Analysis of the mechanism of neurite outgrowth driven by membrane transport and cytoskeletal reorganization

细胞骨架的重组和膜的添加是轴突生长的关键。在这里，我们利用FRET生物传感器研究了膜添加的调节机制。质膜上的TC10活性在伸长生长锥体时下降。TC10的GTP水解通过释放胞外成分Exo70促进含有Rab11和L1的囊泡的胞外融合，从而促进轴突生长。我们为Rab35开发了FRET传感器，它可以促进轴突生长。利用Rab35传感器，我们发现生长锥质膜上的Rab35活性高于轴突和细胞体。蛋白激酶A对Stef的磷酸化对于在经DBcAMP处理的PC12D细胞中的rac1激活和突起生长是至关重要的。我们认为轴突顶端的rac1和cdc42的局部激活是轴突生长所必需的。海马神经元中最长的突起特异性激活的Rap1B除了PI3-激酶外，还通过Rala和Nore1A参与极性的形成。

Cytoskeletal reorganization and the membrane addition are pivotal for neurite outgrowth. Here we studied the regulation-mechanism of membrane addition using FRET biosensors. TC10 activity at the plasma membrane decreased at extending growth cones. GTP hydrolysis of TC10 promotes neurite outgrowth through exocytic fusion of Rab11- and L1-containing vesicles by releasing exocyst component Exo70. We developed FRET sensor for Rab35, which promotes neurite outgrowth. Using Rab35 sensor, we showed that Rab35 activity at plasma membrane of growth cones was higher than that of neurite shafts and cell bodies. Phosphorylation of STEF by PKA is critical for Rac1 activation and neurite outgrowth in dbcAMP-treated PC12D cells. We propose that local activation of Rac1 and Cdc42 at neurite tips is necessary for neurite outgrowth. Longest neurite-specific activation of Rap1B in hippocampal neurons contributes to polarity formation through RalA and Nore1A in addition to PI3-kinase.