The mechanism of ESCRT-mediated surveillance of the nuclear envelope barrier

摘要 所有真核细胞的基因组都被一个保护膜系统--核膜包裹着，它 建立一种选择性屏障，确保细胞核和细胞质的生化区划。 这一屏障的功能障碍在许多病理情况下都被观察到，包括癌症和 神经退行性变。这个应用程序的基本前提是有保护机制， 监测和改善对核膜的扰动，包括对嵌入的核孔复合体的扰动。 我们发表的工作确定了运输所需的内体分选复合体(ESCRT)和 核膜内膜蛋白是监控核膜屏障完整性的关键因素。在这 应用方面，我们通过检测ESCRT在核膜上的作用机制，进一步探讨了ESCRT在核膜上的作用机制 在新的生理环境中的作用：在有丝分裂结束时单个核包膜孔的封闭 裂变型酵母。我们提出了一个全面的和定量的分析身份，组装顺序和 招募到这个核膜封闭部位的所有ESCRT蛋白的拷贝数。我们的初步数据 提示独特的ESCRT蛋白(与内小体上的补体不同)至少在两个 时间上不同的波，每个波都执行潜在的独特功能，将在 求婚。最值得注意的是，由于至少有一些ESCRT似乎对核膜密封是可有可无的，但会导致 有丝分裂过程中核膜空洞的扩张，我们假设ESCRT形成一个灵活的 限制核包膜孔大小以抵消来自延长的纺锤体的力的扣环。 为了测试这个模型，我们将生成工具来测量核包膜孔处的张力，并确定一个 低温聚焦离子束球磨与低温电子耦合制备ESCRT聚合物的高分辨结构 体层摄影术。该提案的成功将为经济、社会和文化权利中心如何运作和 告知与核膜功能相关的生理和病理过程。

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.