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
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.