Localization of HMG chromosomal proteins in the nucleus and cytoplasm by microinjection of functional antibody fragments into living fibroblasts.
We have used microinjection and cell fractionation to localize the chromosomal high mobility group proteins (HMG) in human fibroblasts. Electrophoretic analysis of nuclear and cytoplasmic fractions from the fibroblasts indicates that the concentration of HMG-1,2 in the cytoplasm is 2.9 times larger than in the nucleus indicating that the majority of the cellular HMG-1,2 is present in the cytoplasm. In contrast, HMG-17 remains predominant in the nuclear fraction. We conclude that the cellular distribution of HMG-1,2 is significantly different from that of HMG-17. To avoid possible artifacts due to cell fractionation, fluoresceinated HMG-1 and HMG antibodies were microinjected into living fibroblasts. The cellular distribution of the injected proteins was monitored using fluorescent microscopy. Fluoresceinated HMG-1 microinjected into the cytoplasm moves very rapidly into the nucleus and concentrates in the nucleolus of living human fibroblasts. However, some control non-nuclear proteins also migrated into the nucleus raising the possibility that exogenous injected proteins do not always distribute in the same pattern as the endogenous proteins. The localization of microinjected F(ab)2 fragments derived from anti-HMG-1 was compared to that of microinjected F(ab)2 derived from anti-histones. Whereas the anti-histone F(ab)2 when injected into the cytoplasm migrated into the nucleus, the anti-HMG-1 F(ab)2 remained in the cytoplasm. Microinjection of anti-HMG-17 and anti-histone inhibited transcription in living cells, anti-HMG-1,2 did not. We conclude that HMG-1,2 proteins are present in both the nucleus and cytoplasm of living fibroblasts.
D000906:Antibodies; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000032:analysis*; D002478:Cells, Cultured; D002868:Chromosomal Proteins, Non-Histone / Q000032:analysis* / Q000276:immunology; D003593:Cytoplasm / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000032:analysis; D006609:High Mobility Group Proteins; D006801:Humans; D008856:Microscopy, Fluorescence
D000906:Antibodies; D002868:Chromosomal Proteins, Non-Histone; D006609:High Mobility Group Proteins