Microtubule Severing and Regrowth by Spastin

Spastin 微管切断和再生

基本信息

批准号：
10221743
负责人：
Jonathon Howard
金额：
$ 32.28万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-01 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10221743
关键词：
ATP Hydrolysis ATP phosphohydrolase Affect Animal Model Binding C-terminal Cells Cytoskeleton Degenerative Disorder Disease Engineering Enzymes Excision Family member Fluorescence Fluorescence Microscopy Frequencies Functional disorder Future Gait abnormality Genes Goals Grant Growth Guanosine Triphosphate Hereditary Spastic Paraplegia Human Hydrolysis In Vitro Island Kinetics Knowledge Laboratories Lead Measures Mechanics Mediating Microtubules Modeling Molecular Motor Neurons Mutate Mutation Mutation Analysis Nerve Degeneration Neurodegenerative Disorders Neurons Organism Outcome Pathologic Patients Peptides Plant Model Polymers Proteins Reagent Research Role Spastic Paraplegia Spectrum Analysis Speed Structure Tail Testing Tubulin Work base design experimental study in vivo insight katanin laser tweezer mathematical model molecular mechanics monomer mutant novel reconstitution repaired single molecule spastin therapeutically effective

项目摘要

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.

项目摘要 Spastin 的微管切断和再生遗传性痉挛性截瘫 (HSP) 是一种导致进行性步态的神经退行性疾病 HSP 患者中最常见的突变基因编码微管。切割酶 spastin，可以将微管聚合物切割成更短的片段。微管切断蛋白——spastin、katanin 和 fidgetin——长期以来一直被认为分解细胞微管网络，各种生物体的体内研究表明他们实际上可以增加细胞中微管的数量。建立 spastin 如何调节细胞微管网络的框架，以及如何 spastin 活性的扰动会导致神经变性。最近，通过重建纯化的 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。与其典型的切断活动无关，我们证明了切断和切断的结合。促进微管再生可导致微管数量呈指数级增加基于这项工作，我们捕获了 spastin。通过结合这两种活性来增加细胞微管的数量。然而，人们对如何执行这些功能知之甚少。该项目的总体目标是了解切断的分子机制并结合使用单分子荧光显微镜、力我们的两个具体目标是：（i）剖析分子。 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，预计将产生详细的结果。 spastin 活性的动力学和机械机制，并生成能够促进未来的结构和细胞研究，包括 spastin 的病理生理学研究。