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

真核生物基因组在细胞核中被划分；其边界是称为核膜（NE）的双层膜结构。核膜隔离转录和翻译，保护基因组免受寄生核酸和其他环境危害，调节基因活性，防止DNA损伤和恶性转化。脊椎动物的分裂细胞经历了一个“开放有丝分裂”的过程，在这个过程中，NE打开到这样一个程度，核质身份就丧失了。在染色体分离过程中，NE膜包括许多蛋白质分散到连接的内质网（ER）中。在有丝分裂结束时，当一个完整和封闭的NE重组时，这个过程被逆转。在每个细胞周期之后，以及在各种破坏核包膜的机械压力之后，细胞需要密封核包膜以保持完整的区隔。人类细胞在每次细胞分裂过程中如何打开和封闭核膜的双层膜是一个长期存在的问题，但仍然知之甚少。最近的研究表明，运输III蛋白所需的内体分选复合物（ESCRT-III）参与细胞周期依赖性核膜的重组和修复。ESCRT-III蛋白及其膜重塑功能在古细菌和所有真核生物中都是保守的。它们进一步推动多泡体的形成、包膜病毒的出口以及细胞动力学脱落。ESCRT-III蛋白CHMP7最近被认为在核膜重组过程中发挥重要作用。我们最近报道，在其他esrt - iii成分（如IST1/CHMP8和CHMP2A）被募集之前，核膜蛋白Lem2募集Chmp7作为NE间隙关闭的起始步骤。尽管它们在细胞周期中具有重要的基础作用，但我们对ESCRTIII蛋白在细胞周期中的功能知之甚少。我的博士后研究旨在结构表征i) ESCRT-III机制如何被招募到核膜；ii) ESCRT-III蛋白如何组装在NE处进行膜重塑反应；iii)这个过程在哺乳动物细胞周期中是如何被调节的。在这项工作中，我将使用生化重构、交联质谱复杂表征、低温电镜结构测定和人类细胞全基因组遗传筛选来了解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.