ESCRT and MIT Complexes in Cytokinesis

摘要 当细胞退出有丝分裂时，它们在分裂检查点暂停，以确保有丝分裂程序已经完成 成功地然后，它们通过分裂进行，不可逆地分离两个子细胞。内体分选 转运所需复合物（ESCRT）机制在这两个重要的细胞动力学过程中起着至关重要的作用。 流程.某些ESCRT组分由释放检查点负调控，以防止过早的 放弃。一旦检查点得到满足，ESCRT机器就会在中间体组装，以收缩膜 并执行任务。本提案中的项目旨在揭示关键步骤背后的分子机制 在这些过程中。具体来说，我们将：表征阿利克斯的调节，ALIX是一个关键的ESCRT因子， 的收缩ESCRT-III丝在中间体（AIM 1），确定ESCRT-III丝如何招募MIT结构域- 含有蛋白质的中间体，并定义这些蛋白质在胞质分裂（AIM 2）中的功能，并表征 9个相关的“减数分裂进化枝”AAA ATP酶的结构和功能，它们通过以下方式共同促进减数分裂： 重塑中间体微管和ESCRT-III丝。Subaim 1.1中的结构研究将针对不同的 沿着阿利克斯活化途径的沿着构象状态，目的是了解有丝分裂磷酸化如何活化 阿利克斯参加了一个任务。这些研究将建立在我们以前对阿利克斯的生化和结构分析的基础上 核心结构域，包括游离的和与ESCRT-III配体复合的。Subaim 1.2中的细胞生物学研究将定义 检查点激活延迟阿利克斯向中间体的募集，并测试阿利克斯隔离是否抑制释放。 初步研究已经确定了阿利克斯募集的延迟，并表明检查点激活导致阿利克斯 在细胞质病灶内集中，与其他因子一起需要的脱落和脱落检查点。在 在Subaim 2.1中，我们将鉴定和结构表征25种不同的人MIT结构域蛋白与 它们在12种不同的ESCRT-III蛋白的尾部上的结合位点。迄今为止，这些研究已经揭示了20多个 新的相互作用，并产生了六种结构的ESCRT-III-MIT复合物。Subaim 2.2中的补充研究将 鉴定胞质分裂不同阶段所需的人MIT蛋白并表征其功能。这些方法 已经确定了三种在脱落检查点中发挥重要作用的新MIT蛋白质。结构研究 Subaim 3.1将靶向与相关底物复合的微管切断AAA ATP酶。这些研究将 补充我们最近的高分辨率cryoEM结构的相关Vps 4 AAA ATP酶与ESCRT-III复合 衬底最后，Subaim 3.2中的生物化学研究将研究这些相关酶如何区分 ESCRT-III细丝和微管底物，并测试我们关于这些ATP酶利用共同机制的假设 以转位和展开多肽底物。这些研究将建立在我们的重建和高分辨率的基础上 由ESCRT-III蛋白CHMP 1B和IST 1形成的螺旋双链丝的cryoEM结构， 与细胞膜复合。总之，我们的研究将确定一系列的分子机制， 胞质分裂的基本细胞生物学的中心事件。

ABSTRACT As cells exit mitosis, they pause at the abscission checkpoint to ensure that the mitotic program has been completed successfully. They then proceed through abscission, irreversibly separating the two daughter cells. The Endosomal Sorting Complexes Required for Transport (ESCRT) machinery plays essential roles in both of these important cytokinetic processes. Certain ESCRT components are negatively regulated by the abscission checkpoint to prevent premature abscission. Once the checkpoint is satisfied, the ESCRT machinery then assembles in the midbody to constrict the membrane and carry out abscission. Projects in this proposal are designed to uncover the molecular mechanisms that underlie key steps in these processes. Specifically, we will: characterize the regulation of ALIX, a key ESCRT factor that nucleates assembly of constricting ESCRT-III filaments within the midbody (AIM 1), determine how ESCRT-III filaments recruit MIT domain- containing proteins to the midbody and define how these proteins function in cytokinesis (AIM 2), and characterize the structures and functions of the 9 related “meiotic clade” AAA ATPases that work together to promote abscission by remodeling midbody microtubules and ESCRT-III filaments. Structural studies in Subaim 1.1 will target the different conformational states along the ALIX activation pathway, with the goal of learning how mitotic phosphorylation activates ALIX to participate in abscission. These studies will build on our previous biochemical and structural analyses of the ALIX core domains, both free and in complex with ESCRT-III ligands. Cell biological studies in Subaim 1.2 will define how checkpoint activation delays ALIX recruitment to the midbody and test whether ALIX sequestration inhibits abscission. Preliminary studies have established the delay in ALIX recruitment and shown that checkpoint activation causes ALIX to concentrate within cytoplasmic foci, together with other factors required for abscission and the abscission checkpoint. In Subaim 2.1, we will identify and structurally characterize complexes of the 25 different human MIT domain proteins with their binding sites on the tails of the 12 different ESCRT-III proteins. To date, these studies have revealed more than 20 new interactions and produced six structures of ESCRT-III-MIT complexes. Complementary studies in Subaim 2.2 will identify human MIT proteins required for different stages of cytokinesis and characterize their functions. These approaches have already identified three new MIT proteins with important roles in the abscission checkpoint. Structural studies in Subaim 3.1 will target microtubule severing AAA ATPases in complex with relevant substrates. These studies will complement our recent high resolution cryoEM structure of the related Vps4 AAA ATPase in complex with an ESCRT-III substrate. Finally, biochemical studies in Subaim 3.2 will examine how these related enzymes discriminate between ESCRT-III filaments and microtubule substrates, and test our hypothesis that these ATPases utilize a common mechanism to translocate and unfold polypeptide substrates. These studies will build upon our reconstitution and high resolution cryoEM structure of helical double stranded filaments formed by the ESCRT-III proteins CHMP1B and IST1, both free and in complex with membranes. Taken together, our studies will define the molecular mechanisms that underlie a series of central events in the fundamental cell biology of cytokinesis.