Abstract
The exocyst is a 734-kDa complex essential for development. Perturbation of its function results in early embryonic lethality. Extensive investigation has revealed that this complex participates in multiple biological processes, including protein synthesis and vesicle/protein targeting to the plasma membrane. In this article we report that the exocyst may also play a role in modulating microtubule dynamics. Using monoclonal antibodies, we observed that endogenous exocyst subunits co-localized with microtubules and mitotic spindles in normal rat kidney cells. To test for a functional relationship between the exocyst complex and microtubules, we established an in vitro exocyst reconstitution assay and studied exocyst effect on microtubule dynamics. We found that the exocyst complex reconstituted from eight recombinant exocyst subunits inhibited tubulin polymerization in vitro. Deletion of exocyst subunit sec5, sec6, sec15, or exo70 diminished its tubulin polymerization inhibition activity. Surprisingly, exocyst subunit exo70 itself was also capable of inhibiting tubulin polymerization, although exocyst complex with exo70 deletion did not lose its activity completely. Overexpression of exo70 in NRK cells resulted in microtubule network disruption and the formation of filopodia-like plasma membrane protrusions. The formation of these membrane protrusions was greatly hampered by stabilizing microtubules with taxol. Overexpression of exo84, an exocyst subunit that did not show tubulin polymerization inhibition activity, did not cause this phenotype. Results shown in this article, along with a previous report that localized microtubule instability induces plasma membrane addition, implicates a novel role for the exocyst in modulating microtubule dynamics underlying exocytosis.
Highlights
The exocyst is a 734-kDa complex essential for development
We found that the exocyst complex reconstituted from eight recombinant exocyst subunits inhibited tubulin polymerization in vitro
Exocyst Subunits Exhibit Filamentous Subcellular Distribution That Co-localizes with Microtubules and Mitotic Spindles—To begin investigating a functional relationship between the exocyst complex and microtubules, we studied the in vivo co-localization of endogenous exocyst subunits and microtubules in NRK cells
Summary
Cell Culture—NRK cells were cultured at a density of 5,000 cells/cm on culture dishes coated with 0.1 mg/ml polylysine for transfection and immunocytochemistry experiments. The treated cells were fixed with 4% paraformaldehyde for 20 min at room temperature, permeabilized with 0.1% Triton X-100 for 10 min, washed with phosphate-buffered saline three times and mounted for GFP fluorescence visualization using an inverted fluorescence microscope (Axiovert 200; Zeiss, Thornwood, NY). Three assays were used to monitor the effect of exo on tubulin polymerization: optical scattering at 340 nm, electron microscopy, and SDS-PAGE of microtubules harvested by centrifugation. Negative Staining Transmission Electron Microscopy—To visualize microtubules following exocyst activity assay, the tubulin/exocyst mixture was adsorbed onto formvar/carbon-coated 200-mesh copper grids (EMS, Fort Washington, PA) for 1 min. Polyclonal antibodies against tubulin were purchased from Cytoskeleton Inc. To monitor exocyst subcellular localization, NRK cells were fixed with methanol at room temperature or 37 °C for 1 min. The resulting cell lysates were subjected to SDS-PAGE and transferred to nitrocellulose membranes for Western blot analysis as described [16]
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