SUMMARY The genome of all eukaryotic cells is enclosed by a protective membrane system, the nuclear envelope, which establishes a selective barrier that ensures the biochemical compartmentalization of nucleus and cytoplasm. The malfunctioning of this barrier has been observed in many pathological contexts including cancers and neurodegeneration. The underlying premise of this application is that there are protective mechanisms that monitor and ameliorate perturbations to the nuclear envelope including to embedded nuclear pore complexes. Our published work identified the endosomal sorting complexes required for transport (ESCRT) and integral inner nuclear membrane proteins as key factors that surveil the integrity of the nuclear envelope barrier. In this application, we further explore the mechanism of ESCRT function at the nuclear envelope by examining its function in a new physiological context: the sealing of a single nuclear envelope hole at the end of mitosis in fission yeast. We propose a comprehensive and quantitative analysis of the identity, order of assembly and copy number of all of the ESCRT proteins recruited to this nuclear envelope sealing site. Our preliminary data suggest that unique ESCRT proteins (distinct from the complement at endosomes) are recruited in at least two temporally distinct waves, each of which carries out potentially unique functions to be interrogated in the proposal. Most notably, as at least some ESCRTs appear dispensable for nuclear envelope sealing but lead to an expansion of the nuclear envelope hole during mitosis, we hypothesize that ESCRTs form a flexible grommet that restricts the size of the nuclear envelope hole to counteract forces from the elongating spindle. To test this model, we will generate tools to measure tension at the nuclear envelope hole and determine a high resolution structure of ESCRT polymers using cryo-focused ion beam milling coupled to cryo-electron tomography. The success of the proposal will introduce several new concepts for how ESCRTs function and inform both physiological and pathological processes associated with nuclear envelope function.