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

总结 所有真核细胞的基因组都被保护膜系统--核被膜所包围， 建立了一个选择性屏障，确保细胞核和细胞质的生化区室化。 已经在许多病理学背景中观察到该屏障的故障，包括癌症和癌症。 神经变性本申请的基本前提是存在保护机制， 监测和改善对核膜的扰动，包括对嵌入的核孔复合物的扰动。 我们已发表的工作确定了转运所需的内体分选复合物（ESCRT）和整合 内核膜蛋白是监视核膜屏障完整性的关键因素。在这 应用，我们进一步探讨ESCRT功能的机制，在核膜，通过检查其 在一个新的生理背景下的功能：在有丝分裂结束时， 分裂酵母我们提出了一个全面的和定量的分析身份，秩序的组装， 所有ESCRT蛋白的拷贝数募集到这个核膜密封位点。我们的初步数据 表明独特的ESCRT蛋白（不同于内体的补体）在至少两个 时间上不同的波，每个波都执行在时间上被询问的潜在的独特功能。 提议最值得注意的是，因为至少一些ESCRT似乎不适合核膜密封，但会导致 有丝分裂时核膜孔的扩大，我们假设ESCRTs形成了一个灵活的 限制核膜孔大小以抵消纺锤体伸长力的纤维。 为了测试这个模型，我们将生成工具来测量核膜孔处的张力，并确定一个 利用低温聚焦离子束铣削耦合低温电子的ESCRT聚合物的高分辨率结构 断层扫描该提案的成功将为ESCRT的运作引入几个新的概念， 它提供了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.