GTP Hydrolysis of TC10 Promotes Neurite Outgrowth through Exocytic Fusion of Rab11- and L1-Containing Vesicles by Releasing Exocyst Component Exo70

Abstract

The use of exocytosis for membrane expansion at nerve growth cones is critical for neurite outgrowth. TC10 is a Rho family GTPase that is essential for specific types of vesicular trafficking to the plasma membrane. Recent studies have shown that TC10 and its effector Exo70, a component of the exocyst tethering complex, contribute to neurite outgrowth. However, the molecular mechanisms of the neuritogenesis-promoting functions of TC10 remain to be established. Here, we propose that GTP hydrolysis of vesicular TC10 near the plasma membrane promotes neurite outgrowth by accelerating vesicle fusion by releasing Exo70. Using Förster resonance energy transfer (FRET)-based biosensors, we show that TC10 activity at the plasma membrane decreased at extending growth cones in hippocampal neurons and nerve growth factor (NGF)-treated PC12 cells. In neuronal cells, TC10 activity at vesicles was higher than its activity at the plasma membrane, and TC10-positive vesicles were found to fuse to the plasma membrane in NGF-treated PC12 cells. Therefore, activity of TC10 at vesicles is presumed to be inactivated near the plasma membrane during neuronal exocytosis. Our model is supported by functional evidence that constitutively active TC10 could not rescue decrease in NGF-induced neurite outgrowth induced by TC10 depletion. Furthermore, TC10 knockdown experiments and colocalization analyses confirmed the involvement of Exo70 in TC10-mediated trafficking in neuronal cells. TC10 frequently resided on vesicles containing Rab11, which is a key regulator of recycling pathways and implicated in neurite outgrowth. In growth cones, most of the vesicles containing the cell adhesion molecule L1 had TC10. Exocytosis of Rab11- and L1-positive vesicles may play a central role in TC10-mediated neurite outgrowth. The combination of this study and our previous work on the role of TC10 in EGF-induced exocytosis in HeLa cells suggests that the signaling machinery containing TC10 proposed here may be broadly used for exocytosis.

Membrane Trafficking in Neuronal Development: Ins and Outs of Neural Connectivity

“…Overexpression of the Exo70 subunit can induce filopodia and increase secretion (Gupton and Gertler, 2010; Wang et al, 2004). In embryonic mouse cortical neurons, Exo70 overexpression promotes neuritogenesis, supporting an important role for exocytosis and the exocyst complex in developing neurons (Fujita et al, 2013; Gupton and Gertler, 2010; Vega and Hsu, 2001). Several groups have shown various exocyst components at the tips of extending neurites, further supporting their role in neuritogenesis and neurite extension (Hazuka et al, 1999; Lalli and Hall, 2005; Mehta et al, 2005).…”

“…Expression levels of the exocyst subunit Sec6 correlate temporally with the occurrence of neuritogenesis (Chin et al, 2000). During axon specification, the exocyst complex is integral in both vesicle targeting to the axon (Fujita et al, 2013) and the regulation of cell polarity (He and Guo, 2009). Lalli reported that siRNA depletion of exocyst subunits Sec6, Sec8, or Exo84 inhibits axon specification in rat cortical neurons, as no neurites become positive for the axon marker tau.…”

Rem2 signaling affects neuronal structure and function in part by regulation of gene expression

“…Dlc1 can act as a GTPase activating protein (GAP) for several Rho GTPases including CDC42 and Rho A–C which have been implicated in cytoskeleton regulation [48]. Furthermore, the Rho GTPase Rhoq promotes neurite outgrowth through a Rab11 and Exo70 dependent mechanism [49]. Therefore, repression of Rhoq and Dlc1 expression by Rem2 signaling could represent another mechanism by which Rem2 restricts dendritic complexity.…”

Endosidin2 targets conserved exocyst complex subunit EXO70 to inhibit exocytosis

“…T he EXO70 (exocyst component of 70 kDa) protein is a component of the evolutionarily conserved octameric exocyst complex that tethers post-Golgi vesicles to the plasma membrane before SNARE-mediated membrane fusion (1). As an important component of the exocyst complex that mediates exocytosis, EXO70 regulates, for example, neurite outgrowth, epithelial cell polarity establishment, cell motility, and cell morphogenesis in animal cells (2)(3)(4)(5)(6). In plants, EXO70 proteins participate in polarized pollen tube growth, root hair growth, deposition of cell wall material, cell plate initiation and maturation, defense, and autophagy (7)(8)(9)(10)(11)(12).…”