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 fusion peptides. Epitopes were mapped to four different regions of human lamin A and three different regions of human lamin C. The distribution of A-type lamins was compared with the distribution of the proliferation marker Ki67 in proliferating and quiescent cultures of human dermal fibroblasts (HDFs) using a double indirect immunofluorescence assay. Antibodies that had been mapped to a region of the lamin C tail stained the nuclear envelope of proliferating and quiescent cells equally brightly. In contrast, antibodies recognizing epitopes in the head domain and rod domain of lamins A and C and the tail domain of lamin A stained the nuclear envelope of quiescent cells strongly but reacted poorly or not at all with the nuclear envelope of proliferating cells. Changes in the level of expression of lamins A and C were not detected in immunoblotting assays. However, epitope masking was revealed, and this occurred by two distinct mechanisms. Epitope masking in the head domain 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 domain of lamin A occurred through a physical association between the lamin and chromatin and/or other nuclear proteins. The cell cycle timing of epitope masking was investigated in HDFs that had been restimulated after serum starvation. Extensive epitope masking in restimulated cells only occurred after cells had passed through mitosis. These results are consistent with the hypothesis that rearrangement of A-type lamin filaments, as cells progress from a quiescent to a proliferating state, results in altered lamina associations.
Dyer
J A
JA
The Department of Biological Sciences, The University of Dundee, UK.
Kill
I R
IR
Pugh
G
G
Quinlan
R A
RA
Lane
E B
EB
Hutchison
C J
CJ
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D000911:Antibodies, Monoclonal; D002453:Cell Cycle / Q000502:physiology*; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002843:Chromatin / Q000276:immunology / Q000378:metabolism; D003470:Culture Media; D016895:Culture Media, Serum-Free; D018604:Epitope Mapping; D005260:Female; D005347:Fibroblasts / Q000276:immunology / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D015151:Immunoblotting; D034904:Lamin Type A; D034882:Lamins; D051379:Mice; D008807:Mice, Inbred BALB C; D009687:Nuclear Proteins / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D012867:Skin / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D013268:Stimulation, Chemical; D013997:Time Factors
D000906:Antibodies; D000911:Antibodies, Monoclonal; D002843:Chromatin; D003470:Culture Media; D016895:Culture Media, Serum-Free; D034904:Lamin Type A; D034882:Lamins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; C440308:lamin C