Brown M D, Cornejo B J, Kuhn T B, Bamburg J R
Department of Anatomy and Neurobiology, Colorado State University, Fort Collins, Colorado 80523-1670, USA.
J Neurobiol. 2000 Jun 15;43(4):352-64. doi: 10.1002/1097-4695(20000615)43:4<352::aid-neu4>3.0.co;2-t.
To assess the role of cdc42 during neurite development, cmyc-tagged constitutively active (CA) and dominant negative (DN) cdc42 were expressed in dissociated primary chick spinal cord neurons using adenoviral-mediated gene transfer. Three days after infection, >85% of the neurons in infected cultures expressed cdc42 proteins, as detected by indirect immunofluorescence against cmyc. Growth cones of infected neurons displayed 1.83- (CAcdc42) and 1.93-fold (DNcdc42) higher cmyc immunofluorescence per square micrometer than uninfected controls. CAcdc42 expression stimulated growth cones, almost doubling growth cone size and number of filopodia, and increased neurite growth rates by 65-89%. In neurons plated onto fibronectin, the percent of growth cones with both filopodia and lamellipodia increased from 71 to 92%. Total Texas Red-phalloidin staining in these growth cones doubled, and the percent of growth cones with F-actin localized to peripheral regions increased from 52% in controls to 78% after CAcdc42 expression. Expression of DNcdc42 did not significantly alter growth cone morphology or neurite growth rates. Addition of soluble laminin to spinal cord neurons resulted in the identical phenotype as CAcdc42 expression, including changes in growth cone morphology, F-actin localization, and neurite growth rates. Significantly, expression of DNcdc42 blocked the effects of laminin on growth cones. These results show that cdc42 promotes neurite outgrowth and filopodial and lamellipodial formation in growth cones and suggests that cdc42 and laminin share a common signaling pathway during neurite development. Addition of laminin to CAcdc42-expressing neurons is inhibitory to growth cones, indicating that laminin also may activate some other pathways.
为了评估细胞分裂周期蛋白42（cdc42）在神经突发育过程中的作用，利用腺病毒介导的基因转移技术，将带有cmyc标签的组成型激活（CA）和显性负性（DN）cdc42在原代鸡脊髓神经元中进行表达。感染三天后，通过针对cmyc的间接免疫荧光检测发现，感染培养物中超过85%的神经元表达了cdc42蛋白。感染神经元的生长锥每平方微米的cmyc免疫荧光比未感染的对照高1.83倍（CAcdc42）和1.93倍（DNcdc42）。CAcdc42的表达刺激了生长锥，使生长锥大小和丝状伪足数量几乎增加了一倍，并使神经突生长速率提高了65 - 89%。在接种到纤连蛋白上的神经元中，同时具有丝状伪足和片状伪足的生长锥百分比从71%增加到92%。这些生长锥中总的德克萨斯红鬼笔环肽染色增加了一倍，并且F - 肌动蛋白定位于周边区域的生长锥百分比从对照中的52%增加到CAcdc42表达后的78%。DNcdc42的表达并未显著改变生长锥形态或神经突生长速率。向脊髓神经元中添加可溶性层粘连蛋白导致了与CAcdc42表达相同的表型，包括生长锥形态、F - 肌动蛋白定位和神经突生长速率的变化。重要的是，DNcdc42的表达阻断了层粘连蛋白对生长锥的影响。这些结果表明，cdc42促进神经突生长以及生长锥中丝状伪足和片状伪足的形成，并提示在神经突发育过程中cdc42和层粘连蛋白共享一条共同的信号通路。向表达CAcdc42的神经元中添加层粘连蛋白对生长锥具有抑制作用，表明层粘连蛋白也可能激活一些其他通路。
