Regulation of cytokinesis by Class I Rab11-Family Interacting Proteins

有丝分裂后，一个称为细胞质分裂的多步骤过程物理地分离了两个子细胞的细胞质。它从解理面的规范开始。一个可收缩的肌动球蛋白环随后在皮层聚集，并促进质膜沟的侵入，直到形成一个非常致密的结构，称为中间体。最后，中间体被分解，一个促进分离的专门机制被招募，诱导子细胞的物理分离。***在之前的工作中，我们涉及了两个蛋白在果蝇S2细胞中调控细胞分裂：Rip11，果蝇中唯一的I类Rab11-Family相互作用蛋白（FIP），以及肌醇多磷酸5-磷酸酶ocl。我们和其他研究人员发现，果蝇ocl及其哺乳动物同源基因在有丝分裂过程中对控制磷脂酰肌醇-4,5-二磷酸（PI(4,5)P2）水平至关重要。***FIPs是连接Rab蛋白的支架蛋白，Rab蛋白是调节囊泡运输的小gtp酶，与马达等效应器相连。FIPs最初被鉴定为Rab11结合蛋白，但它们可以与不同的Rab11家族成员相互作用。我们发现Rip11是细胞分裂所必需的，独立于它与Rab11的结合。***在后续工作中，我们发现Rip11缺失对S2细胞分裂过程中的Rab35和ocl都有影响。Rab35通过定位ocl和MICAL1清除中间的肌动蛋白，使其脱落，在细胞分裂中起关键作用。此外，我们发现Rip11的同源物之一Rab11-FIP1 （FIP1）的缺失会延迟U2OS细胞的细胞分裂，类似于ocl或Rab35的缺失。总的来说，我们的初步结果表明Rip11和FIP1调节Rab35的活性。我们的首要目标是表征Rip11和FIP1在细胞分裂过程中的精确分子功能。***为了更深入地了解Rip11和FIP1的分子功能和调控，我们将结合基于候选物和蛋白质组学的方法。在第一个目标中，我们将首先确定哪些Rab(s)和motor(s)与Rip11相互作用以促进S2细胞的细胞分裂，我们将测试它们是否直接参与S2细胞的细胞分裂。在第二个目标中，我们将首先更详细地描述FIP1缺失对细胞分裂的影响。然后，我们将同步有丝分裂进程，并使用邻近生物素化和蛋白质组学来确定FIP1在细胞分裂过程中的潜在相关伙伴。最后，我们将通过生化和成像方法验证这些候选物。我们还将通过消耗U2OS细胞中的候选物质，并通过监测细胞分裂和细胞质分裂决定因素的分布来确定它们是否需要细胞质分裂。***我们的项目将促进我们对调节细胞分裂的蛋白质时空分布的机制的理解。此外，它将提供果蝇和人类细胞中I类FIPs的详细特征。

After mitosis, a multistep process called cytokinesis physically separates the cytoplasm of the two daughter cells. It begins with the specification of the cleavage plane. A contractile actomyosin ring then assembles at the cortex and promotes the ingression of the plasma membrane furrow until a very compact structure called the midbody is formed. Finally, the midbody is resolved and a specialized machinery that promotes abscission is recruited, inducing the physical separation of the daughter cells.***In previous work, we have involved two proteins in the regulation of cytokinesis in Drosophila S2 cells: Rip11, the sole Class I Rab11-Family Interacting proteins (FIP) in flies, and the Inositol polyphosphate 5-phosphatase OCRL. We and others showed that Drosophila OCRL and its mammalian orthologue are critical for controlling the level of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) during mitosis.***FIPs are scaffold proteins that link Rab proteins, which are small GTPases that regulate vesicular transport, to effectors such as motors. FIPs were initially identified as Rab11 binding proteins, but they can interact with different Rab family members. We showed that Rip11 is required for cytokinesis independently of its binding to Rab11.***In follow-up work, we found that Rip11 depletion affects both Rab35 and OCRL during the division of S2 cells. Rab35 is critical role for cytokinesis by localizing OCRL and MICAL1 to clear the midbody of actin, allowing abscission. Furthermore, we found that the depletion of Rab11-FIP1 (FIP1), one of the orthologue of Rip11, delays the cytokinesis of U2OS cells similarly to the loss of OCRL or Rab35. Overall, our preliminary results suggest that Rip11 and FIP1 regulate the activity of Rab35. Our overarching goal is to characterize the precise molecular function of Rip11 and FIP1 during cytokinesis.***To gain more insights into the molecular function and the regulation of Rip11 and FIP1, we will combine candidate-based and proteomic approaches. In the first aim, we will first determine which Rab(s) and motor(s) interact with Rip11 to promote cytokinesis in S2 cells and we will test if they are directly involved in S2 cells cytokinesis. In the second aim, we will first characterize the effect of FIP1 depletion on cytokinesis in greater details. Then, we will synchronize mitotic progression and use proximity biotinylation and proteomics to identify potential relevant partners of FIP1 during cytokinesis. Finally, we will validate these candidate by using biochemical and imaging methods. We will also determine if they are required for cytokinesis by depleting the candidates in U2OS cells and by monitoring cell division and the distribution of determinant of cytokinesis.***Our project will advance our understanding of the machinery that regulates the spatiotemporal distribution of proteins regulating cytokinesis. Furthermore, it will provide a detailed characterization of class I FIPs both in Drosophila and in human cells.