The role of membrane protein phase separation in nuclear membrane sealing and fusion

膜重塑是生命所必需的。脊椎动物有丝分裂末期细胞核的自组装证明了细胞重塑细胞膜的效率和健壮程度，甚至到了用溶解的碎片构建整个细胞器的地步。我们最近的工作表明，有丝分裂结束时的核膜改革是通过将膜蛋白相分离与细胞膜融合机制运输所需的内体分选复合体(ESCRT-III)的工作结合起来，通过将膜蛋白相分离与细胞膜融合机制中运输所需的内体分选复合体(ESCRT-III)相结合来进行的。在这项提议中，我们旨在研究尚未确定的结构和生物物理原理，即浓缩的含有内在无序结构域(IDD)的膜蛋白LEM2和MAN1招募结构化的ESCRT-III机械蛋白CHMP7形成细丝，密封并最终融合核膜，完成核重组。我们将在膜上重组LEM2/MAN1-ESCRT-III途径组件，以使用低温电子显微镜(Cryo-EM)和生物物理来研究它们的性质。已建立的监测有丝分裂中的核自组装的分析方法将使我们能够在体内验证我们的模型。越来越多的工作将LEM2-ESCRT-III途径，以及潜在的MAN1与各种年龄相关的病理联系起来，强调了它对细胞生理学的关键作用及其治疗潜力。因此，建立第一个蛋白质相分离如何控制核膜组装的机械框架不仅将解决膜细胞器自组织的基本问题，而且还将具有医学意义。

Membrane remodeling is essential to life. The self-assembly of the nucleus at the end of vertebrate mitosis exemplifies how efficiently and robustly cells reshape membranes, even up to the point of building an entire organelle from solubilized pieces. Our recent work suggests that nuclear membrane reformation at the end of mitosis proceeds via the complete fusion of incoming sheets and tubules of the endoplasmic reticulum (ER) on the surface of decondensing chromatin – a prerequisite for nuclear compartmentalization – by coupling membrane protein phase separation to the work of the cell’s membrane fusion machinery endosomal sorting complexes required for transport-III (ESCRT-III). In this proposal, we aim to investigate the yet undefined structural and biophysical principles by which the condensed intrinsically disordered domain (IDD)-containing membrane proteins LEM2 and MAN1 recruit the structured ESCRT-III machinery protein CHMP7 to form filaments and seal and finally fuse the nuclear membrane to complete nuclear reformation. We will reconstitute the LEM2/MAN1-ESCRT-III pathway components on membranes to study their properties using cryo electron microscopy (cryo-EM) and biophysics. Established assays monitoring nuclear self-assembly in mitosis will allow us to validate our models in vivo. A growing body of work links the LEM2-ESCRT-III pathway, and potentially MAN1, to a variety of age-related pathologies underscoring its critical role for cellular physiology and its therapeutic potential. Generating a first mechanistic framework of how protein phase separation controls nuclear membrane assembly will hence not only address fundamental questions of membrane organelle self-organization but will also be of medical relevance.