Regulation of cytokinesis by Class I Rab11-Family Interacting Proteins

有丝分裂后，一个多步骤的过程称为胞质分裂物理分离的两个子细胞的细胞质。它从解理面的规格开始。然后，收缩性肌动球蛋白环在皮层聚集，并促进质膜沟的进入，直到形成一个非常紧凑的结构，称为中间体。最后，中间体被分解，一种促进分裂的专门机制被招募，诱导子细胞的物理分离。在以前的工作中，我们已经涉及两个蛋白质在果蝇S2细胞胞质分裂的调节：Rip 11，唯一的I类Rab 11家族相互作用蛋白（FIP）在苍蝇，和肌醇多磷酸5-磷酸酶OCRL。我们和其他人表明，果蝇OCRL及其哺乳动物直向同源物对于有丝分裂期间控制磷脂酰肌醇-4，5-二磷酸（PI（4，5）P2）的水平至关重要。FIP是将Rab蛋白（其是调节囊泡转运的小GTP酶）连接到效应子（如马达）的支架蛋白。FIPs最初被鉴定为Rab 11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Rip 11是胞质分裂所必需的，与Rab 11的结合无关。在后续工作中，我们发现Rip 11缺失在S2细胞分裂过程中影响Rab 35和OCRL。Rab 35通过定位OCRL和MICAL 1清除肌动蛋白的中间体，允许胞质分裂，在胞质分裂中起关键作用。此外，我们发现Rip 11的直系同源物之一Rab 11-FIP 1（FIP 1）的缺失延迟了U2 OS细胞的胞质分裂，类似于OCRL或Rab 35的缺失。总体而言，我们的初步结果表明Rip 11和FIP 1调节Rab 35的活性。我们的首要目标是描述Rip 11和FIP 1在胞质分裂过程中的精确分子功能。为了更深入地了解Rip 11和FIP 1的分子功能和调控，我们将结合联合收割机候选人和蛋白质组学方法。在第一个目标中，我们将首先确定哪些Rab和马达与Rip 11相互作用以促进S2细胞中的胞质分裂，并且我们将测试它们是否直接参与S2细胞胞质分裂。在第二个目标中，我们将首先更详细地描述FIP 1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细胞质分裂过程中FIP 1的潜在相关伙伴。最后，我们将使用生物化学和成像方法来验证这些候选人。我们还将通过耗尽U2 OS细胞中的候选物并通过监测细胞分裂和胞质分裂决定子的分布来确定它们是否是胞质分裂所需的。*我们的项目将促进我们的理解机制，调节蛋白质的时空分布调节胞质分裂。此外，它将提供在果蝇和人类细胞中的I类FIPs的详细表征。

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.