Regulation of membrane remodelling during cell division

运输所需的内体分选复合体(ESCRT)机制构成了一个高度保守的异构体蛋白质复合体家族(ESCRT-0、ESCRT-I、ESCRT-II和ESCRT-III)，它们协调整个细胞的膜重塑过程[1]。ESCRT共同调节细胞分裂过程中的关键事件，如核膜有丝分裂重组和细胞动力脱落[2]。ESCRT介导的膜重构过程的中心是ESCRT-III的作用，ESCRT-III是一种收缩膜的微丝形成复合体[3]。从历史上看，ESCRT复合体最初的特征是它们在协调泛素化蛋白质的内体分类方面的作用[4]。然而，ESCRT-III在缺乏内膜结构的古生物中的运行表明，ESCRT-III的原型作用是在细胞分裂期间发挥作用[5]。在细胞分裂期间，后生动物微管组织中心(MTOC)，即中心体，协调微管的组装，并产生两极有丝分裂纺锤体的相反两极，共同促进DNA分离[6]。在有丝分裂期间，复制的DNA被有丝分裂纺锤体的微管纤维分开，这需要动态重塑核膜以提供进入染色体的途径[7]。核膜的有丝分裂重组发生在染色体分离到细胞的相反两极之后，这是由ESCRT-III和微管切断蛋白spastin促进的[8]。在细胞质分裂的最后阶段，子代细胞在中体保持连接，这是一个由来自有丝分裂纺锤体的反平行微管紧密连接的暂时性桥梁[9]。在这里，微管的切断是由痉挛蛋白介导的，而ESCRT-III被募集到中体并随后成核进入螺旋细丝，导致细胞运动在靠近中体的次生收缩部位脱落[10，11]。因此，中心体和有丝分裂纺锤体的活动是核膜有序重组和胞质分裂及时脱落的核心，这需要ESCRT-III来调节它们的维持和协助它们的功能[12]。此外，ESCRT-III亚基的耗尽改变了中心体和纺锤体的极数，导致染色体分离缺陷，表现为异常的核形态[12]。最近，我们发现CC2D1B是CC2D1/LGD家族中一个特征不佳的蛋白质家族成员，它在有丝分裂过程中调节ESCRT-III在重整核膜上的聚合，防止过早招募，否则将导致膜密封机制失败和穿透纺锤体微管的移除[13]。CC2D1A，一种相关的蛋白质，也被证明在体外可以阻止ESCRT-III的聚合[14]。CC2D1A通过CDK1进行有丝分裂的磷酸化，CDK1是有丝分裂的主要调节者，并在细胞周期中防止中心粒过早分裂[15，16]。我们有初步证据表明，CC2D1A在细胞分裂过程中作为ESCRT-III聚合和活性的时空调节因子。我们发现CC2D1A在有丝分裂过程中定位于细胞质，并特异性地定位于有丝分裂纺锤体的两极。在CC2D1A缺失的细胞中，我们观察到ESCRT-III亚基在有丝分裂纺锤体周围的异位定位，以及微管生长异常，这与异常的细胞形态一致。此外，我们发现CC2D1A的耗尽与脱落时间的延迟有关，但从我们的初步研究中，我们不能得出任何对ESCRT-III聚合的影响。我们假设CC2D1A通过协调ESCRT-III的聚合，在细胞分裂过程中作为ESCRT-III活性的时空调节因子。此外，我们假设CDK1磷酸化可能引起的构象变化介导了CC2D1A的功能。

The Endosomal Sorting Complex Required for Transport (ESCRT) machinery constitutes a family of highly conserved heteromeric protein complexes (ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III), which orchestrate membrane remodelling processes throughout the cell [1]. Together ESCRTs mediate key events during cell division such as mitotic reformation of the nuclear envelope and cytokinetic abscission [2]. Central to ESCRT-mediated membrane remodelling processes is the action of ESCRT-III, a filament forming complex which constricts membranes [3]. Historically, the ESCRT complexes were first characterised for their role in coordinating endosomal sorting of ubiquitinated proteins [4]. However, operation of ESCRT-III in Archaea, which lack endomembrane structures, suggests the archetypal role of ESCRTs was to function during cell division [5]. During cell division the metazoan microtubule-organising centre (MTOC), the centrosome, coordinates assembly of microtubules and generates opposing poles of the bipolar mitotic spindle which together facilitate DNA segregation [6]. During mitosis, duplicated DNA is separated by microtubule fibres of the mitotic spindle, which requires dynamic remodelling of the nuclear envelope to provide access to chromosomes [7]. Mitotic reformation of the nuclear envelope occurs following segregation of chromosomes to opposite poles of the cell, which is facilitated by ESCRT-III and the microtubule-severing protein spastin [8]. During the final stages of cytokinesis, daughter cells remain connected at the midbody, a transient bridge packed tightly with antiparallel microtubules derived from the mitotic spindle [9]. Here, microtubule severing is mediated by spastin while recruitment of ESCRT-III to the midbody and its subsequent nucleation into helical filaments directs cytokinetic abscission at a secondary constriction site adjacent to the midbody [10, 11]. Therefore, central to orderly reformation of the nuclear envelope and timely cytokinesis abscission are the action of the centrosome and the mitotic spindle which require the action of ESCRT-III to regulate their maintenance and assist with their function [12]. Furthermore, depletion of ESCRT-III subunits alters centrosome and spindle pole numbers, resulting in defects in chromosome segregation materialising in aberrant nuclear morphologies [12]. Recently we showed that CC2D1B, a poorly characterised member of the CC2D1/Lgd family of proteins, regulates ESCRT-III polymerisation at the reforming nuclear envelope during mitosis, preventing premature recruitment which would otherwise lead to failure in the membrane sealing mechanism and removal of penetrating spindle microtubules [13]. CC2D1A, a related protein, has also been shown to prevent ESCRT-III polymerisation in vitro [14]. CC2D1A undergoes mitotic phosphorylation by CDK1, a master regulator of mitosis, and prevents premature centriole splitting during the cell cycle [15, 16]. We have preliminary evidence that suggests CC2D1A acts as a spatiotemporal regulator of ESCRT-III polymerisation and activity during cell division. We found that CC2D1A localises to the cytoplasm and specifically to the poles of the mitotic spindle during mitosis. In CC2D1A-depleted cells, we observed ectopic localisation of ESCRT-III subunits around the mitotic spindle in addition to dysregulated microtubule growth, which coincided with aberrant cellular morphologies. Furthermore, we found that depletion of CC2D1A was associated with delays in abscission timing, but we could not conclude any impact on ESCRT-III polymerisation from our preliminary investigation. We hypothesise that CC2D1A functions as a spatiotemporal regulator of ESCRT-III activity around the mitotic spindle during cell division by coordinating ESCRT-III polymerisation. Furthermore, we postulate that conformational changes, potentially induced by CDK1 phosphorylation, mediate CC2D1A function.