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

倒形福明 2 (INF2) 介导的 ER 和线粒体影响机制

• 批准号: 418076373
• 负责人: Dr. Frieda Kage
• 金额: --
• 依托单位: Department of Biochemistry and Cell Biology
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/418076373?language=en
• 关键词: Mechanism; inverted; formin; INF2; mediated

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.

线粒体具有非常复杂的特征形态。为了维持线粒体功能，融合和裂变等控制过程至关重要。一个关键成分是 GTPase Drp1，它在线粒体周围寡聚并收缩。已知裂变优先发生在内质网-线粒体接触位点，但内质网刺激线粒体裂变的机制尚不清楚。最近的结果表明，内质网结合的肌动蛋白组装因子 INF2 对于线粒体裂变非常重要。 INF2 聚合肌动蛋白丝以两种方式刺激线粒体裂变：1）通过增加 Drp1 募集到线粒体裂变位点； 2) 通过刺激内质网到线粒体的钙转移，线粒体钙的增加引起线粒体内膜的收缩。我的项目解决了这个发展机制模型中尚未解答的关键问题。首先，哪些肌动蛋白丝群是线粒体裂变的关键？ INF2 引起至少三类聚合肌动蛋白的组装：裂变位点本身的细丝、沿着线粒体运行的细丝以及大量细胞质中的细丝。我将使用荧光标记的 CRISPR 敲入模型来开发通过活细胞显微镜定位 INF2 和肌动蛋白群体的特异性探针。其次，INF2 的 ER 定位在线粒体裂变中的确切作用是什么？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 同工型，以及人工将 INF2 靶向其他细胞器。第三，肌球蛋白II在线粒体裂变中起什么作用？先前的研究表明，肌球蛋白 II 是线粒体分裂中线粒体外膜和内膜动力学所必需的，但其与 INF2 聚合肌动蛋白的确切关系尚不清楚。此外，尚不清楚三种非肌肉肌球蛋白 II 蛋白（肌球蛋白 IIA、B 或 C）中哪一种是重要的。我将开发敲除和荧光敲入模型来检查肌球蛋白 II 在线粒体裂变过程中的作用和定位。第四，其他肌动蛋白结合蛋白如何促进线粒体动力学？ 其他实验室的证据表明，cortactin、cofilin 和 Arp2/3 复合物在组装线粒体相关肌动蛋白中发挥作用，并且这些因素可能有助于裂变。我将使用敲除系和活细胞显微镜动态定位来测试这些蛋白质在 INF2 介导的肌动蛋白组装和线粒体裂变中的相关性。