MARCKS Phosphorylation in Growth Cone Signaling

描述（由申请人提供）：驱避作用需要生长锥体的部分不对称塌陷，以有效地“引导”发育中的神经突到达其目标。确定崩溃反应背后的分子事件将提供对神经突寻路的更好理解，这是一个在功能性神经系统发育中至关重要的过程。本研究的主要目的是阐明信号蛋白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塌陷所需的粘附位点拆卸中的作用，2)表征生长锥中主要的PKC底物，豆芽糖酰化，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）将用于确定已知粘附位点蛋白、MARCKS和生长锥内粘附区域之间的空间关系。生化分离生长锥粘附位点将用于分析MARCKS磷酸化对粘附复合物完整性的影响。通过观察驱蚊诱导的marks -绿色荧光蛋白（GFP）融合蛋白分布的变化，可以表征生长锥内marks的动态。异常PKC信号的功能后果将通过观察来自表达显性阴性、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.