$\textit{In Cellulo}$ Analysis of the Nuclear Envelope by Cryo-Electron Tomography

Abstract

Nuclear lamins are type V intermediate filament proteins which form a dense filamentous meshwork at the inner nuclear membrane of all metazoan cells. Together with multiple lamin binding proteins and other factors, nuclear lamins form the nuclear lamina. In mammals, four different lamin isoforms, A, C, B1 and B2 are expressed. Primarily, nuclear lamins play a vital role in nuclear mechanics but they also contribute to many processes, such as Chromatin organization, DNA repair and gene regulation. Mutations in the gene encoding lamin A and C, LMNA, have been linked to a wide variety of diseases including muscular dystrophies and accelerated aging syndrome, termed laminopathies. However, the cellular and molecular mechanisms behind these diseases are still poorly understood. Therefore, revealing the structure of lamin filaments, the nuclear lamina organization, and the interactions between lamins and chromatin is fundamental for understanding the molecular basis of laminopathies. In this thesis, I applied cryo-electron tomography and genetic manipulation of cells, to study the structure of the nuclear envelope. By using a focused ion beam (FIB)-milling for sample preparation we were able to image the nuclear lamins in their native cellular environment, together with all interacting partners. In the first part of my work, we show how the laminopathic mutation H222P in lamin A/C, has no fundamental structural effect on the nuclear lamina or lamin filaments but that it alters the chromatin organization at the nuclear envelope and the dimensions of the whole nuclei. In a second study we determined the exact locations of the nucleosomes at the nuclear envelope by sub-tomogram averaging. This enabled us to measure the local nucleosome concentrations in relation to their distance to the nuclear lamina. Furthermore, we show how the removal of either A- or B-type lamins alter both the nucleosome concentration at the nuclear envelope and the number of filaments in the nuclear lamina.