MECHANISMS FOR THE FUNCTION AND REGULATION OF KATANIN

 描述(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、维护和重塑的，以使细胞能够执行不同的功能、改变形状和适应不断变化的条件。聚合物数量、长度和周转的调控是这一问题的核心，而微管切断蛋白katanin已成为这一任务的关键蛋白质，其在神经元发育、减数分裂和有丝分裂纺锤体的组装和功能、细胞迁移、纤毛组装和拆解以及细胞形态发生中的重要作用证明了这一点。然而，我们对katanin活性的位置、时间和结果的控制机制的了解非常有限。为了解决这一缺陷，我们提出了以下三个具体目标：(1)将katanin靶向特定微管位置的机制是什么？我们假设，p80调节亚基通过优先结合相交的微管几何形状来靶向katanin的p60催化亚基，而脱靶的p60 katanin结合被微管相关蛋白抑制。为了验证这一假设，我们将使用体外重建、冷冻-EM和细胞研究相结合的方法：(I)确定将p60 katanin招募到微管交叉位点的充要条件；(Ii)阐明p60 katanin在微管切断过程中的结构和构象；(Iii)确定MAP65蛋白如何保护微管免受katanin活性的影响。(2)p60 katanin切断活性的调节机制是什么？我们假设，p60 katanin活性受到多种调控机制的严格控制，以根据细胞环境提供细微差别的控制。为了验证这一假说，我们将关注两个新发现的调节蛋白，RIC1和SPR2，它们被认为分别控制p60活性的速度和位置。我们将使用体外重建和活体成像来研究RIC1如何促进p60 katanin的切割活性，以及SPR2如何调节p60 katanin对微管交叉部位的访问。(3)切断的微管末端的动力学调节机制是什么？我们假设，微管加端跟踪蛋白通过调节新的加端的动态，在决定微管切断的结果中发挥主要作用。为了验证这一假设，我们将重点研究一种名为SPR1的+TIP，初步数据表明，它是切断微管的“拯救因子”。我们将使用动态微管的体外重建来确定SPR1是否以及如何促进因切断而产生的微管和末端的挽救；并使用活细胞实验来测试通过切断的微管放大是否驱动光诱导的阵列重定向，以及细胞是否通过蛋白分解调节SPR1的水平来调节切断的微管的挽救程度。

 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.