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The Fine Points of Exocyst Dynamics

Posted by on Monday, December 17, 2018 in Discoveries.

Exocytic vesicles fuse with the plasma membrane (PM) through the action of SNARE proteins, which in turn, are delivered to the vesicles by multisubunit protein tethering complexes. One such complex is the exocyst, comprising two tetrameric subcomplexes, SC1 (composed of SEC3, SEC5, SEC6, and SEC8) and SC2 (composed of SEC10, SEC15, EXO70, and EXO84). Prior studies in cells from various organisms had led to a confusing picture regarding the dynamics of this complicated protein assembly, leading Vanderbilt Basic Sciences investigator Ian Macara, his postdoctoral fellow Syed Mukhtar Ahmed, and his lab to apply CRISPR-Cas9 technology and advanced fluorescence microscopy approaches to obtain a detailed, high resolution view of exocyst function. They started by using CRISPR-Cas to create murine breast epithelial cell lines expressing SEC3, SEC5, SEC6, SEC8, and EXO70 tagged at the C-terminus with green fluorescent protein (GFP). They showed that immunoprecipitation of any one of these proteins using the GFP tag led to capture of all eight exocyst components. In addition, immunoprecipitation of EXO70-GFP, but only this protein, led to recovery of the SNARE protein SNAP23. Furthermore, if they used shRNA to knockdown expression of one of the non-tagged exocyst subunit, then GFP-dependent immunoprecipitation of a tagged subunit no longer captured the members of the subcomplex to which the missing protein belonged. These results indicated that, although each subcomplex required all four of its own components for assembly, it did not require the presence of the other subcomplex. Total internal reflection fluorescence microscopy (TIRFM) enabled the researchers to observe the approach of exocytic vesicles to and their fusion with the PM. Results indicated that vesicles reached the PM approximately 14 s prior to fusion, and similar timing between approach and vesicle fusion was observed for the exocyst subunits. These findings suggested that many of subunits arrive already bound to the vesicles. Following vesicle fusion, all of the subunits dissociated from the PM after about 1.4 s with the exception of SEC3, which dissociated after only 0.4 s. Using cells expressing a GFP-tagged exocyst subunit along with SEC5 that had been tagged with a red fluorescent protein allowed the application of two-channel time-lapse TIRFM to monitor the timing of arrival of two different subunits to the PM. In general, subunits belonging to the same subcomplex arrived together, but those in different subcomplexes arrived at slightly different times, with SC2-associated proteins most often arriving before those associated with SC1. Single molecule counting of proteins in cells expressing two differently tagged fluorescent subunits at once enabled the researchers to determine that, at any one time, approximately 65% of subunits were assembled in octamers, while about 25% were associated in tetramers and 10% were free in the cytosol. The one exception was SEC3, of which about 40% was unassociated with either tetramers or octamers. Together, the results demonstrate that the exocyst is a highly dynamic complex formed from two tetrameric subcomplexes, each of which assembles and travels to the PM independently, where they associate with incoming vesicles. The investigators proposed a model in which the octameric complex assembles on vesicles at or near the PM, possibly from subunits that are already vesicle-associated. Complete assembly enables the delivery of SNARE-associated proteins via EXO70, ultimately leading to vesicle fusion. Release of SEC3 occurs soon thereafter, possibly serving as a first step toward octamer dissociation. The data help to clarify major ongoing questions in exocyst function and lay the groundwork for future experiments to help delineate the role of the individual subunits.

The work is published in the journalNature Communications [S. M. Ahmed, et al. Nat. Comm., (2018) 9, 5140].

 

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