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

Nuclear lamins and nuclear pore complexes (NPCs) are major components of the nuclear \nenvelope in metazoan cells. The objectives of this thesis were first to study interactions \nbetween nuclear lamins and the nuclear pore protein Nup153 (Chapters 2 and 3) and second \nto determine lamin assembly conditions of the Xenopus oocytes LIII in vitro (Chapter \n4). Nuclear lamins are major constituents of the nuclear lamina underlying the nuclear periphery \nalong with inner nuclear membrane proteins. The nuclear lamina provides stability \nand determines the nuclear architecture and spacing of the NPCs. NPCs form supramolecular \nassemblies that regulate nucleocytoplasmic transport. An overview of the functional \naspects associated with the nuclear lamina and NPCs in health and disease is provided \nin Chapter 1. In depth analysis of the interaction of nuclear lamins with the nucleoporin \nNup153 is revealed in Chapters 2 and 3. Using in vitro solution binding assays as well \nas immunoprecipitation assays, in chapter 2 we show direct associations between Nup153 \nand nuclear lamins. This work is explored even further in chapter 3 using binding assays \nand immunofluorescence microscopy as well as immunoprecipitation assays; we examined \nthe interaction in the presence of lamin related mutations. Finally, in chapter 4 we established \nbuffer conditions for LIII assembly in vitro, analysed by electron microscopy (EM) \nusing glycerol spraying/low-angle rotary metal shadowing and negative staining. Our \nresults presented in this thesis contribute to expand our current knowledge of the interactions \nof the NPCs with the nuclear lamins, as well as to increase our understanding of the \nimpact of mutations in lamins that can cause laminopathies. In addition, the studies on the \nassembly conditions of LIII provide a vehicle for further characterisation of the influence \nof binding partners and the importance of lamin sub-fragments on the formation of higher \norder assemblies. \n