Comparative molecular physiology of mammalian formins

描述（由申请人提供）：虽然很明显细胞骨架元件（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，但这些作用发生的机制尚不确定。在上一个资助期间，我的实验室揭示了哺乳动物的蛋白质INF 2令人兴奋的细胞功能。重要的是，INF 2作为两个C-末端剪接变体存在，它们基本上作为不同的蛋白质起作用。一种剪接变体INF 2-CAAX被异戊烯化并与内质网（ER）紧密结合。我们发现INF 2-CAAX在线粒体分裂中起作用。INF 2-CAAX在ER/线粒体界面聚合肌动蛋白丝，并且INF 2介导的肌动蛋白聚合刺激动力蛋白样GTd 1 Drp 1向线粒体的募集，导致分裂。第二个INF 2剪接变体INF 2- nonCAAX不被异戊烯化，并且在细胞质中以肌动蛋白依赖性网状模式存在。INF 2-nonCAAX在高尔基体区域动态肌动蛋白“补丁”的生成中起作用，我们的研究结果表明，这些补丁在高尔基体和其他细胞器的分裂中起作用。总之，我们的研究结果表明，INF 2是一种新的肌动蛋白为基础的膜分裂因子。在此期间，我们研究了INF 2对肌动蛋白的影响如何转化为线粒体分裂。目的1阐明INF 2影响肌动蛋白动力学的生化机制。INF 2是一种不寻常的生物化学物质。虽然大多数formin加速肌动蛋白聚合，但INF 2加速聚合和解聚。INF 2具有强的切断纤维丝的活性。在这个目标中，我们阐明INF 2的切断和解聚机制，使用TIRF显微镜，原子力显微镜和生化技术。我们还使用诱变来确定用于切断/解聚的重要序列，并将这些突变体用于目的2和3中的实验。目的2解决了INF 2使膜变形的机制。基于我们的初步结果显示线粒体分裂需要肌球蛋白活性，我们假设INF 2产生的细丝是肌球蛋白介导的收缩的轨迹。在这个目标中，我们使用固定细胞和活细胞显微镜来测试这个模型。目标3涉及INF 2监管。虽然其他formin受自抑制调节，但我们的初步数据表明，INF 2抑制需要额外的分子。此外，我们有证据表明Drp 1可能是INF 2-CAAX的抑制因子。我们使用纯化的蛋白质和一种新的无细胞测定系统来阐明Drp 1对INF 2和肌动蛋白的影响，以及鉴定INF 2-nonCAAX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.