ESCRT-III and MIT Protein Complexes in Cytokinesis

描述（由申请人提供）：转运所需的内体分选复合物（ESCRT）在细胞动力学分裂中起着保守和重要的作用，细胞动力学分裂是细胞分裂的最后一步，它将子细胞物理分离，并可能在癌症发展过程中出错。ESCRT-III蛋白的同源家族通过形成似乎收缩细胞间桥并促进解离的细丝在此过程中起关键作用。ESCRT-III丝还招募包含MIT结构域的蛋白质，我们假设这些蛋白质提供了胞质分裂所需的大量关键但基本上未表征的活动。为了在分子细节上定义这些过程，我们将分析12种人类ESCRT-III和21种MIT蛋白的结构、相互作用和胞质分裂功能。 我们已经确定了晶体结构的ESCRT-III蛋白，IST 1，在其封闭的构象，并获得了高分辨率cryoEM结构的螺旋共聚物形成的IST 1和它的ESCRT-III结合伙伴，CHMP1B。这是第一次这样的重建，它揭示了一个显着的装配，其中两个ESCRT-III亚基采用完全不同的结构。双链螺旋丝包括闭合的IST 1亚基的外链和高度延伸的CHMP 1B亚基的内链。我们现在将测试一系列基于结构的ESCRT-III丝形成和功能的机制假设，重点是亚基构象灵活性的重要性。IST1也可以形成膜相关的双链螺旋丝，从脂质体中挤出膜小管，我们将在生化细节中描述这种膜重塑反应。 MIT ATP酶VPS4和Spastin在胞质分裂期间通过结合到聚合的ESCRT-III亚基的暴露的C-末端尾部内的MIT相互作用基序（MIM）而定位于细胞间桥。不太好的特点MIT蛋白具有不同的效应域，包括激酶，蛋白酶，和去泛素化酶，我们建议，一个复杂的网络MIM-MIT相互作用招募这些活动的细胞间桥在胞质分裂中发挥作用。为了支持这个想法，我们有 确定了三种完全不同的ESCRT-III-MIT复合物的溶液结构，并表明MIT激酶ULK3在胞质分裂中起作用，被募集到细胞间桥，并可以磷酸化ESCRT-III亚基。我们已经结晶了ULK3 MIT-IST 1相互作用复合物，现在将表征其结构和相关的生物化学，然后扩展我们的研究，包括所有人类MIT蛋白的结构和ESCRT-III相互作用的全球分析。 最后，我们将使用基于细胞的测定来验证和扩展我们的生化和结构研究，并筛选整套人类MIT蛋白质的胞质分裂功能。总之，这些研究将表征ESCRT-III蛋白的基本活性;即，形成细丝，募集MIT蛋白在胞质分裂中发挥作用，并促进膜分裂。我们的研究还将确定在胞质分裂中起作用的MIT蛋白的子集，阐明它们的结构生物学，并揭示它们是如何被招募的。

DESCRIPTION (provided by applicant): The endosomal sorting complexes required for transport (ESCRT) play a conserved and essential role in cytokinetic abscission, the final step of cell division that physically separates daughter cells and can go awry during cancer development. The homologous family of ESCRT-III proteins plays key roles in this process by forming filaments that appear to constrict the intercellular bridge and promote abscission. ESCRT-III filaments also recruit MIT domain-containing proteins that we hypothesize provide a host of critical, but largely uncharacterized activities required for cytokinesis. To define these processes in molecular detail, we will analyze the structures, interactions and cytokinesis functions of the 12 human ESCRT-III and 21 MIT proteins. We have determined the crystal structure of the ESCRT-III protein, IST1, in its closed conformation and obtained a high resolution cryoEM structure of a helical co-polymer formed by IST1 and its ESCRT-III binding partner, CHMP1B. This is the first such reconstruction, and it reveals a remarkable assembly in which the two ESCRT-III subunits adopt radically different structures. The double-stranded helical filament comprises an outer strand of closed IST1 subunits and an inner strand of highly extended CHMP1B subunits. We will now test a series of structure-based mechanistic hypotheses for ESCRT-III filament formation and function, focusing on the importance of subunit conformational flexibility. IST1 can also form membrane-associated, double-stranded helical filaments that extrude membrane tubules from liposomes, and we will characterize this membrane remodeling reaction in biochemical detail. The MIT ATPases VPS4 and Spastin localize to intercellular bridges during cytokinesis by binding to MIT interaction motifs (MIM) within the exposed C-terminal tails of polymerized ESCRT-III subunits. Less well characterized MIT proteins have diverse effector domains that include kinases, proteases, and deubiquitinases, and we propose that a complex network of MIM-MIT interactions recruits these activities to the intercellular bridge to function in cytokinesis. In support of this idea, we have determined solution structures of three quite distinct ESCRT-III-MIT complexes and shown that an MIT kinase, ULK3, functions in cytokinesis, is recruited to intercellular bridges, and can phosphorylate ESCRT-III subunits. We have crystallized the ULK3 MIT-IST1 interaction complex and will now characterize its structure and associated biochemistry and then expand our studies to include global analyses of the structures and ESCRT-III interactions of all human MIT proteins. Finally, we will use cell-based assays to validate and extend our biochemical and structural studies and to screen the entire set of human MIT proteins for cytokinesis functions. Together, these studies will characterize the fundamental activities of ESCRT-III proteins; i.e., forming filaments, recruiting MIT proteins to function in cytokinesis, and facilitating membrane fission. Our studies will also identify the subset of MIT proteins that function in cytokinesis, elucidate their structural biology, and reveal how they are recruited.