Molecular Mechanisms of Semaphorin/Plexin-mediated Cytoskeletal Reorganization

描述(申请人提供)：这个项目的目标是描述一种新的生物学机制，它直接应用于肌动蛋白细胞骨架的调节-神经细胞行为的基础结构，包括形态、极性、粘连、突起延长、运动性、导航、连通性和可塑性。为了改变它们的大小、形状和连通性，神经元需要肌动蛋白组装成长丝。调整这些肌动蛋白细丝的长度和组织(F-肌动蛋白)指定了运动的方向，使细胞能够精确地相互连接和通信。现在已经确定了许多控制肌动蛋白动态的细胞外信号，但我们对这些存在于细胞外的信号如何在细胞内发挥确切作用知之甚少。信号素(Semaphorins，SIMA)是这些引导信号中最大的家族之一，它们通过对F-肌动蛋白产生不稳定的影响来调节细胞行为，包括F-肌动蛋白的丢失和新的F-肌动蛋白聚合能力的降低。重要的是，最近的突破已经确定了细胞表面受体和细胞内蛋白对于Sema介导的肌动蛋白效应是必不可少的，但我们仍然知之甚少，直接调节F-肌动蛋白对Semas的反应的分子机制。为了确定这些分子和机制，我们已经确定了与SEMA受体丛蛋白相关的蛋白质，包括一个新的胞浆蛋白家族，称为MICALs。在无脊椎动物中有一个Mical基因，在哺乳动物中有三个Mical基因，它们控制轴突引导、突触发生、树突修剪和其他由Semas/Plexins介导的形态变化。事实上，我们最近发表的结果表明，Mical在Semas/Plexins和肌动蛋白细胞骨架的修饰之间提供了长期追求的直接联系。我们发现，Mical直接分解F-肌动蛋白，并且对于调节Semas/Plexins下游的肌动蛋白动力学是必要和充分的。这些新的结果提供了一个潜在的逻辑，通过它可以在空间和时间上精确地实现Sema介导的肌动蛋白细胞骨架的重组：通过直接Sema-Plexin激活新的肌动蛋白分解因子Mical。有趣的是，MICAL还含有氧化还原酶(Redox)酶部分，我们的结果强烈表明Mical利用其氧化还原活性来改变F-肌动蛋白，首次暗示了特定的氧化还原信号事件在肌动蛋白细胞骨架调节中的作用。因此，我假设Mical酶是一类新的系统发育保守的肌动蛋白分解因子家族，它们利用先前未知的可逆氧化还原信号机制来直接调节肌动蛋白的动力学。为了验证这一假设，我建议将遗传学、细胞培养和细胞生物学方法结合起来，使用无脊椎动物和脊椎动物模型系统，以及使用纯化的Mical和肌动蛋白进行生化、结构和高分辨率成像分析。了解这一不寻常的酶家族，MICAL(不同于任何已被表征的蛋白质)是如何导致F-肌动蛋白分解的，将揭示调控神经细胞生物学和行为的新策略。

DESCRIPTION (provided by applicant): The goals of this project are to characterize a new biological mechanism that has direct application to the regulation of the actin cytoskeleton - the structure underlying neural cell behaviors including morphology, polarity, adhesion, process elongation, motility, navigation, connectivity, and plasticity. In order to change their size, shape, and connectivity, neurons require actin proteins to assemble together into long filaments. Adjusting the length and organization of these actin filaments (F-actin) specifies the direction of movement and enables cells to precisely connect and communicate with one another. A number of extracellular cues have now been identified that control actin dynamics, but we know little of how these signals present outside of cells exert their precise effects within cells. Semaphorins (Semas) are one of the largest families of these guidance cues and they regulate cellular behaviors by eliciting destabilizing effects on F-actin that include a loss of F- actin and the decreased ability to polymerize new F-actin. Importantly, recent breakthroughs have identified cell-surface receptors and intracellular proteins that are essential for Sema-mediated effects on actin but we still know little of the molecular mechanisms that directly regulate F-actin in response to Semas. To identify these molecules and mechanisms we have identified proteins that associate with the Sema receptor Plexin, including a novel family of cytosolic proteins called the MICALs. There is one MICAL gene in invertebrates and three MICAL genes in mammals and they control axon guidance, synaptogenesis, dendritic pruning, and other morphological changes mediated Semas/Plexins. Indeed, our recently published results reveal that MICAL provides a long-sought-after direct link between Semas/Plexins and the modification of the actin cytoskeleton. We find that MICAL directly disassembles F-actin and is both necessary and sufficient for regulating actin dynamics downstream of Semas/Plexins. These new results provide an underlying logic through which Sema- mediated reorganizations of the actin cytoskeleton can be precisely achieved in space and time: through direct Sema-Plexin activation of the novel actin disassembly factor MICAL. Interestingly, MICALs also contain an oxidoreductase (Redox) enzymatic moiety and our results strongly suggest that MICAL utilizes its Redox activity to alter F-actin, implicating for the first time a role for specific Redox signaling events in actin cytoskeletal regulation. Therefore, I hypothesize that MICAL enzymes are a novel family of phylogenetically conserved actin disassembly factors that utilize a previously uncharacterized reversible Redox signaling mechanism to directly regulate actin dynamics. To test this hypothesis, I propose to combine genetics, cell culture, and cell biological approaches using both invertebrate and vertebrate model systems with biochemical, structural, and high-resolution imaging assays utilizing purified MICAL and actin proteins. Understanding how this unusual family of enzymes, the MICALs (which are unlike any proteins that have ever been characterized) causes F-actin to disassemble will reveal new strategies to regulate neural cell biology and behavior.