Regulating Microtubule Severing Physically and Chemically

项目摘要 这个新的提案应用程序的目标是揭示物理和化学调节 控制微管切断酶katanin的方案。Katanin是一种AAA+酶， 六聚化以从微管丝中除去微管蛋白二聚体， 灯丝切断当在高水平时，在细胞中不受调节，katanin可以破坏整个细胞， 微管网络，因此关闭它是一个必不可少的控制旋钮。katanin can过度活跃 导致微管聚合物完全丧失，但活性不足与发育有关 大脑缺陷和纤毛病变这项工作的中心假设是， 控制卡他宁的机制实际上调节卡他宁六聚体寡聚化。我们事先 工作表明低聚是Katanin的限速步骤。我们寻求利用 定量荧光显微镜直接测试的假设，寡聚化控制 通过物理和化学手段切断。具体而言，我们会探讨规管 使用新的光敏二聚化结构域驱动活细胞中的katanin浓度 通过探索以下目的来局部地测定katanin浓度：（1）定量两种 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)使用体外重建 微管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的磷酸化状态对katanin的结合、寡聚化和切断的影响。 (3)微管蛋白的羧基末端尾已被证明是一个代码，以控制许多 微管相关蛋白和酶。切割酶没有什么不同， 已知需要羧基末端尾部来切断微管。我们的初步数据显示 katanin的调节与其他酶不同。我们将使用一个切断 抑制测定和单分子计数以定量katanin结合和作用于 各种羧基末端尾部序列的微管。 实现所提出的目标将创造一种新的微管破坏工具， 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，拟议的研究将揭示新的信息， 微管断裂可以通过控制katanin寡聚化来调节 状态这个katanin活性的关键步骤可能是创造小分子的切入点 微管切断酶和其他AAA+酶的抑制剂，这些酶对 宿主的基本细胞功能，包括蛋白质稳态，DNA重组， 复制和修复。

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.