Peković, Vanja (2005) The role of A-Type lamins and LAP2a in cellular ageing of human fibroblasts in vitro. Doctoral thesis, Durham University.
Mutations in LMNA gene have been linked to a number of age-related tissue-specific diseases and premature ageing syndromes termed laminopathies. The finding that A- type lamins affect longevity and maintenance of a number of somatic tissues makes them potential candidates in ageing of normal tissues. At the cellular level, ageing is manifested by an accumulation of irreversibly arrested senescent cells. Premature senescence is also hallmark of premature ageing syndromes. A-type lamins and lamina- associated polypeptide 2 α (LAP2a) bind to and tether retinoblastoma protein (Rb) in the nucleus, a main regulator of senescence pathway. In order to explore the role of A- type lamins and LAP2a in normal ageing and disease, I studied these proteins during ageing of wild type human fibroblasts in vitro. My results show that protein expression level of lamins and LAP2s does not change during ageing of wild-type fibroblasts in vitro. Nonetheless, fibroblasts aged in vitro acquire a range of nuclear morphological changes characteristic of laimnopathy fibroblasts. These nuclear 'laminopathy' phenotypes are accompanied by changes in peripheral heterochromatin, accumulation of lamins А/С in the nucleoplasm, aggregation of LAP2a and decreased Rb phosphorylation. Interestingly, acquisition of such abnormal nuclear changes correlates with premature senescent arrest in five laminopathy cell lines studied. In contrast, although a down-regulation of LAP2a expression and Rb dephosphorylation accompany a reversible quiescent arrest, quiescent fibroblasts do not acquire alterations in nuclear morphology and instead show increased association of lamins at the NE. A-type and B- type lamms become more insoluble in quiescent cells, which may be a cause of a loss of nuclear anchorage of LAP2a and Rb as well as an increased solubility of LAP2ß. On the contrary, during biochemical fractionation of aged fibroblasts, lamin A, but not lamin B2， becomes altered in a manner which makes it more unstable (i.e. prone to degradation) which leads to accumulation of a soluble lamin A proteolytic fragment whilst lamin с and LAP2a become lost from detergent/salt-resistant nucleoskdeton. Protein sequence analysis revealed that both lamm A and LAP2a contain number of cysteine residues in their C-terminal specific tails. Glutathione blot assay and immunoprecipitation under non-reducing conditions showed that A-type lamins and LAP2a become s-glutathiolated in senescent cells, which may cause their decreased binding seen on immunoprecipitation. Both lamin A and LAP2a show an age-dependent accumulation of monomeric protein under non-reducing conditions, which տ early passage cells leads to formation of non-native disulphide cross-links in these proteins. Interestingly, neither of the four laminopathy cell lines studied show an increased accumulation of monomeric lamin A as compared to their age-matched controls. Aged and lamin A/C-deficient laminopathy fibroblasts display a targeted loss of nucleoplasmic but not speckle-associated forms of Rb. These Rb speckles associate with sites of DNA damage in both senescent and laminopathy cells and may be involved in DNA damage signalling or post-transcriptional regulation of gene expression. sRNAi knockdown of LAP2a also leads to decreased phosphorylation and a loss of nucleoplasmic forms of Rb, which correlates with cell cycle arrest. I propose a model for ageing of human fibroblasts in vitro whereby oxidative modification of A-type lamin filaments induces changes in their structural conformation leading to destabilisation and a less efficient assembly at the nuclear envelope. In addition, aberrant telomere targeting of oxidatively modified LAP2a in senescent cells or aggregated LAP2a m laminopathy cells would lead to destabilised telomere structures, telomere uncapping and/or chromosome fusions which would trigger Rb-mediated DNA damage-induced senescent arrest.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||09 Sep 2011 09:53|