Comparative molecular physiology of mammalian formins

描述(由申请人提供)：尽管细胞骨架元素(肌动蛋白和微管)在细胞内细胞器的动力学和完整性中发挥作用是显而易见的，但这些作用发生的机制尚不确定。在上一次资助期间，我的实验室揭示了哺乳动物Forin蛋白INF2令人兴奋的细胞功能。重要的是，INF2以两个C末端剪接变体的形式存在，本质上是作为不同的蛋白质。一种剪接变异体INF2-CAAX是异丙基化的，与内质网(ER)紧密结合。我们证明了INF2-CAAX在线粒体分裂中的作用。INF2-CAAX在内质网/线粒体界面聚合肌动蛋白细丝，INF2介导的肌动蛋白聚合刺激动力蛋白样GTP酶Drp1重新聚集到线粒体，导致分裂。第二个INF2剪接变异体INF2-non CAAX不是异丙基化的，在细胞质中以肌动蛋白依赖的网状图案存在。INF2-non CAAX在高尔基体区动态肌动蛋白“斑块”的产生中起作用，我们的结果表明这些斑块在高尔基体和其他细胞器的分裂中起作用。总之，我们的结果表明INF2是一种基于肌动蛋白的新的膜分裂因子。在这个授权期，我们研究了INF2的S对肌动蛋白的影响是如何转化为线粒体分裂的。目的1探讨INF2影响肌动蛋白动力学的生化机制。INF2在生物化学上是一种不寻常的福尔明。虽然大多数福尔马林促进肌动蛋白聚合，但INF2同时加速聚合和解聚。对于解聚细丝，InF2具有很强的断丝活性。为此，我们利用TIRF显微镜、原子力显微镜和生化技术对InF_2的S切割和解聚机理进行了研究。我们还使用突变来确定切断/解聚的重要序列，并将使用这些突变体进行AIMS 2和AIMS 3的实验。AIM 2解决了INF2使膜变形的机制。根据我们的初步结果表明线粒体的分裂需要肌球蛋白的活性，我们假设INF2产生的细丝是肌球蛋白介导的收缩的轨迹。为了达到这个目的，我们使用固定细胞显微镜和活细胞显微镜来测试这个模型。目标3涉及INF2调节。虽然其他的福尔马林是通过自身抑制来调节的，但我们的初步数据表明，INF2的抑制需要额外的分子。此外，我们有证据表明，Drp1可能是INF2-CAAX的抑制因子。我们使用纯化的蛋白质和一种新的无细胞检测系统来阐明Drp1的S对INF2和肌动蛋白的影响，以及寻找INF2-non CAAX的抑制剂。

DESCRIPTION (provided by applicant): While it is clear that cytoskeletal elements (actin and microtubules) play roles in the dynamics and integrity of intracellular organelles, the mechanisms by which these actions occur are uncertain. In the last grant period, my laboratory revealed exciting cellular functions for the mammalian formin protein INF2. Importantly, INF2 exists as two C-terminal splice variants that essentially act as distinct proteins. One splice variant, INF2-CAAX, is prenylated and tightly bound to endoplasmic reticulum (ER). We showed that INF2-CAAX functions in mitochondrial fission. INF2-CAAX polymerizes actin filaments at the ER/mitochondrial interface, and INF2-mediated actin polymerization stimulates recruitment of the dynamin-like GTPase Drp1 to mitochondria, leading to fission. A second INF2 splice variant, INF2- nonCAAX, is not prenylated and is found in an actin-dependent meshwork-like pattern in the cytoplasm. INF2-nonCAAX plays a role in generation of dynamic actin "patches" in the Golgi region, and our results suggest that these patches act in fission of Golgi and other organelles. Overall, our results suggest that INF2 is a novel actin-based membrane fission factor. In this grant period, we study how INF2's effects on actin translate into mitochondrial fission. Aim 1 addresses the biochemical mechanism by which INF2 influences actin dynamics. INF2 is an unusual formin biochemically. While most formins accelerate actin polymerization, INF2 accelerates both polymerization and depolymerization. To depolymerize filaments, INF2 possesses a potent filament severing activity. In this aim, we elucidate INF2's severing and depolymerization mechanism using TIRF microscopy, Atomic Force Microscopy and biochemical techniques. We also use mutagenesis to determine important sequences for severing/depolymerization, and will use these mutants for experiments in Aims 2 and 3. Aim 2 addresses the mechanism by which INF2 deforms membranes. Based on our preliminary results showing that mitochondrial fission requires myosin activity, we hypothesize that INF2-generated filaments are tracks for myosin-mediated contraction. In this aim, we use fixed-cell and live-cell microscopy to test this model. Aim 3 addresses INF2 regulation. While other formins are regulated by auto-inhibition, our preliminary data suggest that an additional molecule is required for INF2 inhibition. Furthermore, we have evidence that Drp1 might be an inhibitory factor for INF2-CAAX. We use purified proteins and a novel cell-free assay system to elucidate Drp1's effect on INF2 and on actin, as well as to identify inhibitors for INF2-nonCAAX.