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。这要求要么通过核膜(NE)运输，要么至少部分击穿。关于NE的完整性，对于单个真核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，即闭合的、开放的和半开放/闭合的，在各种Opisthokonta中都存在。此外，在古质体纲中也有开放和闭合有丝分裂的有机体。因此，最后一个真核生物共同祖先(LECA)的情况是未知的。到目前为止，只有在动物和真菌中才对有丝分裂过程中NE组织和NE通透性/NE破坏的关系进行了深入的研究。在研究得很好的模式生物中，阿米巴原虫Dictyostelials是独一无二的，因为它拥有以祖先层为基础的核层，以及具有完整NE的半闭合有丝分裂。这里还不清楚有丝分裂的NE是通过核孔复合体的修饰，还是通过在复制中心体插入NE时形成的窗孔，还是通过两种机制的组合而通透。中心体窗孔的参与将是一种前所未有的新机制。这种机制的存在得到了Dictyostelius菌株的观察支持，该菌株缺乏中心体蛋白CP75，似乎在NE通透性和随后的纺锤体形成方面存在缺陷。了解去甲肾上腺素是如何在有丝分裂过程中被渗透以允许纺锤体组装的，这将使我们更接近于了解LECA的情况。我们的工作计划将阐明NE修饰的时空事件，包括中心体插入NE和NPC修饰，以及选定的分子参与者在这些过程中的作用。无中心粒的网柄网柄菌中心体的复制与去甲肾上腺素修饰紧密相连。因此，本项目要研究的时空事件是：(1)放射状间期微管细胞骨架连同微管成核的中心体冠一起丧失，(2)剩余的中心体核心结构进入NE并伴随后者的通透性，(3)中心体核心在失去含有CP75的中心层后分裂成两部分，(4)通过部分解体NPC使NE进一步通透化。通过对表达合适荧光标记蛋白的双标菌株进行活细胞成像，我们将为这些事件建立一个时间表。CP75和相关的中心体NPC蛋白Nup53在这一过程中的可能作用将通过对合适的Dictyostelius突变株的分析和蛋白质相互作用的研究来研究。最后，我们将寻找ESCRT复合体在中心体插入NE过程中所起作用的迹象。

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.