ESCRT-III and MIT Protein Complexes in Cytokinesis

描述（由申请人提供）：运输所需的内体分选复合物（ESCRT）在细胞动力学脱落中起着保守而重要的作用，细胞动力学脱落是细胞分裂的最后一步，物理分离子细胞，在癌症发展过程中可能出错。ESCRT-III蛋白同源家族在这一过程中发挥关键作用，形成丝，收缩细胞间桥并促进脱落。ESCRT-III细丝也招募含有MIT结构域的蛋白质，我们假设这些蛋白质为细胞分裂提供了许多关键的，但在很大程度上未被表征的活性。为了从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、相互作用和细胞分裂功能。我们已经确定了ESCRT-III蛋白IST1在其封闭构象中的晶体结构，并获得了IST1与其ESCRT-III结合伙伴CHMP1B形成的螺旋共聚物的高分辨率低温电镜结构。这是第一次这样的重建，它揭示了一个显着的组装，其中两个ESCRT-III亚基采用完全不同的结构。双链螺旋丝包括封闭的IST1亚基的外链和高度延伸的CHMP1B亚基的内链。我们现在将测试一系列基于结构的ESCRT-III细丝形成和功能的机制假设，重点关注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细丝，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。在细胞分裂过程中，MIT atp酶VPS4和Spastin通过结合ESCRT-III亚基暴露的c端尾部的MIT相互作用基序（MIM）定位于细胞间桥。不太明确的MIT蛋白具有多种效应域，包括激酶、蛋白酶和去泛素酶，我们认为MIM-MIT相互作用的复杂网络将这些活性招募到细胞间桥梁中，在细胞质分裂中发挥作用。为了支持这个观点，我们有

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.