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

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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.