Molecular basis of ESCRT-mediated sealing of the nuclear envelope.

真核生物的基因组在核中被分割，其边界是一个被称为核膜(NE)的双膜结构。核膜将转录和翻译分开，保护基因组不受寄生核酸和其他环境危害的影响，调节基因活动，并因此防止DNA损伤和恶性转化。脊椎动物的分裂细胞经历了“开放有丝分裂”的过程，在这个过程中，NE开放到如此程度，以至于失去了核质的特性。在染色体分离过程中，NE膜及其许多蛋白质分散进入相连的内质网(ER)。当一个完整和封闭的NE重整时，这个过程在有丝分裂退出时是相反的。在每个细胞周期之后，在各种破坏核膜的机械应激源之后，细胞需要密封核膜，以保持完整的区隔。人类细胞如何在每次细胞分裂过程中打开和密封核膜的双膜，这是一个长期存在的问题，至今仍知之甚少。最近的研究表明，转运III所需的内体分选复合体(ESCRT-III)蛋白参与了细胞周期依赖性的核膜重组和修复。ESCRT-III蛋白及其膜重塑功能在古生物和所有真核生物中都是保守的。它们进一步推动多囊泡体的形成，包膜病毒的排出，以及细胞动学脱落。ESCRT-III蛋白CHMP7最近被认为在核膜重塑过程中发挥重要作用。我们最近报道，内核膜蛋白Lem2招募Chmp7作为NE缝隙关闭的起始步骤，然后招募其他ESCRT-III组分，如IST1/CHMP8和CHMP2A。尽管ESCRT-III蛋白在细胞周期中非常重要，但我们对ESCRT-III蛋白在细胞周期中的作用知之甚少。我的博士后研究旨在从结构上描述i)ESCRT-III机制是如何被招募到核膜上的；ii)ESCRT-III蛋白是如何组装在NE执行膜重塑反应的；以及iii)这一过程在哺乳动物细胞周期中是如何调控的。在这项工作中，我将使用生化重组、交联质谱复杂表征、低温电子显微镜结构确定和人类细胞全基因组遗传筛选，以了解LEM2、CHMP7和ESCRT-III机制在核膜部位的作用。

The eukaryotic genome is compartmentalized in the nucleus; whose boundary is a double-membrane structure called the nuclear envelope (NE). The nuclear envelope segregates transcription from translation, shields the genome from parasitic nucleic acids and other environmental hazards, regulates gene activity, and prevents DNA damage and malignant transformation as a consequence. Dividing cells of vertebrates go through the process of “open mitosis” in which the NE opens up to such a degree that nucleoplasmic identity is lost. NE membranes including many of their proteins disperse into the connected endoplasmic reticulum (ER) during chromosomes segregation. The process is reversed during mitotic exit when an intact and closed NE reforms. After each cell cycle—and following a variety of mechanical stressors that damage the nuclear envelope—cells need to seal the nuclear envelope to maintain intact compartmentalization. How human cells open and seal the double membrane of the nuclear envelope in the course of each cell division is a long-standing question that still remains poorly understood. Recent studies demonstrated that endosomal sorting complexes required for transport III (ESCRT-III) proteins are involved in the cell cycle-dependent reformation and repair of the nuclear envelope. ESCRT-III proteins and their membrane remodeling functions are conserved in archaea and all eukaryotes. They further drive the formation of multi-vesicular bodies, and egress of enveloped viruses, as well as cytokinetic abscission. The ESCRT-III protein CHMP7 has recently been implicated to play a major role in the process of nuclear envelope reformation. We have recently reported that the inner nuclear membrane protein Lem2 recruits Chmp7 as the initiating step in NE gap closure before other ESCRT-III components, like IST1/CHMP8 and CHMP2A are recruited. Despite their fundamental importance during the cell cycle, we understand very little about how ESCRTIII proteins function during stages.My postdoctoral studies aim to structurally characterize i) how the ESCRT-III machinery is recruited to the nuclear envelope; ii) how ESCRT-III proteins assemble to perform membrane remodeling reactions at the NE; and iii) how this process is regulated during the mammalian cell cycle. In course of this work I will use biochemical reconstitutions, complex characterization by cross-linking mass spectrometry, structure determination by cryo-electron microscopy and genome wide genetic screens in human cells to understand the role of LEM2, CHMP7 and the ESCRT-III machinery at the site of the nuclear envelope.