Interphase Chromosomes in Replicative Senescence: Chromosome Positioning as a Senescence Biomarker and the Lack of Nuclear Motor-Driven Chromosome Repositioning in Senescent Cells

This study demonstrates, and confirms, that chromosome territory positioning is altered in primary senescent human dermal fibroblasts (HDFs). The chromosome territory positioning pattern is very similar to that found in HDFs made quiescent either by serum starvation or confluence; but not completely. A few chromosomes are found in different locations. One chromosome in particular stands out, chromosome 10, which is located in an intermediate location in young proliferating HDFs, but is found at the nuclear periphery in quiescent cells and in an opposing location of the nuclear interior in senescent HDFs. We have previously demonstrated that individual chromosome territories can be actively and rapidly relocated, with 15 min, after removal of serum from the culture media. These chromosome relocations require nuclear motor activity through the presence of nuclear myosin 1β (NM1β). We now also demonstrate rapid chromosome movement in HDFs after heat-shock at 42°C. Others have shown that heat shock genes are actively relocated using nuclear motor protein activity via actin or NM1β (Khanna et al., 2014; Pradhan et al., 2020). However, this current study reveals, that in senescent HDFs, chromosomes can no longer be relocated to expected nuclear locations upon these two types of stimuli. This coincides with a entirely different organisation and distribution of NM1β within senescent HDFs

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

This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University LondonInterphase 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