Dynamic elastic behavior of alpha-satellite DNA domains visualized in situ in living human cells.
We have constructed a fluorescent alpha-satellite DNA-binding protein to explore the motile and mechanical properties of human centromeres. A fusion protein consisting of human CENP-B coupled to the green fluorescent protein (GFP) of A. victoria specifically targets to centromeres when expressed in human cells. Morphometric analysis revealed that the alpha-satellite DNA domain bound by CENPB-GFP becomes elongated in mitosis in a microtubule-dependent fashion. Time lapse confocal microscopy in live mitotic cells revealed apparent elastic deformations of the central domain of the centromere that occurred during metaphase chromosome oscillations. These observations demonstrate that the interior region of the centromere behaves as an elastic element that could play a role in the mechanoregulatory mechanisms recently identified at centromeres. Fluorescent labeling of centromeres revealed that they disperse throughout the nucleus in a nearly isometric expansion during chromosome decondensation in telophase and early G1. During interphase, centromeres were primarily stationary, although motility of individual or small groups of centromeres was occasionally observed at very slow rates of 7-10 microns/h.
Department of Cell Biology, Scripps Research Institute, La Jolla, California 92037, USA.
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Shelby
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Hahn
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Sullivan
D001324:Autoantigens; D002460:Cell Line; D002503:Centromere; D051936:Centromere Protein B; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004276:DNA, Satellite / Q000032:analysis*; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007399:Interphase; D008164:Luminescent Proteins; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence; D008870:Microtubules; D008938:Mitosis; D011993:Recombinant Fusion Proteins
D001324:Autoantigens; C497926:CENPB protein, human; D051936:Centromere Protein B; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004276:DNA, Satellite; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins