MARCKS Phosphorylation in Growth Cone Signaling

描述(由申请人提供)：排斥作用需要生长锥体部分的、不对称的坍塌，以有效地将发育中的轴突“引导”到其目标。确定塌陷反应背后的分子事件将提供对轴突寻路的更多了解，轴突寻路是功能神经系统发育中的一个基本重要过程。这项研究的主要目的是阐明信号素介导的生长锥塌陷的信号事件。本研究将1)分析蛋白激酶C(PKC)信号在信号素诱导的生长锥崩溃所需的黏附位点分解中的作用；2)表征生长锥中主要的PKC底物--肉豆蔻酰化的富含丙氨酸的C激酶底物(MARCKS)的分布和动态。共聚焦免疫荧光显微镜和反射干涉显微镜(RIM)将用于确定已知的粘附点蛋白、Marcks和生长锥体内粘附区之间的空间关系。生长锥黏附位点的生化分离将被用来分析Marcks磷酸化对黏附复合体完整性的影响。Marcks在生长锥内的动态将通过观察驱避剂诱导的Marcks-绿色荧光蛋白(GFP)融合蛋白分布的变化来表征。异常的PKC信号的功能后果将通过观察表达显性阴性、磷酸化缺陷的Marcks突变体的DRG神经元的生长锥体的转向反应来确定。

DESCRIPTION (provided by applicant): Repellent action requires a partial, asymmetric collapse of the growth cone to effectively "steer" the developing neurite to its target. Determination of the molecular events that underlie the collapse response will provide a greater understanding of neurite pathfinding, a process of fundamental importance in the development of the functional nervous system. The main goal of the proposed research is to elucidate the signaling events of semaphorin-mediated growth cone collapse. This research will 1) analyze the role of protein kinase C (PKC) signaling in the disassembly of adhesion sites required for semaphorin-induced growth cone collapse and 2) characterize the distribution and dynamics of the predominant PKC substrate in growth cones, the myristoylated, alanine-rich C kinase substrate (MARCKS). Confocal immunofluorescence microscopy and reflection interference microscopy (RIM) will be used to determine the spatial relationships between known adhesion site proteins, MARCKS, and adhesive areas within the growth cone. Biochemical isolation of growth cone adhesion sites will be used to analyze the effect of MARCKS phosphorylation on adhesion complex integrity. MARCKS dynamics within the growth cone will be characterized by observing repellent-induced changes in the distribution of a MARCKS-green fluorescent protein (GFP) fusion protein. Functional consequences of aberrant PKC signaling will be determined by observing the turning response of growth cones from DRG neurons expressing a dominant-negative, phosphorylation-deficient MARCKS mutant.