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Title: Interactions between nuclear lamins and their binding partners in EDMD fibroblasts
Author: Alvarez-Reyes, Mauricio
ISNI:       0000 0001 3418 9196
Awarding Body: Durham University
Current Institution: Durham University
Date of Award: 2003
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Lamins are components of the nuclear lamina and are divided in A and B-types, which interact with proteins of the inner nuclear membrane like emerin. Mutations in emerin (X-linked) and A-type lamins (Autosomal Dominant) has been linked to the Emery-Dreifuss Muscular Dystrophy (EDMD), which conduced to the hypothesis that these two proteins might interact in the nucleus. I examined the interaction between A and B-type lamins with emerin using a panel of deletion mutants of lamin Bl and full-length lamins A, C and B1 in a yeast two-hybrid assay, where emerin interacted with all lamins and the preferred region of interaction was the globular tail domain of lamin Bl. Ectopic expression of tagged proteins in human dermal fibroblasts confirmed that emerin remains attached to the inner nuclear envelope through its association with lamin 81, as aggregation of tagged A-type lamins did not miss localize endogenous emerin or lamin Bl. In addition, methanol-acetone fixation showed higher number of cells presenting characteristic morphological abnormalities called "honeycombs". A-type lamins and their associated protein emerin co- localized in these structures. Lamin Bl depletion from the honeycombs was accompanied by depletion of nuclear pore complexes. In the honeycombs, A- type lamins segregated from the B-type lamins, forming homo-filaments. On the other hand, AD-EDMD cell lines showed a characteristic pattern as a high sub-population of cells presented nesprin 1 (amino-terminal) in stress fibres co- localizing with a-S-Actin fibres, which was enhanced by growth inhibition induced by serum starvation. Re-stimulation of fibroblasts by normal serum concentrations increased the appearance of honeycombs by up to 2.5 fold in the AD-EDMD cell, lines. Late passage cultures of AD-EDMD entered a senescence state reminiscent of the induced quiescence state induced by serum starvation. Finally, differential allelic expression was evidenced using a specific set of ARMS-primers in the cell lines studied, indicative of transcript imbalance, and bioinformatics analysis demonstrated the presence of SNPs in the coding region of the wild type LMNA gene. The results of these study confirm that lamins interact with emerin and suggest that the interacting region is the tail domain of lamins; honeycomb structures might have a biological meaning in patient cells; other proteins might be involved in EDMD, like nesprins; and heterozygosis is presented with transcript imbalance, which might have a negative impact in the correct assembly of the nuclear lamina.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available