The Exocyst Complex Associates with Microtubules to Mediate Vesicle Targeting and Neurite Outgrowth

Abstract

During neuronal development, vesicles are targeted to the growth cone to promote neurite outgrowth and synaptogenesis. The Exocyst complex is an essential macromolecule in the secretory pathway that may play a role in vesicle targeting. Although it has been shown that this complex is enriched in rat brain, the molecular mechanism underlying its function is largely unknown. Here, we report that the Exocyst complex coimmunoprecipitates with microtubules from total rat brain lysate. Additionally, the Exocyst complex subcellular localization changes on neuronal differentiation. In undifferentiated pheochromocytoma (PC12) cells, this complex is associated with microtubules at the microtubule organizing center. However, in differentiated PC12 cells and cultured hippocampal neurons, the Exocyst complex and microtubules extend to the growing neurite and colocalize at the growth cone with synaptotagmin. Inhibition of the NGF-activated MAP kinase pathway blocks the Exocyst complex and microtubule redistribution, abolishing neurite outgrowth and promoting cytosolic accumulation of secretory vesicles. Consistently, the overexpression of Exocyst sec10 subunit mutant blocks neurite outgrowth. These results indicate that the Exocyst complex targets secretory vesicles to specific domains of the plasma membrane through its association with the microtubules, promoting neurite outgrowth.

A dual role for IQGAP1 in regulating exocytosis

“…Antibodies for IQGAP1 and IQGAP2 were from Upstate Biotech, for IQGAP3 were from Novus Biologicals, and those for EXO70 and SEPT2 were previously described (Vega and Hsu, 2001;Vega and Hsu, 2003). Mouse anti-rSec8 was from Stressgen Biotechnology and HA from Covance.…”

“…The GST-EXO70 and GST-SEPT2 fusion proteins were previously described (Vega and Hsu, 2001;Vega and Hsu, 2003). These were expressed in Escherichia coli BL21 (DE3) and sonicated in buffer S (20 mM Tris, pH 8, 2 mM EDTA, 2 mM DTT, 150 mM NaCl, 0.1% Tween 20).…”

Membrane Organization and Dynamics in Cell Polarity

“…In mammalian cells, although the exocyst mostly localizes to internal membrane compartments such as Golgi and recycling endosomes Ang et al 2003;Fölsch et al 2003;Oztan et al 2007), it is recruited to designated regions of the plasma membrane where active exocytosis and membrane expansion occur. For example, in neurons undergoing differentiation or synapse formation, the exocyst is enriched at the expanding neurites and discrete puncta along axons where synapses are forming (Hsu et al 1996;Kee et al 1997;Hazuka et al 1999;Vega and Hsu, 2001;Brown et al 2001;Murthy et al 2003). In migrating cells, the exocyst is recruited to the plasma membrane at the leading edge (Zuo et al 2006;Rosse et al 2006).…”

Phosphatidylinositol 4,5-Bisphosphate Mediates the Targeting of the Exocyst to the Plasma Membrane for Exocytosis in Mammalian Cells

“…However, they are recruited to the PM during a number of cellular processes. For example, in epithelial cells, the exocyst is recruited to the adherens junction region upon cell-cell contact, where it mediates protein and membrane addition at the basolateral domain (Grindstaff et al, 1998;Yeaman et al, 2001); in developing neurons, the exocyst is localized to the growing neurites, where it mediates membrane expansion (Hazuka et al, 1999;Vega and Hsu, 2001); during cell migration, the exocyst is recruited to the leading edges of the PM (Rosse et al, 2006;Zuo et al, 2006).…”