ESCRT and MIT Complexes in Cytokinesis

摘要 当细胞退出有丝分裂时，它们会在脱落检查点暂停，以确保有丝分裂程序已经完成 成功了。然后，它们通过脱落进行，不可逆转地分离两个子细胞。内窥体分选 运输所需的复合体(ESCRT)机制在这两个重要的细胞动力学中都扮演着重要的角色 流程。某些ESCRT组件受到分离检查点的负面调节，以防止早产 离位。一旦检查点被满足，ESCRT机器就会在中体内组装以压缩膜 并进行剥离。该提案中的项目旨在揭示构成关键步骤的分子机制。 在这些过程中。具体地说，我们将：表征Alix的调节，这是一个关键的ESCRT因子，它是组装的核心 在中体内收缩ESCRT-III细丝(AIM 1)，确定ESCRT-III细丝如何招募MIT结构域- 包含蛋白质到中体，并定义这些蛋白质如何在细胞质分裂中发挥作用(AIM 2)，并表征 9个相关的“减数分裂分支”AAA-ATPase的结构和功能 重建中体微管和ESCRT-III细丝。Subaim 1.1中的结构研究将针对不同的 Alix激活途径的构象状态，目的是了解有丝分裂磷酸化是如何激活的 别力克斯参与离任。这些研究将建立在我们之前对Alix的生化和结构分析的基础上 核心区，既有游离的，也有与ESCRT-III配体形成复合体的。Subaim 1.2中的细胞生物学研究将如何定义 检查点激活延迟Alix向中体的募集，并测试Alix隔离是否抑制脱落。 初步研究已经确定Alix招募的延迟，并表明检查点激活导致Alix 集中在细胞质内，以及脱落所需的其他因素和脱落检查点。在……里面 Subaim 2.1，我们将鉴定和结构表征25个不同的人类MIT结构域蛋白与 它们在12种不同ESCRT-III蛋白尾部的结合部位。到目前为止，这些研究揭示了20多个 新的相互作用，并产生了ESCRT-III-MIT络合物的六种结构。Subaim 2.2中的补充研究将 确定细胞质分裂的不同阶段所需的人类MIT蛋白，并表征其功能。这些方法 已经确定了三个在脱落检查点具有重要作用的新的MIT蛋白。中国的结构研究 Subaim 3.1将在与相关底物的复合体中靶向切割AAA ATPase的微管。这些研究将 用ESCRT-III补充我们最新的相关Vps4 AAA ATPase的高分辨低温EM结构 底物。最后，Subaim 3.2中的生化研究将检查这些相关酶如何区分 ESCRT-III细丝和微管底物，并检验我们的假设，即这些ATPase利用一个共同的机制 移位和展开多肽底物。这些研究将建立在我们的重组和高分辨率的基础上 ESCRT-III蛋白CHMP1B和IST1形成的螺旋双链微丝的低温电子显微镜结构 与膜形成复合体。综上所述，我们的研究将定义一系列 胞质分裂基础细胞生物学中的中心事件。

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.