Molecular Mechanisms of Semaphorin/Plexin-mediated Cytoskeletal Reorganization

这个项目的目标是破译调节肌动蛋白和微管细胞骨架的机制， 神经细胞行为的基础结构，包括形态学、极性、粘附、突起伸长， 能动性、导航性、连通性和可塑性。为了改变它们的大小、形状和连接性，神经元需要 肌动蛋白和微管蛋白组装成长的聚合物（分别为F-肌动蛋白和微管）- 现在已经确定了许多细胞外刺激物，它们可以改变这些细胞的组装和组织， 细胞骨架结构然而，我们仍然不知道这些细胞外信号是如何对细胞产生精确影响的。 细胞骨架为了更好地了解这些机制，我的实验室一直专注于最大的家庭之一， 脑信号蛋白（Semaphorins，Semas）是细胞外信号的一种， 对F-肌动蛋白和微管的影响。我们的策略是使用模式生物和筛选 寻找在信号转导级联反应中起作用的蛋白质的方法， 丛状蛋白受体。在我们已经鉴定的蛋白质中，有一个新的细胞内蛋白质家族，称为 MICAL是Sema/丛蛋白信号转导所需的。在我的实验室里工作 该R 01的循环揭示了MICALs采用以前未知的氧化还原信号系统， 控制肌动蛋白细胞骨架。也就是说，我们发现Mical是一种新的F-肌动蛋白分解因子， 我们的结果表明，Sema/丛蛋白介导的肌动蛋白细胞骨架重组可以精确地 通过激活麦克风在空间和时间上实现。我们还发现MICAL属于一类 氧化还原酶（Redox）的酶和Mical利用其Redox酶活性改变性质， F-actin。我们的工作已经确定，Mical使用F-肌动蛋白作为直接底物和后底物， 氧化肌动蛋白上的保守氨基酸，同时分解F-肌动蛋白， 聚合法此外，我们还发现，这种Sema/Escherichia coli/Mical介导的肌动蛋白氧化还原调节是可逆的， (by一种名为SelR/MsrB的蛋白质），这种特异性的可逆氧化还原肌动蛋白调节系统 神经元和非神经元组织中的多种不同的生物过程。因此，我假设 Sema/丛状蛋白引导线索利用由Mical和SelR组成的可逆氧化还原信号传导机制， 直接和时空协调细胞骨架重塑以驱动细胞形式和功能。我提议 为了验证这一假设，我们跟踪了几条初步观察线， Sema/丛蛋白/Mical介导的细胞骨架重组的分子机制，包括1）特定类型 F-actin/F-actin网络对Sema/Escherichia/Mical效应最敏感，2）分子相互作用 允许Sema/Plexins协调肌动蛋白和微管细胞骨架的分解，3） - 允许Sema/Escherichia/Mical细胞骨架效应在时空上放大的配体/受体系统，以及 4)保护肌动蛋白丝免受Sema/Plex/Mical效应的特定肌动蛋白调节蛋白。

The goals of this project are to decipher the mechanisms that regulate the actin and microtubule cytoskeletons, the structures underlying neural cell behaviors including morphology, polarity, adhesion, process elongation, motility, navigation, connectivity, and plasticity. To change their size, shape, and connectivity, neurons require actin and tubulin proteins to assemble together into long polymers (F-actin and microtubules, respectively) – and numerous extracellular stimuli have now been identified that alter the assembly and organization of these cytoskeletal structures. Yet, we still know little of how these extracellular cues exert their precise effects on the cytoskeleton. To better understand these mechanisms, my lab has been focusing on one of the largest families of extracellular cues, the Semaphorins (Semas) – which alter neuronal behaviors by eliciting destabilizing effects on both F-actin and microtubules. Our strategy has been to use model organisms and screening approaches to search for proteins that work in the signal transduction cascade utilized by Semas and their Plexin receptors. Among the proteins that we have identified, is a new family of intracellular proteins called the MICALs that are required for Sema/Plexin signal transduction. Now, work in my lab during the previous funding cycle of this R01 has revealed that the MICALs employ a previously unknown Redox signaling system to control the actin cytoskeleton. Namely, we have found that Mical is a novel F-actin disassembly factor – and our results reveal that Sema/Plexin-mediated reorganizations of the actin cytoskeleton can be precisely achieved in space and time through activation of Mical. We have also found that the MICALs belong to a class of oxidoreductase (Redox) enzymes and that Mical employs its Redox enzymatic activity to alter the properties of F-actin. Our work has gone on to identify that Mical uses F-actin as a direct substrate and post- translationally oxidizes conserved amino acids on actin, simultaneously dismantling F-actin and decreasing polymerization. Moreover, we find that this Sema/Plex/Mical-mediated Redox regulation of actin is reversible (by a protein called SelR/MsrB) – and that this specific reversible Redox actin regulatory system directs multiple different biological processes in neuronal and non-neuronal tissues. Therefore, I hypothesize that Sema/Plexin guidance cues utilize a reversible Redox signaling mechanism composed of Mical and SelR to directly and spatiotemporally coordinate cytoskeletal remodeling to drive cellular form and function. I propose to test this hypothesis by following-up on several lines of preliminary observations that illuminate critical molecular mechanisms of Sema/Plexin/Mical-mediated cytoskeletal reorganization including 1) specific types of F-actin/networks of F-actin that are most responsive to Sema/Plex/Mical effects, 2) molecular interactions that allow Sema/Plexins to coordinate the disassembly of the actin and microtubule cytoskeletons, 3) ligand/receptor systems that allow Sema/Plex/Mical cytoskeletal effects to be magnified spatiotemporally, and 4) specific actin regulatory proteins that protect actin filaments from Sema/Plex/Mical effects.