Mechanism of inverted formin 2 (INF2)-mediated effects on ER and mitochondria

线粒体具有高度复杂的形态特征。为了维持线粒体功能，控制过程如融合和分裂是必不可少的。一个关键的组成部分是GTADRP 1，它围绕并限制了GTADRP 1。已知分裂优先发生在ER-线粒体接触位点，但ER刺激线粒体分裂的机制尚不清楚。最近的研究结果表明，ER结合的肌动蛋白组装因子，INF 2，是重要的线粒体分裂。INF 2聚合的肌动蛋白丝以两种方式刺激线粒体分裂：1）通过增加Drp 1募集到线粒体分裂位点;和2）通过刺激ER到线粒体钙转移，增加的线粒体钙引起线粒体内膜的收缩。我的项目解决了这个发展中的机械模型中的关键未回答的问题。首先，什么样的肌动蛋白纤维是线粒体分裂的关键？INF 2导致至少三类聚合的肌动蛋白的组装：在分裂位点本身的细丝，沿着沿着肌动蛋白的细丝，以及大量胞质溶胶中的细丝。我将使用荧光标记的CRISPR敲入模型来开发特异性探针，用于通过活细胞显微镜定位INF 2和肌动蛋白群体。第二，在线粒体分裂的背景下，INF 2的ER定位的确切作用是什么？为了解决这个问题，我将采用两种方法：特异性消除ER相关的CAAX亚型和人工靶向INF 2到其他细胞器。第三，肌球蛋白II在线粒体分裂中起什么作用？以前的工作表明，肌球蛋白II是所需的外和内线粒体膜动力学在线粒体分裂，但其确切的关系与INF 2聚合肌动蛋白是未知的。此外，目前还不清楚三种非肌肉肌球蛋白II蛋白（肌球蛋白IIA，B或C）是重要的。我将开发敲除和荧光敲入模型来研究肌球蛋白II在线粒体分裂中的作用和定位。第四，其他肌动蛋白结合蛋白如何影响线粒体动力学？ 来自其他实验室的证据表明，corprin，cofilin和Arp 2/3复合物在组装与细胞相关的肌动蛋白中发挥作用，并且这些因素可能有助于裂变。我将使用敲除线和动态定位活细胞显微镜检查，以测试这些蛋白质在INF 2介导的肌动蛋白组装和线粒体分裂的相关性。

Mitochondria possess a characteristic morphology of remarkably high complexity. For maintenance of mitochondrial function, control processes such as fusion and fission are essential. One key component is the GTPase Drp1, which oligomerizes around and constricts the mitochondrion. Fission is known to preferentially occur at ER-mitochondrial contact sites, but the mechanism by which the ER stimulates mitochondrial fission is unclear. Recent results show that an ER-bound actin assembly factor, INF2, is important for mitochondrial fission. INF2-polymerized actin filaments stimulate mitochondrial fission in two ways: 1) by increasing Drp1 recruitment to mitochondrial fission sites; and 2) by stimulating ER-to-mitochondrial calcium transfer, with the increased mitochondrial calcium causing contractions of the inner mitochondrial membrane. My project addresses key unanswered questions in this developing mechanistic model. First, what population(s) of actin filaments are key to mitochondrial fission? INF2 causes assembly of at least three classes of polymerized actin: filaments at the fission site itself, filaments that run along the mitochondrion, and filaments in the bulk cytosol. I will use fluorescently-tagged CRISPR knock-in models to develop specific probes for localizing INF2 and actin populations by live-cell microscopy. Second, what is the precise role of INF2´s ER-localization in the context of mitochondrial fission? To address this question, I will follow two approaches: specific elimination of the ER-associated CAAX isoform and artificial targeting of INF2 to other organelles. Third, what role does myosin II play in mitochondrial fission? Previous work has shown that myosin II is required for both outer and inner mitochondrial membrane dynamics in mitochondrial fission, but its precise relationship with INF2-polymerized actin is unknown. Furthermore, it is unclear which of the three non-muscle myosin II proteins (myosin IIA, B or C) is/are important. I will develop knock-out and fluorescent knock-in models to examine myosin II roles and localization during mitochondrial fission. Fourth, how do other actin binding proteins contribute to mitochondrial dynamics? Evidence from other laboratories has suggested that cortactin, cofilin, and Arp2/3 complex play roles in assembling mitochondrially-associated actin, and that these factors might contribute to fission. I will use knock-out lines and dynamic localization by live-cell microscopy to test the relevance of these proteins in INF2-mediated actin assembly and mitochondrial fission.