Regulation of cytokinesis by Class I Rab11-Family Interacting Proteins

有丝分裂后，一个称为胞质分裂的多步骤过程在物理上将两个子细胞的细胞质分开。它从解理平面的规范开始。然后，一个可收缩的肌球蛋白环在皮质聚集，促进质膜皱纹的内向，直到形成一个非常紧密的结构，称为中体。最后，中体被分解，并招募了一种专门的机制来促进脱落，诱导子细胞的物理分离。*在之前的工作中，我们涉及了两种蛋白质对果蝇S2细胞胞质分裂的调节：Rip11，果蝇中唯一的I类Rab11家族相互作用蛋白(FIP)和肌醇多聚磷酸5-磷酸酶OCRL。我们和其他人发现，果蝇OCRL及其哺乳动物同源物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。*FIP是将Rab蛋白连接到效应器(如马达)的支架蛋白。Rab蛋白是调节囊泡运输的小GTP酶。FIPS最初被鉴定为Rab11结合蛋白，但它们可以与不同的Rab家族成员相互作用。我们发现Rip11是胞质分裂所必需的，而不是与Rab11结合。*在后续工作中，我们发现Rip11缺失在S2细胞分裂过程中同时影响Rab35和OCRL。Rab35是胞质分裂的关键角色，通过定位OCRL和MICAL1来清除中体的肌动蛋白，允许脱落。此外，我们发现Rip11的同源物之一Rab11-FIP1(FIP1)的缺失类似于OCRL或Rab35的缺失，延迟了U2OS细胞的胞质分裂。总体而言，我们的初步结果表明Rip11和FIP1调节Rab35的活性。我们的首要目标是确定Rip11和FIP1在细胞质分裂过程中的确切分子功能。*为了更深入地了解Rip11和FIP1的分子功能和调控，我们将结合候选和蛋白质组方法。在第一个目标中，我们将首先确定哪些Rab(S)和Motor(S)与Rip11相互作用促进S2细胞的胞质分裂，我们将测试它们是否直接参与S2细胞的胞质分裂。在第二个目标中，我们将首先更详细地描述FIP1缺失对胞质分裂的影响。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在胞质分裂过程中的潜在相关伙伴。最后，我们将使用生化和成像方法来验证这些候选基因。我们还将通过耗尽U2OS细胞中的候选细胞并通过监测细胞分裂和胞质分裂决定因素的分布来确定它们是否是胞质分裂所必需的。*我们的项目将促进我们对调节胞质分裂蛋白质时空分布的机制的理解。此外，它还将提供果蝇和人类细胞中I类FIP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.