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.

项目摘要 该新提案应用的目的是揭示物理和化学调节 方案以控制微管几种酶Katanin。 katanin是一种AAA+酶 六聚体以从微管丝中去除微管蛋白二聚体，导致 灯丝几个。高水平且在细胞中不受监管时，Katanin会破坏整个 微管网络，因此将其关闭是必不可少的控制旋钮。 katanin的活动过度可以 导致微管聚合物的完全损失，但不足与发育有关 大脑和纤毛病的缺陷。拟议工作的中心假设是 控制Katanin的机制实际上调节了Katanin己酸六聚体的低聚。我们的先验 工作表明，寡聚是Katanin的限制步骤。我们试图使用 定量荧光显微镜直接检验了寡聚控制的假设 通过物理和化学方法切断。具体来说，我们将探讨 使用新型的光敏二聚化结构域中的活细胞中的Katanin浓度驱动 通过探索以下目的，katanin浓度局部：（1）量化这两种目标 Katanin浓度和微管丝密度随时间的函数将启用 Katanin首次在细胞中的生物化学。 （2）使用体外重构 微管切断和一种新颖的单分子计数技术，我们将检查效果 丝氨酸131的磷酸化状态在结合，寡聚和katanin切断。 （3）微管蛋白羧基末端尾部已被证明是控制许多的代码 微管相关的蛋白质和酶。切断的酶没有什么不同，并且 已知需要几个微管的羧基端尾。我们的初步数据显示 Katanin的调节与其他切断酶不同。我们将使用切断 抑制测定和单分子计数以量化Katanin结合并作用于 各种羧基末端尾序的微管。 完成提议的目标将创建一个新型的微管破坏工具 用于多种细胞和有机测定中，以控制 微管网络。此外，拟议的研究将揭示有关如何 微管切断可以通过控制Katanin寡聚来调节细胞中 状态。 Katanin活性的关键步骤可能是创建小分子的入口点 微管切开酶和其他AAA+酶的抑制剂，对A很重要 包括蛋白质稳态，DNA重组，包括蛋白质稳态的基本细胞功能的宿主， 复制和维修。