Ca and Rho GTPase Control of the Neuronal Cytoskeleton

描述（由申请人提供）：Rho gtpase是一种分子开关，在非神经元细胞的细胞粘附、极性和细胞骨架蛋白动力学控制中发挥着很好的特征作用。尽管在功能上涉及轴突引导和神经再生，神经元Rho GTPase细胞生物学尚不清楚。为了解决这一差距，我们最近使用了多模式荧光斑点显微镜（FSM），它允许直接测量细胞骨架蛋白动力学，来研究化学驱避剂溶血磷脂酸（LPA）引起的Rho依赖性反应。我们发现LPA处理增加了生长锥内“肌动蛋白弧”和肌动蛋白束结构的收缩性，并提供证据表明这种收缩性驱动了Rho/Rho激酶依赖性生长锥的收缩。肌动蛋白弧的收缩性似乎与肌凝蛋白II有关，因为它取决于肌凝蛋白轻链激酶和肌凝蛋白轻链磷酸酶的活性。其他研究表明Rho GTPase和Ca活性可以调节轴突对单个配体的引导反应的极性。尽管这些发现引起了轴突引导和神经再生领域的关注，但对反应转换的细胞生物学机制知之甚少。我们有初步的证据表明，背景Rac活性的增加将LPA收缩反应转化为神经突起的生长和发展。有趣的是，这种LPA诱导的生长伴随着细胞内Ca的增加和肌动蛋白弧收缩性的丧失，即与没有Rac激活时观察到的相反。我们建议结合荧光斑点显微镜和Ca成像定量评估生长锥中的肌动蛋白、微管和钙动力学，以研究Rac活性明显改变LPA响应极性的细胞骨架和信号传导机制。我们还将描述肌球蛋白II在这些反应中的作用，并进行相关的超微结构研究，以更好地定义生长锥中这一重要分子马达的细胞生物学。我们的工作假设是，Rac和Rho gtpase可以通过对MT动力学的特定影响来调节配体激活反应的功能输出，从而影响ER Ca储存的定位并调节Ca释放地形和/或释放灵敏度。这一假设将在LPA以及Ephrin和Slit配体的背景下得到验证，后两种配体通过激活中枢神经系统中的Rho来发挥其化学排斥反应。

DESCRIPTION (provided by applicant): Rho GTPases are molecular switches that play well characterized roles in cell adhesion, polarity, and control of cytoskeletal protein dynamics in non-neuronal cells. Although functionally implicated in axon guidance and nerve regeneration, neuronal Rho GTPase cell biology is far less well understood. To address this gap, we recently used multi-mode Fluorescent Speckle Microscopy (FSM), which allows direct measurement of cytoskeletal protein dynamics, to investigate Rho dependent responses evoked by the chemorepellant agent, lysophosphatidic acid (LPA). We discovered that LPA treatment increased the contractility of "actin arc" and actin bundle structures within the growth cone and provided evidence that this contractility was driving Rho/Rho Kinase dependent growth cone retractions. Actin arc contractility appears to involve Myosin II since it depends on Myosin Light Chain Kinase and Myosin Light Chain phosphatase activities. Other work suggests Rho GTPase and Ca activity can modulate the polarity of axon guidance responses to a single ligand. Although these findings have caught the attention of axon guidance and nerve regeneration fields, the cell biological mechanisms underlying response switching are poorly understood. We have preliminary evidence that increasing background Rac activity converts LPA retraction responses to neurite growth and advance. Interestingly, such LPA evoked growth is accompanied by increases in intracellular Ca and loss of actin arc contractility -i.e. the opposite of what is observed without Rac activation. We propose to combine use of Fluorescent Speckle Microscopy and Ca Imaging to quantitatively assess actin, microtubule, and Calcium dynamics in growth cones to investigate the cytoskeletal and signaling mechanisms underlying apparent switching of LPA response polarity by Rac activity. We will also characterize the role of Myosin II in these responses and do correlative ultrastructural studies to better define the cell biology of this important molecular motor in the growth cone. Our working hypothesis is that Rac and Rho GTPases can modulate the functional output of ligand activated responses via specific effects on MT dynamics which in turn affect localization of ER Ca stores and regulate the Ca release topography and/or release sensitivity. This hypothesis will be tested in the context of LPA as well as Ephrin and Slit ligands, the latter two being known to exert their chemorepellant responses via activation of Rho in the CNS.