Permeabilization of the Dictyostelium nuclear envelope during semi-closed mitosis

• Applicant: Professor Dr. Ralph Gräf
• Subject Area: Cell Biology ; Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2017 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 391560682

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.

• DFG Programme: Research Grants