Item

Abstract

The defining attribute of the eukaryotic cell is the compartmentalization of genetic material inside the cell’s nucleus. This is possible by the existence of a double lipid bilayer: the nuclear envelope (NE). This compartmentalization enables the eukaryotic cells to separate transcription and translation both spatially and temporally. The NE comprises two membranes: the outer nuclear membrane (ONM) that is continuous with the endoplasmic reticulum and the inner nuclear membrane (INM) that is characterized by a distinct protein composition and can make contact with the chromatin and the nuclear lamina. The ONM and the INM fuse to form pores where the nuclear pore complexes (NPCs) reside. NPCs act as gatekeepers of the nucleus and are involved in nucleo-cytoplasmic transport. They also act as a physical barrier thereby maintaining distinct composition of the nucleus and the cytoplasm. NPCs are proteinaceous macromolecular assemblies ranging from 40-60 Mega Daltons in size. Despite their enormous size, NPCs are composed of approx. 30 proteins referred as nucleoporins or Nups. Most nucleoporins are organized into distinct sub complexes that act as building blocks for NPC assembly. The NPC can be roughly dissected into two parts, the structural backbone of the pore that is composed of structural nucleoporins, which establish the basic scaffold of the pore and act as anchor proteins to recruit other nucleoporins. The other portion is the functional region of the pore that is composed of mainly the FG repeats containing nucleoporins that form the central channel of the pore thereby imparting functional properties like transport and exclusion competency. In animals undergoing open mitosis the NE and the NPCs are disassembled in prophase and they must reassemble from disassembled soluble pore components and membranes as the cell enters interphase to re-establish an intact nucleus. This thesis aims to functionally characterize the role of one of the sub-complexes that is the Nup93 complex, in NPC assembly and function. Nup93 is one of the structural complexes of the NPC. It is positioned in the pore at a region where it establishes a link between membranes of the NE and the NPC. Nup93 complex is composed of five nucleoporins: Nup53, Nup93, Nup155, Nup188 and Nup205. It was previously shown in the lab that Nup93 exists as part of two distinct complexes: Nup93-Nup188 and the Nup93-Nup205. Individually neither the Nup93-Nup188 nor the Nup93-Nup205 complexes were essential for NPC assembly. We were curious to find out if both the complex together were not essential for NPC assembly, as they have been speculated to have redundant roles in the pore. For this we took advantage of the Xenopus laevis egg extract system (in vitro nuclear assembly reactions) and by various cell biological and biochemical assays showed that the conserved nucleoporin Nup93 is essential for NPC/NE formation and functions. Immunodepleting Nup93 (thereby co depleting Nup205 and Nup188) lead to a block in NPC and NE formation and this was rescued by addition of the full-length recombinant Nup93. Hence Nup93 could compensate for the loss of both Nup93-Nup188 and Nup93-Nup205 complexes and thereby establishing the evidence that both major components of the Nup93 complex, Nup188 and Nup205 together are not crucial for post-mitotic NPC assembly. We also found that the C-terminus of Nup93 was sufficient and essential for formation of the structural backbone of the pore. It does so by strengthening the interaction between two major structural components of the pore Nup53 and Nup155. We believe that this is a key event in the formation of the scaffold/structural skeleton of the pore and hence in post-mitotic NPC assembly. Via the N-terminus, Nup93 recruits the Nup62 complex and other FG repeats containing nucleoporins to the pore, which form the central channel of the pore. Through the central channel of the pore nucleo-cytoplasmic transport occurs and a permeability barrier is established thereby maintaining distinct environments of the nucleus and cytosol in the cell. In this way, Nup93 connects both the structural and functional regions of the NPC and serves as a decisive and critical link between the two portions. We could also show that the middle domain of Nup93 is dispensable for NPC assembly. This study also sheds light on the order of recruitment of different nucleoporins in post-mitotic NPC assembly. It places Nup93 at a crucial and strategic position wherein it serves as a link between the structural and functional parts of the pore. Our findings also elucidated the role of different domains of Nup93 and their contribution in NPC assembly and function.