Regulating Microtubule Severing Physically and Chemically

项目摘要/摘要 这一新提案申请的目标是揭示物理和化学监管方案 为了控制微管切断酶，卡塔宁。Katanin是一种AAA酶，它在体内形成六聚体 为了从微管细丝中去除微管蛋白二聚体，导致细丝断裂。何时在 高水平，在细胞内不受调控，katanin可以破坏整个微管网络，从而使 它关闭是一个必不可少的控制旋钮。Katanin活性过高可导致微管完全丧失 聚合物，但活动不足与大脑发育缺陷和纤毛疾病有关。这个 拟议工作的中心假设是，控制katanin的机制实际上调节 卡塔宁六聚体齐聚。我们以前的工作表明，齐聚反应是一种速度限制 卡塔宁上台。我们试图使用定量荧光显微镜直接检验这一假说。 这种寡聚作用通过物理和化学手段控制着切割。具体来说，我们将 用一种新的光敏二聚体探索活细胞中katanin浓度的调节 通过探索以下目标来推动局部katanin浓度的领域：(1)量化 作为时间函数的Katanin浓度和微管细丝密度都将使 首次在细胞内进行生物化学检测。(2)微管的体外重建 切断和一种新的单分子计数技术，我们将检查 丝氨酸131在卡丹宁结合、寡聚和切断时的磷酸化状态。(3) 微管蛋白羧基末端尾巴已被证明作为一种密码控制许多与微管相关的 蛋白质和酶。断裂酶没有什么不同，已知需要羧基- 末端尾巴切断微管。我们的初步数据显示，katanin的调节是明显的 从其他的切断酶中分离出来。我们将使用分离抑制试验和单分子计数来 量化katanin结合和作用于不同羧基末端尾巴的微管的能力 序列。 实现所提出的目标将创造一种新型的微管破裂工具，可用于 控制微管位置和密度的各种细胞和生物分析方法 网络。此外，拟议的研究将揭示有关微管切断如何 通过控制katanin齐聚状态在细胞内进行调控。这对卡塔宁来说至关重要的一步 活性可能是为微管切断酶创造小分子抑制剂的一个切入点 以及其他对包括蛋白质在内的许多基本细胞功能都很重要的AAA酶 动态平衡、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.