CELL-CYCLE CHANGES IN A-TYPE LAMIN ASSOCIATIONS DETECTED IN HUMAN DERMAL FIBROBLASTS USING MONOCLONAL-ANTIBODIES

A new panel of anti-A-type lamin monoclonal antibodies was generated. Epitope mapping was performed by immunoblotting against GST-lamin fusi on peptides. Epitopes were mapped to four different regions of human l amin A and three different regions of human lamin, C. The distribution of A-type lamins was compared with the distribution of the proliferat ion marker Ki67 in proliferating and quiescent cultures of human derma l fibroblasts (HDFs) using a double indirect immunofluorescence assay. Antibodies that had been mapped to a region of the lamin C tail stain ed the nuclear envelope of proliferating and quiescent cells equally b rightly. In contrast, antibodies recognizing epitopes in the head doma in and rod domain of lamins A and C and the tail domain of lamin A sta ined the nuclear envelope of quiescent cells strongly but reacted poor ly or not at all with the nuclear envelope of proliferating cells. Cha nges in the level of expression of lamins A and C were not detected in immunoblotting assays. However, epitope masking was revealed, and thi s occurred by two distinct mechanisms. Epitope masking in the head dom ain of lamins A and C occurred as a result of protein phosphorylation. Epitope masking in the rod domain of lamins A and C and in the tail d omain of lamin A occurred through a physical association between the l amin and chromatin and/or other nuclear proteins. The cell cycle timin g of epitope masking was investigated in HDFs that had been restimulat ed after serum starvation. Extensive epitope masking in restimulated c ells only occurred after cells had passed through mitosis. These resul ts ape consistent with the hypothesis that rearrangement of A-type lam in filaments, as cells progress from a quiescent to a proliferating st ate, results in altered lamina associations.