ESCRT and MIT Complexes in Cytokinesis

细胞分裂中的 ESCRT 和 MIT 复合物

基本信息

批准号：
10442697
负责人：
CHRISTOPHER P. HILL
金额：
$ 35.08万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-07-01 至 2023-07-18
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10442697
关键词：
ATP phosphohydrolase Alleles Binding Binding Proteins Binding Sites Biochemical Biological Biopolymers C-terminal Cell Proliferation Cell division Cells Cellular biology Chimera organism Chromosomes Complement Complex Cryoelectron Microscopy Cytokinesis DNA Damage Dipeptides Ensure Enzymes Event Excision Exhibits Filament Foundations Funding Genomic Instability Goals Human Learning Ligands Link Mammalian Cell Meiosis Membrane Microtubules Mitosis Mitotic Modeling Molecular Molecular Conformation N-terminal Normal Cell Ovarian Pathogenicity Pathway interactions Peptide Hydrolases Phosphorylation Phosphotransferases Play Polymers Positioning Attribute Process Protein Family Proteins Publishing Regulation Residual state Resolution Role Series Sorting - Cell Movement Structure Substrate Specificity Surveys Tail Tertiary Protein Structure Testing Work cancer cell cofactor daughter cell design enzyme activity enzyme substrate experimental study falls katanin novel therapeutic intervention polypeptide premature prevent programs protein function reconstitution recruit response spastin three dimensional structure

项目摘要

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.

抽象的当细胞退出有丝分裂时，它们会在脱落检查点停顿，以确保有丝分裂程序已完成然后，他们通过脱落，不可逆转地将两个子细胞分开。内体分类运输（ESCRT）机械所需的复合物在这两种重要的细胞力学中都起着重要作用过程。某些ESCRT组件受脱落检查点负调节，以防止过早脱落。一旦满足检查站，ESCRT机械就会在中体中组装以收缩膜并进行脱落。该提案中的项目旨在发现关键步骤的分子机制在这些过程中。具体而言，我们将：表征ALIX的调节，ALIX是核素组装的关键ESCRT因子在中体内收缩的ESCRT-III丝（AIM 1），确定ESCRT-III丝如何募集MIT域 - 将蛋白质包含到中体，并定义这些蛋白质在细胞因子中的作用（AIM 2），并表征 9个相关“减数分裂进化枝” AAA ATPases的结构和功能，共同促进脱离重塑中体微管和ESCRT-III丝。 Subaim 1.1的结构研究将针对不同沿ALIX激活途径的构象状态，目的是学习有丝分裂磷酸化如何激活 ALIX参与脱落。这些研究将基于我们以前对ALIX的生化和结构分析核心结构域，无论是与ESCRT-III配体的复杂域。 Subaim 1.2中的细胞生物学研究将定义检查点激活将ALIX募集延迟到中体，并测试ALIX固存是否抑制了脱落。初步研究已经确定了ALIX募集的延迟，并表明检查点激活使ALIX达到将浓缩物浓缩在细胞质灶中，以及脱落和脱落检查点所需的其他因素。在 Subaim 2.1，我们将在结构上识别并结构表征25种不同人类MIT域蛋白的复合物它们的结合位点在12种不同的ESCRT-III蛋白的尾部。迄今为止，这些研究揭示了20多个新的相互作用并产生了六个结构的ESCRT-III-MIT复合物。 Subaim 2.2中的互补研究将确定人类MIT蛋白需要不同阶段的细胞因子蛋白并表征其功能。这些方法已经确定了三种在脱落检查点中具有重要作用的新的MIT蛋白。结构研究 Subaim 3.1将靶向与相关底物复合物中的aaA atpases的微管。这些研究会补充我们最近与ESCRT-III复合物中相关VPS4 AAA ATPase的高分辨率冷冻结构基材。最后，Subaim 3.2中的生化研究将研究这些相关酶如何区分 ESCRT-III丝和微管底物，并测试我们的假设，即这些ATPases利用了共同的机制易位和展开多肽底物。这些研究将以我们的重组和高分辨率为基础由ESCRT-III蛋白CHMP1B和IST1形成的螺旋双链细丝的冷冻结构，均为自由和与机制复杂。综上所述，我们的研究将定义一系列的分子机制细胞因子基本细胞生物学的中心事件。