Regulating Microtubule Severing Physically and Chemically

物理和化学调控微管切断

• 批准号: 10202821
• 负责人: JENNIFER L ROSS
• 金额: $ 45万
• 依托单位: SYRACUSE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10202821
• 关键词: Address; Binding; Biochemistry; Biological Assay; Biological Process; Biology; Biophysics; Brain; Cell Division Process; Cell division; Cell physiology; Cells; Chemicals; Cilia; Code; Complex; Cytoskeleton; Data; Defect; Development; Dimerization; Engineering; Environment; Enzymes; Family; Filament; Fluorescence Microscopy; Genetic Recombination; Goals; Health; Human body; Imaging Techniques; In Vitro; Interdisciplinary Study; Investigation; Kidney; Knowledge; Lead; Light; Link; Liver; Location; Maintenance; Meiosis; Microtubule-Associated Proteins; Microtubules; Mission; Modification; Molecular; Morphogenesis; Nomenclature; Organ; Peer Review; Phosphorylation; Polymers; Positioning Attribute; Post-Translational Protein Processing; Problem Solving; Process; Publications; Regulation; Reproducibility; Research Personnel; Scheme; Science; Serine; Tail; Techniques; Testing; Time; Tissues; Training; Training Activity; Tubulin; Universities; Work; base; cell motility; ciliopathy; density; dimer; experimental study; innovation; katanin; light microscopy; monomer; neuron development; next generation; novel; physical science; proteostasis; reconstitution; repaired; single molecule; skills; small molecule inhibitor; tool; undergraduate student

Project Summary/Abstract The goal of this new proposal application is to uncover the physical and chemical regulatory schemes to control the microtubule severing enzyme, katanin. Katanin is a AAA+ enzyme that hexamerizes in order to remove tubulin diimers from the microtubule filament resulting in filament severing. When at high levels, and unregulated in cells, katanin can destroy the entire microtubule network, thus turning it off is an essential control knob. Overactivity of katanin can lead to complete loss of microtubule polymer, but underactivity is linked with developmental defects in the brain and ciliopathies. The central hypothesis of the proposed work is that the mechanisms to control katanin actually regulate katanin hexamer oligomerization. Our prior work indicated that oligomerization is the a rate-limiting step for katanin. We seek to use quantitative fluorescence microscopy to directly test the hypothesis that oligomerization controls severing through physical and chemical means. Specifically, we will explore the regulation of katanin concentration in live cells using a novel light-sensitive dimerization domain to drive katanin concentration locally by exploring the following aims: (1) Quantification of both the katanin concentration and the microtubule filament density as a function of time will enable biochemistry in the cell for the first time for katanin. (2) Using in vitro reconstitution of microtubule severing and a novel single molecule counting technique, we will examine the effect of the phosphorylation state of serine 131 on binding, oligomerization, and severing by katanin. (3) The tubulin carboxy-terminal tail has been shown to act as a code to control many microtubule-associated proteins and enzymes. Severing enzymes are no different and are known to require the carboxy- terminal tail to sever microtubules. Our preliminary data shows that katanin’s regulation is distinct from other severing enzymes. We will use a severing inhibition assay and single molecule counting to quantify the ability to katanin to bind and act on microtubules of various carboxy-terminal tail sequences. Accomplishing the proposed aims will create a novel microtubule disruption tool that could be used in a variety of cellular and organismal assays to control the location and density of the microtubule network. Further, the proposed studies will reveal new information on how microtubule severing can be regulated in cells through controlling the katanin oligomerization state. This crucial step for katanin activity may be an entry-point for creating small molecule inhibitors for microtubule severing enzymes and other AAA+ enzymes that are important for a host of essential cellular functions including protein homeostasis, DNA recombination, replication, and repair.

项目摘要/摘要 这项新建议申请的目的是发现物理和化学调节方案 为了控制微管几种酶，katanin。 Katanin是一种AAA+酶，六聚体 为了从微管丝中去除微管蛋白二聚体，导致丝几个。何时 高水平且在细胞中不受管制，Katanin可以破坏整个微管网络，从而转动 它是一个必不可少的控制旋钮。 Katanin的过度活动性会导致微管完全丧失 聚合物，但不足的活动与大脑和纤毛病中的发育缺陷有关。 提议的工作的中心假设是控制Katanin的机制实际上受调节 katanin己酸酯寡聚化。我们先前的工作表明，低聚是限制速率 Katanin的一步。我们寻求使用定量荧光显微镜直接检验假设 这种低聚通过物理和化学方法控制第六。具体来说，我们会的 使用新型的光敏二聚化探索活细胞中Katanin浓度的调节 通过探索以下目的来驱动Katanin浓度的域：（1）定量 Katanin浓度和微管丝密度随时间的函数都将实现 Katanin首次在细胞中的生物化学。 （2）使用微管的体外重构 切断和一种新颖的单分子计数技术，我们将研究 丝氨酸131的磷酸化状态在结合，寡聚化和Katanin上的磷酸化状态。 （3） 微管蛋白羧基末端尾部已被证明是控制许多微管相关的代码 蛋白质和酶。切断的酶没有什么不同，已知需要羧基 末端尾部至微管。我们的初步数据表明，Katanin的监管是不同的 来自其他切断的酶。我们将使用切断的抑制测定法和单分子计数 量化Katanin结合并作用于各种羧基末端尾的微管的能力 序列。 完成提议的目标将创建一个新颖的微管破坏工具 各种细胞和有机纹状体控制微管的位置和密度 网络。此外，拟议的研究将揭示有关微管断开如何切断的新信息 通过控制Katanin寡聚状态在细胞中受到调节。 Katanin的关键步骤 活性可能是为微管切断酶创建小分子抑制剂的入口点 以及其他对于包括蛋白质（蛋白质）的大量必需细胞功能很重要的AAA+酶 稳态，DNA重组，复制和修复。