Permeabilization of the Dictyostelium nuclear envelope during semi-closed mitosis

在有丝分裂过程中，微管蛋白二聚体，纺锤体组装因子和其他蛋白质需要进入染色体。这需要通过核膜（NE）或其至少部分破裂的运输。关于NE的完整性，在单个真核细胞超群内的有丝分裂类型似乎没有共同的方案。例如，所有类型的有丝分裂，即封闭，开放和半开放/封闭，在各种后鞭毛纲中发现。此外，在原质虫中，有开放和闭合有丝分裂的生物。因此，在最后的真核生物共同祖先（LECA）的情况是未知的。到目前为止，NE组织和NE透化/NE分解之间的关系，有丝分裂期间已被彻底研究，只有在后鞭毛纲，即动物和真菌。在广泛研究的模式生物中，网囊变形虫是独特的，因为它具有基于祖先核纤层蛋白的核纤层，以及具有完整NE的半封闭有丝分裂。在此，不清楚有丝分裂NE是否通过核孔复合物（NPC）的修饰，或通过在复制中心体插入NE期间形成的窗孔，或通过两种机制的组合而变得透化。中心体窗孔的参与将是一个前所未有的新机制。这种机制的存在是支持的Dictyosteoprotein菌株的中心体蛋白CP 75，这似乎是有缺陷的NE透化和随后的纺锤体形成耗尽的观察。了解NE是如何在有丝分裂过程中透化的网骨藻，使纺锤体组装将使我们更接近的LECA的情况的理解。我们的工作计划将阐明NE修饰的时空事件，关于中心体插入NE和NPC修饰，以及在这些过程中所选择的分子球员的作用。无中心粒的网骨藻中心体的复制与NE修饰紧密偶联。因此，本项目中要研究的时空事件是：（1）放射状间期微管细胞骨架和微管成核中心体冠的丢失，（2）剩余的中心体核心结构进入NE，沿着后者的透化，（3）中心体核心在含有CP 75的中心层丢失后分裂为两部分，（4）通过NPC的部分分解使NE进一步透化。通过表达合适的荧光标记蛋白的双标记菌株的活细胞成像，我们将为这些事件建立时间轴。CP 75和相关的中心体NPC-蛋白Nup 53在这个过程中的假定作用将通过分析合适的突变Dictyosteelium菌株和蛋白质相互作用的研究。最后，我们将寻找ESCRT复合物在中心体插入NE过程中的作用的迹象。

During mitosis, tubulin dimers, spindle-assembly factors and other proteins need to get access to the chromosomes. This requires either transport through the nuclear envelope (NE) or its at least partial breakdown. With regard to the integrity of the NE there appears no common scheme for the types of mitosis within the individual eukaryotic supergroups. For instance, all types of mitosis, i.e. closed, open and semi-open/closed, are found in various Opisthokonta. Also, among the Archeaplastida there are organisms with open and closed mitosis. Thus, the situation in the last eukaryotic common ancestor (LECA) is unknown. So far, the relationship between NE organization and NE permeabilization/NE breakdown during mitosis has been studied thoroughly only in Opisthokonta, i.e. animals and fungi. Among the well-studied model organisms the amoebozoan Dictyostelium is unique, as it possesses a nuclear lamina based on an ancestral lamin, and a semi-closed mitosis with an intact NE. Here it is not clear whether the mitotic NE becomes permeabilized through modification of nuclear pore complexes (NPCs), or through the fenestrae that are formed during insertion of the duplicating centrosome into the NE, or by a combination of both mechanisms. An involvement of the centrosomal fenestrae would be an unprecedented novel mechanism. The existence of such a mechanism is supported by observations in a Dictyostelium strain depleted of the centrosomal protein CP75, which appeared to be defective in NE permeabilization and subsequent spindle formation. Understanding how the NE is permeabilized during mitosis in Dictyostelium to allow spindle assembly will bring us closer to an understanding of the situation in the LECA. Our work program will elucidate the spatio-temporal events of NE modification regarding centrosome insertion into the NE and NPC modification, and the roles of selected molecular players in these processes. Duplication of the centriole-free Dictyostelium centrosome is tightly coupled to NE modification. Thus, the spatio-temporal events to be investigated in this project are (1) loss of the radial interphase microtubule cytoskeleton together with the microtubule-nucleating centrosomal corona, (2) entry of the remaining centrosomal core structure into the NE along with permeabilization of the latter,(3) splitting of the centrosomal core into two parts upon loss of its central layer containing CP75, (4) further permeabilization of the NE by partial disassembly of NPCs. Through live cell imaging of double labeled strains expressing suitable fluorescent marker proteins, we will establish a timeline for these events. The putative roles of CP75 and the associated centrosomal NPC-protein Nup53 in this process will be investigated by analysis of suitable mutant Dictyostelium strains and by protein interaction studies. Finally, we will look for indications for a role of the ESCRT complex during centrosome insertion into the NE.