MECHANISMS FOR THE FUNCTION AND REGULATION OF KATANIN

 描述（由申请人提供）：我们研究的长期目标是了解微管阵列是如何创建，维持和重塑的，以使细胞能够执行不同的功能，改变形状并适应不断变化的条件。调节聚合物的数量，长度和营业额是这个问题的核心和微管切断蛋白katanin已成为一个关键的蛋白质为这项任务证明了其在神经元发育，减数分裂和有丝分裂纺锤体组装和功能，细胞迁移，纤毛组装和拆卸，和细胞形态发生的重要作用。然而，我们对控制katanin活动的位置，时间和结果的机制的了解非常有限。为了解决这个问题，我们提出了以下三个具体目标：（1）什么是靶向卡他宁的具体微管网站的机制？我们假设p80调节亚基通过优先结合交叉微管几何结构靶向katanin的p60催化亚基，并且微管相关蛋白抑制p60 katanin的脱靶结合。为了验证这一假设，我们将使用体外重建，冷冻EM和细胞研究的组合：（i）确定必要和充分的条件，招募p60 katanin微管交叉位点;（ii）阐明的结构和构象的p60 katanin，有和没有p80，在微管切断;和（iii）确定如何MAP 65蛋白保护微管对katanin活性。(2)调节p60 katanin切断活性的机制是什么？我们假设p60 katanin活性被多种调节机制紧紧控制，以根据细胞环境提供细微的控制。为了验证这一假设，我们将集中在两个新发现的调节蛋白，RIC 1和SPR 2，这是建议控制的速度和位置的p60活性分别。我们将使用体外重建和活体成像来研究RIC 1如何增强p60 katanin的切断活性，以及SPR 2如何调节p60 katanin进入微管交叉位点。(3)是什么机制调节断裂的微管末端的动力学？我们假设微管加端跟踪蛋白通过调节新加端的动态在确定微管切断的结果中起主要作用。为了验证这一假设，我们将集中在一个+TIP称为SPR 1，这是牵连作为一个“拯救因子”的微管切断我们的初步数据。我们将使用动态微管的体外重建来确定SPR 1是否以及如何促进通过切断产生的微管加末端的拯救;并使用活细胞实验来测试通过切断的微管扩增是否驱动光诱导的阵列重定向，以及细胞是否通过蛋白水解调节SPR 1的水平以调节切断的微管的拯救程度。

 DESCRIPTION (provided by applicant): The long-term goal of our research is to understand how microtubule arrays are created, maintained and remodeled to enable cells to perform different functions, change shape and adapt to changing conditions. Regulation of polymer number, length and turnover lies at the heart of this problem and the microtubule severing protein katanin has emerged as a key protein for this task as evidenced by its important role in neuronal development, meiotic and mitotic spindle assembly and function, cell migration, ciliary assembly and disassembly, and cell morphogenesis. However, we have very limited knowledge of the mechanisms controlling the location, timing and outcome of katanin activity. To address this shortcoming, we propose the following three specific aims: (1) What are the mechanisms for targeting katanin to specific microtubule sites? We hypothesize that the p80 regulatory subunit targets the p60 catalytic subunit of katanin by preferentially binding to intersecting microtubule geometries and that off-target p60 katanin binding is inhibited by microtubule-associated proteins. To test this hypothesis, we will use a combination of in vitro reconstitution, cryo-EM and cellular studies to: (i) determine the necessary and sufficient conditions to recruit p60 katanin to microtubule crossover sites; (ii) elucidate the structure and conformation of p60 katanin, with and without p80, during microtubule severing; and (iii) determine how the MAP65 protein protects microtubules against katanin activity. (2) What are the mechanisms regulating p60 katanin severing activity? We hypothesize that p60 katanin activity is held tightly in check by multiple regulatory mechanisms to provide nuanced control depending on the cellular context. To test this hypothesis, we will focus on two newly identified regulatory proteins, RIC1 and SPR2, which are proposed to control the rate and location of p60 activity respectively. We will use in vitro reconstitution and live imaging to examine how RIC1 boosts the severing activity of p60 katanin, and how SPR2 regulates access of p60 katanin to microtubule crossover sites. (3) What are the mechanisms regulating the dynamics of severed microtubule ends? We hypothesize that microtubule plus-end tracking proteins play a major role in determining the outcome of microtubule severing by regulating the dynamics of the new plus-ends. To test this hypothesis, we will focus on a +TIP called SPR1, which is implicated as a "rescue factor" for severed microtubules by our preliminary data. We will use in vitro reconstitution with dynamic microtubules to determine whether and how SPR1 promotes rescue of microtubule plus ends created by severing; and use live cell experiments to test whether microtubule amplification via severing drives light-induced array reorientation and whether cells regulate the level of SPR1 via proteolysis to modulate the extent of rescue of severed microtubules.