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

真核生物基因组在细胞核中划分；其边界是称为核膜（NE）的双膜结构。核膜将转录与翻译分开，保护基因组免受寄生核酸和其他环境危害的影响，调节基因活性，并防止 DNA 损伤和恶性转化。脊椎动物的分裂细胞经历“开放有丝分裂”的过程，其中NE开放到核质特性丢失的程度。在染色体分离过程中，NE 膜（包括许多蛋白质）分散到相连的内质网 (ER) 中。当完整且闭合的 NE 重新形成时，该过程在有丝分裂退出期间逆转。在每个细胞周期之后，以及在各种损害核膜的机械应激源之后，细胞需要密封核膜以维持完整的区室化。人类细胞在每次细胞分裂过程中如何打开和密封核膜的双层膜是一个长期存在的问题，目前仍然知之甚少。最近的研究表明，转运 III (ESCRT-III) 蛋白所需的内体分选复合物参与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细菌和所有真核生物中都是保守的。它们进一步驱动多囊泡体的形成、包膜病毒的排出以及细胞分裂的脱落。最近发现 ESCRT-III 蛋白 CHMP7 在核膜重组过程中发挥着重要作用。我们最近报道，在招募其他 ESCRT-III 组件（如 IST1/CHMP8 和 CHMP2A）之前，内核膜蛋白 Lem2 招募 Chmp7 作为 NE 间隙闭合的起始步骤。尽管 ESCRT-III 蛋白在细胞周期中具有根本重要性，但我们对 ESCRT-III 蛋白在各阶段如何发挥作用知之甚少。我的博士后研究旨在从结构上表征 i) ESCRT-III 机制如何被招募到核膜； ii) ESCRT-III 蛋白如何组装以在 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调节的。在这项工作的过程中，我将使用生物化学重建、交联质谱的复杂表征、冷冻电子显微镜的结构测定和人类细胞中的全基因组遗传筛选来了解 LEM2、CHMP7 和 ESCRT-III 机制在核膜位点的作用。

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.