Lamin A and lamin C form homodimers and coexist in higher complex forms both in the nucleoplasmic fraction and in the lamina of cultured human cells.
We have investigated and quantified the nuclear A-type lamin pool from human HeLa S3 suspension cells with respect to their distribution to detergent soluble and insoluble fractions. We devised a sequential extraction protocol and found that maximally 10% of A-type lamins are recovered in the soluble fraction. Notably, lamin C is enriched in low detergent fractions and only with 0.5% Nonidet P-40 lamin A and C are recovered in ratios nearly equivalent to those found in whole cell extracts and in the lamina fraction. Authentic nucleoplasmic proteins such as LAP2a, pRB and p53 are co-extracted to a large part together with the A-type lamins in these fractions. By sucrose density centrifugation we revealed that the majority of lamins co-sedimented with human IgG indicating they form rather small complexes in the range of dimers and slightly larger complexes. Some lamin A - but not lamin C - is obtained in addition in a much faster sedimenting fraction. Authentic nuclear proteins such as PCNA, p53 and LAP2a were found both in the light and the heavy sucrose fractions together with lamin A. Last but not least, immunoprecipitation experiments from both soluble fractions and from RIPA lysates of whole cells revealed that lamin A and lamin C do not form heterodimers but segregate practically completely. Correspondingly, immunofluorescence microscopy of formaldehyde-fixed cells clearly demonstrated that lamin A and C are localized at least in part to distinct patches within the lamina. Hence, the structural segregation of lamin A and C is indeed retained in the nuclear envelope to some extent too.
Division of Molecular Genetics, German Cancer Research Center (DKFZ), INF280, D-69120 Heidelberg, Germany.
Thorsten
T
Kolb
Kendra
K
Maass
Michaela
M
Hergt
Ueli
U
Aebi
Harald
H
Herrmann
D000906:Antibodies / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D034904:Lamin Type A / Q000032:analysis* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008938:Mitosis; D009687:Nuclear Proteins / Q000032:analysis
D000906:Antibodies; D034904:Lamin Type A; D009687:Nuclear Proteins; C440308:lamin C