed et al, 2018). In plants, the ROP/RAC effector INTERACTOR OF CONSTITUTIVE ACTIVE ROPs 1 (ICR1) was shown to influence Sec3 localization (Lavy et al, 2007; Hazak et al, 2010).…”