FRET imaging in nerve growth cones reveals a high level of RhoA activity within the peripheral domain
FRET imaging in nerve growth cones reveals a high level of RhoA activity within the peripheral domain
复制标题
DOI：
10.1016/j.molbrainres.2005.05.030
复制
发表时间：
2005-10-03
期刊：
影响因子：
--
通讯作者：
Matsuda, M
中科院分区：
文献类型：
--
作者：
Nakamura, T;Aoki, K;Matsuda, M
rho家族gtpase在神经元形态发生的调控中起核心作用。例如，在生长球果中，Rho gtpase将细胞外刺激转化为结构变化，如丝状足和板足。虽然人们普遍认为Racl/Cdc42和RhoA分别是神经突起生长的正调控因子和负调控因子，但rho家族成员在神经元形态发生中的作用可能会根据细胞环境而改变。目前，这种复杂性背后的机制在很大程度上是未知的。在生长球果中，这部分是由于缺乏有关活性Rho gtpase分布的信息。在这里，我们使用基于荧光/福斯特共振能量转移的探针，可视化了在层粘胶蛋白诱导的DRG神经元和N1E-115神经母细胞瘤细胞生长锥推进过程中RhoA/Rac1/Cdc42的活性。Rac1和Cdc42活性在生长锥外周结构域（p结构域）较高。活性的Rac1在整个p结构域被均匀检测到，而Cdc42的活性则向生长锥边缘逐渐增加。在突出生长锥外围RhoA下调的模型中，我们发现p区RhoA活性高于中央区和轴突轴，并且生长锥延伸部分RhoA活性维持在较高水平。在溶血磷脂酸处理的N1E-115细胞中，发育良好的神经突和生长锥显示RhoA激活，但保持其延伸的形态，直到它们被吸引到收缩的体细胞。另一方面，C3外泌酶对RhoA活性的抑制导致生长球果中肌动蛋白束的丢失或变形。因此，轴上的RhoA激活导致神经突缩回，而p结构域的高RhoA活性对于保持神经生长锥的扩散形态是必要的。(c) 2005 Elsevier B.V.版权所有
Rho-family GTPases play a central role in the regulation of neuronal morphogenesis. In growth cones, for example, Rho GTPases transduce extracellular stimuli into structural changes such as filopodia and lamellipodia. Although it is generally accepted that Racl/Cdc42 and RhoA are positive and negative regulators of neurite outgrowth, respectively, the role of each Rho-family member in neuronal morphogenesis may change according to the cell context. At present, the mechanism underlying this complexity is largely unknown. In growth cones, this is partly due to a lack of information on the distribution of active Rho GTPases. Here, we visualized RhoA/Rac1/Cdc42 activities during laminin-induced growth cone advance of DRG neurons and N1E-115 neuroblastoma cells using probes based on fluorescence/Forster resonance energy transfer. The Rac1 and Cdc42 activities were high in the peripheral domain (P-domain) of growth cones. Active Rac1 was uniformly detected throughout the P-domain, whereas Cdc42 activity increased gradually toward the growth cone edge. Against a model involving RhoA down-regulation at the periphery of protruding growth cones, we found that the RhoA activity was higher in the P-domain than in the central domain and axon shaft, and that a high level of RhoA activity was maintained in the extending part of growth cones. In lysophosphatidic acid-treated N1E-115 cells, well-developed neurites with growth cones showed RhoA activation, but sustained their extended morphology until they were drawn toward the contracting somata. On the other hand, suppression of RhoA activity by C3 exoenzyme led to loss or deformation of actin bundles in the growth cones. Thus, RhoA activation in the shaft results in neurite retraction, whereas high RhoA activity in the P-domain is necessary to retain the spread morphology of nerve growth cone. (c) 2005 Elsevier B.V. All rights reserved.