Chromosome dynamics and molecular motor proteins in the interphase nuclei of proliferating and non-proliferating cells

Abstract

Interphase chromosome dynamics is an important area of research related to the control of genomic function and gene regulation at the level of the 3D conformation and mobility of chromosomes. This also has relevance to cellular states which can show differences in chromosome organisation at interphase, when proliferating and non-proliferating cells are compared. These comparative studies are important to understanding the regulation of cell proliferation and processes such as ageing. The work in this thesis has taken the aspect of a broad approach to address the exploration of chromosome dynamics. This exploration has involved both extensive laboratory work and in silico analyses to reveal possible candidate proteins involved in chromosome mobility. A main hypothesis in this project is that there are fundamental differences between proliferating and non-proliferating cells with regards to the functions of nuclear myosin motor proteins and this would be reflected in the functions of these proteins, specifically related to chromosome mobility. Further to this, a key aspect of this work is related to the hypothesis that in non-proliferating cells chromosome mobility in response to stimuli is impaired, and this is caused by the relevant nuclear myosins not functioning as they would in proliferating cells. Using the technique of 2D Fluorescence In-Situ Hybridisation (2D FISH), the dynamic mobility of interphase chromosomes was studied in the nuclei of human dermal fibroblast cells, in order to gain further understanding regarding their responses to stress stimuli in the form of serum inhibited conditions and heat shock. Immmunofluorescence studies were performed to determine the patterns and frequency of myosin proteins (MYO5B, MYO16 and MYO18B) in proliferating and non-proliferating cells. Another aspect of the search for possible candidate proteins was an in silico bioinformatics exploration, to find not only other myosins but also actin related proteins and other classes of proteins that may be part of the overall mechanism of interphase chromosome mobility. In this work a heat shock assay in human dermal fibroblast cells has been developed with chromosome 11. In addition to this a novel chromosome 11 relocation has been found in response to heat shock and importantly, it has been found that in non-proliferating cells this relocation of chromosome 11 in response to heat shock does not occur. The results also show that the nuclear staining characteristics and frequency of the various myosin proteins studied show significant differences when compared in proliferating and non-proliferating cells. Using the bioinformatics approach other interesting candidate proteins were identified with possible potential to be involved with the process of chromosome mobility. A possible role for myosin 5b has been implicated in the mobility of splicing speckles, as shown by co-localisation with splicing speckles for the first time. A suggestion is made by inference, that older cells may possibly also have diminished chromosome relocation potential compared with younger cells.