Regulating Microtubule Severing Physically and Chemically

项目概要/摘要 这个新的提案应用程序的目标是揭示物理和化学监管计划 来控制微管切割酶katanin Katanin是一种AAA+酶， 以便从微管丝中除去微管蛋白二聚体，导致丝切断。当 高水平，在细胞中不受调节，katanin可以破坏整个微管网络， 它关闭是一个基本控制旋钮。katanin的过度活性可导致微管的完全丧失 聚合物，但活动不足与大脑发育缺陷和纤毛病有关。的 这项研究的中心假设是，控制katanin的机制实际上是调节 katanin六聚体寡聚化。我们以前的工作表明，低聚反应是一个速率限制 步为katanin。我们寻求使用定量荧光显微镜直接测试的假设 低聚化通过物理和化学手段控制切断。具体来说，我们将 利用一种新的光敏二聚体来探索活细胞中katanin浓度的调节 通过探索以下目标，在局部驱动katanin浓度：（1）定量 katanin浓度和微管丝密度作为时间的函数将使得 这是卡他宁第一次在细胞内进行生物化学研究。(2)利用微管的体外重建 切断和一种新的单分子计数技术，我们将研究的影响， 丝氨酸131在结合、寡聚化和被katanin切断时的磷酸化状态。(3)的 微管蛋白羧基末端尾已被证明作为一个代码，以控制许多微管相关的 蛋白质和酶。切割酶没有什么不同，并且已知需要羧基- 末端尾部切断微管。我们的初步数据表明，katanin的调节是独特的， 与其他酶分离。我们将使用切断抑制测定和单分子计数， 量化Katanin结合和作用于各种羧基末端尾微管的能力 序列的 实现所提出的目标将创造一种新的微管破坏工具，可用于 各种细胞和有机体测定，以控制微管的位置和密度 网络此外，拟议中的研究将揭示有关微管切断如何 在细胞中通过控制katanin寡聚化状态进行调节。卡他宁迈出的关键一步 活性可能是产生微管切断酶的小分子抑制剂的切入点 和其他AAA+酶，这些酶对包括蛋白质在内的许多基本细胞功能都很重要 稳态、DNA重组、复制和修复。

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.