Characterisation of the molecular links between the nuclear pore complex and the nuclear lamins and reconstitution of the "Xenopus" oocyte lamin assembly "in vitro"

Abstract

Nuclear lamins and nuclear pore complexes (NPCs) are major components of the nuclear envelope in metazoan cells. The objectives of this thesis were first to study interactions between nuclear lamins and the nuclear pore protein Nup153 (Chapters 2 and 3) and second to determine lamin assembly conditions of the Xenopus oocytes LIII in vitro (Chapter 4). Nuclear lamins are major constituents of the nuclear lamina underlying the nuclear periphery along with inner nuclear membrane proteins. The nuclear lamina provides stability and determines the nuclear architecture and spacing of the NPCs. NPCs form supramolecular assemblies that regulate nucleocytoplasmic transport. An overview of the functional aspects associated with the nuclear lamina and NPCs in health and disease is provided in Chapter 1. In depth analysis of the interaction of nuclear lamins with the nucleoporin Nup153 is revealed in Chapters 2 and 3. Using in vitro solution binding assays as well as immunoprecipitation assays, in chapter 2 we show direct associations between Nup153 and nuclear lamins. This work is explored even further in chapter 3 using binding assays and immunofluorescence microscopy as well as immunoprecipitation assays; we examined the interaction in the presence of lamin related mutations. Finally, in chapter 4 we established buffer conditions for LIII assembly in vitro, analysed by electron microscopy (EM) using glycerol spraying/low-angle rotary metal shadowing and negative staining. Our results presented in this thesis contribute to expand our current knowledge of the interactions of the NPCs with the nuclear lamins, as well as to increase our understanding of the impact of mutations in lamins that can cause laminopathies. In addition, the studies on the assembly conditions of LIII provide a vehicle for further characterisation of the influence of binding partners and the importance of lamin sub-fragments on the formation of higher order assemblies.