Regulating Microtubule Severing Physically and Chemically

物理和化学调控微管切断

• 批准号: 10580392
• 负责人: JENNIFER L ROSS
• 金额: $ 1.32万
• 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/10580392
• 关键词: Binding; Biochemistry; Biological Assay; Brain; Cell division; Cell physiology; Cells; Chemicals; Code; Cytoskeleton; Data; Defect; Development; Dimerization; Enzymes; Family; Filament; Fluorescence Microscopy; Genetic Recombination; Goals; Human body; In Vitro; Lead; Light; Link; Location; Microtubule-Associated Proteins; Microtubules; Phosphorylation; Polymers; Regulation; Research Personnel; Scheme; Serine; Tail; Techniques; Testing; Time; Tubulin; Work; cell motility; ciliopathy; density; katanin; neuron development; novel; proteostasis; reconstitution; repaired; single molecule; small molecule inhibitor; tool

PROJECT SUMMARY 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.

项目概要 这项新提案申请的目标是揭示物理和化学监管 控制微管切断酶剑宁的方案。 Katanin 是一种 AAA+ 酶， 六聚化以从微管丝中去除微管蛋白二聚体，从而产生 长丝切断。当剑素水平高且在细胞中不受调节时，它可以破坏整个细胞 微管网络，因此关闭它是一个重要的控制旋钮。剑宁过度活跃可以 导致微管聚合物完全丧失，但活性不足与发育有关 大脑缺陷和纤毛病。所提出的工作的中心假设是 控制剑宁的机制实际上调节剑宁六聚体寡聚化。我们之前的 研究表明低聚是剑宁的限速步骤。我们寻求使用 定量荧光显微镜直接检验寡聚控制的假设 通过物理和化学手段进行切断。具体来说，我们将探索监管 使用新型光敏二聚化结构域驱动活细胞中的剑蛋白浓度 通过探索以下目标来局部测定剑宁浓度：(1) 两者的量化 剑蛋白浓度和微管丝密度作为时间的函数将使得 首次在细胞内生化研究剑素。 (2) 使用体外重构 微管切断和新颖的单分子计数技术，我们将检查其效果 丝氨酸 131 的磷酸化状态在结合、寡聚化和被剑蛋白切断时的变化。 (3) 微管蛋白羧基末端尾部已被证明可以作为控制许多 微管相关蛋白和酶。切断酶没有什么不同，并且是 已知需要羧基末端尾部来切断微管。我们的初步数据显示 剑素的调节与其他切断酶不同。我们将使用一个切断 抑制测定和单分子计数，以量化剑素结合和作用的能力 各种羧基末端尾序列的微管。 实现拟议的目标将创建一种新颖的微管破坏工具，该工具可以 用于各种细胞和有机体测定，以控制细胞的位置和密度 微管网络。此外，拟议的研究将揭示有关如何 可以通过控制剑宁寡聚化来调节细胞中的微管切断 状态。剑素活性的这一关键步骤可能是创建小分子的切入点 微管切断酶和其他 AAA+ 酶的抑制剂，这些酶对于 重要细胞功能的宿主，包括蛋白质稳态、DNA 重组、 复制、修复。