Microtubule Severing and Regrowth by Spastin

微管切断和痉挛再生的项目总结 遗传性痉挛截瘫(HSP)是一种导致进行性步态的神经退行性疾病。 无序。HSP患者中发现的最常见的突变基因编码微管- 切断酶痉挛蛋白，这种酶可以将微管聚合物切割成更短的片段。而当 微管切断蛋白--spastin、katanin和fidgetin--长期以来一直被认为是 在各种生物体中的活体研究表明，分解细胞微管网络 它们实际上可以增加细胞中微管的数量。我们的长期目标是 为spastin如何调节细胞微管网络以及如何 痉挛蛋白活性的紊乱会导致神经元变性。 最近，通过重组纯化的spastin的活性，我们发现该蛋白 具有促进切断的微管再生的新活性。本活动是 独立于其规范的切断活动。我们证明了切断和切断的结合 促进微管再生可导致微管再生的数量呈指数增加。 微管和微管聚合体的数量。基于这项工作，我们假设痉挛 通过结合这两种活性来增加细胞微管的数量。如何痉挛 然而，执行这些功能的人却知之甚少。 本项目的总体目标是了解断头的分子机制。 和再生结合使用的单分子荧光显微镜，力 光谱学和数学模型。我们的两个具体目标是：(I)剖析分子 痉挛蛋白依赖的微管切断的机制，以及(Ii)测试相互竞争的模型 痉挛蛋白促进微管再生。这些目标的完成预计将产生详细的 痉挛蛋白活性的动力学和机械机制，并产生将 促进未来的结构和细胞研究，包括痉挛的病理生理学。

PROJECT SUMMARY Microtubule Severing and Regrowth by Spastin Hereditary spastic paraplegia (HSP) is a neurodegenerative disease that causes progressive gait disorder. The most commonly mutated gene found in HSP patients encodes the microtubule- severing enzyme spastin, which can sever microtubule polymers into shorter fragments. While microtubule severing proteins—spastin, katanin and fidgetin—have been long thought to disassemble the cellular microtubule network, in vivo studies in various organisms have shown that they can actually increase the number of microtubules in cells. Our long-term goal is to establish a framework for how spastin regulates cellular microtubule networks, and how perturbation of spastin activity leads to neuronal degeneration. Recently, by reconstituting the activity of purified spastin, we discovered that the protein possesses a novel activity that promotes the regrowth of severed microtubules. This activity is independent of its canonical severing activity. We showed that the combination of severing and microtubule regrowth promotion can lead to an exponential increase in the number of microtubules and the amount of tubulin polymer. Based on this work, we hypothesize that spastin increases the amount of cellular microtubules by combining these two activities. How spastin perform these functions, however, is poorly understood. The overall objective of this project is to understand the molecular mechanisms of severing and regrowth using a combination of single-molecule fluorescence microscopy, force spectroscopy and mathematical modelling. Our two specific aims are: (i) dissect the molecular mechanics of spastin-dependent microtubule severing, and (ii) test competing models for how spastin promotes microtubule regrowth. Completion of these aims is expected to yield detailed kinetic and mechanical mechanisms for spastin’s activities and to generate reagents that will facilitate future structural and cellular studies, including on the pathophysiology of spastin.