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、极性、粘附性、突起伸长、 机动性、导航性、连通性和可塑性。为了改变它们的大小、形状和连通性，神经元需要 肌动蛋白和微管蛋白组装成长聚合物(分别为F-肌动蛋白和微管)- 现在已经确定了许多改变这些细胞的组装和组织的细胞外刺激 细胞骨架结构。然而，我们仍然知之甚少，这些细胞外信号是如何对 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一个最大的家庭 在细胞外信号中，信号素(Semas)通过引发不稳定因素来改变神经元的行为 对F-肌动蛋白和微管的影响。我们的策略一直是使用模式生物和筛选 寻找在SIMAS及其受体所利用的信号转导通路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 Sema/Plexin信号转导所需的MICAL。现在，在我之前的资助期间，在我的实验室工作 这个R01的循环揭示了MICAL使用了一种以前未知的氧化还原信号系统来 控制肌动蛋白的细胞骨架。也就是说，我们发现Mical是一种新的F-肌动蛋白分解因子-并且 我们的结果表明，Sema/Plexin介导的肌动蛋白细胞骨架的重组可以精确地 通过激活Mical在空间和时间上实现。我们还发现，MICAL属于一个类别 氧化还原酶(Redox)的活性，而Mical利用其氧化还原酶的活性来改变性质 F-肌动蛋白。我们的工作继续确定Mical使用F-肌动蛋白作为直接底物和后-肌动蛋白。 翻译氧化肌动蛋白上的保守氨基酸，同时分解F-肌动蛋白并降低 聚合反应。此外，我们发现这种由Sema/Plex/Mical介导的肌动蛋白氧化还原调节是可逆的 (通过一种名为SelR/MSRB的蛋白质)-这种特定的可逆氧化还原肌动蛋白调控系统 神经性和非神经性组织中的多种不同的生物过程。因此，我假设 SEMA/Plexin引导信号利用由Mical和SelR组成的可逆氧化还原信号机制 直接和时空协调细胞骨架重塑，以驱动细胞形态和功能。我建议 通过跟踪几行初步观察来检验这一假设，这些初步观察阐明了关键的 Sema/Plexin/Mical介导的细胞骨架重组的分子机制包括：1)特定类型 对Sema/Plex/Mical效应最敏感的F-肌动蛋白/F-肌动蛋白网络，2)分子相互作用 允许肌动蛋白/丛蛋白协调肌动蛋白和微管细胞骨架的分解。 配体/受体系统，允许Sema/Plex/Mical细胞骨架效应在时空上被放大，以及 4)特殊的肌动蛋白调节蛋白，保护肌动蛋白细丝免受Sema/Plex/Mical效应的影响。

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.