Microtubule Severing and Regrowth by Spastin

Spastin的微管断裂和再生 遗传性痉挛性截瘫（HSP）是一种神经退行性疾病，导致进行性步态 disorder.在HSP患者中发现的最常见的突变基因编码微管- 切断酶spastin，其可以将微管聚合物切断成更短的片段。而 微管切割蛋白--痉挛蛋白、卡他宁蛋白和坐立不安蛋白--长期以来一直被认为 分解细胞微管网络，在各种生物体中的体内研究表明， 它们可以增加细胞中微管的数量。我们的长期目标是 建立一个框架，如何痉挛调节细胞微管网络，以及如何 痉挛素活性的扰动导致神经元变性。 最近，通过重组纯化的spastin的活性，我们发现， 具有促进切断的微管再生长的新活性。本次活动是 独立于其规范切断活动。我们发现切断和 微管再生促进可导致微管数目的指数增加。 微管和微管蛋白聚合物的量。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。怎么痉挛 然而，人们对这些功能的理解却很少。 这个项目的总体目标是了解切断的分子机制， 和再生长的结合使用单分子荧光显微镜，力 光谱学和数学建模。我们的两个具体目标是：（一）解剖分子 spastin依赖性微管切断的机制，以及（ii）测试竞争模型如何 spastin促进微管再生。这些目标的完成预计将产生详细的 Spastin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，包括对痉挛的病理生理学。

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.