File name: cyclinB1.mov
Authors: Tokuko Haraguchi, Jonathon Pines and Yasushi Hiraoka
Playback rate: 1 frame/second for first 3 frames, 4 frames/second for the others
Legend of the movie: This movie shows the dynamic behaviors of human cyclinB1-GFP (green) and chromosomes (red) during mitosis in living human (HeLa) cells. Cyclin B1-GFP was localized in the cytoplasm and to centrosomes in interphase cells. During prophase, cyclin B1-GFP accumulated into the nucleus. Upon nuclear envelope breakdown, cyclinB1-GFP relocalized from the nucleus to the centrosomes and to the mitotic spindle. Finally, when the fluorescence of cyclin B1-GFP started to diminish, mitotic chromosome segregation took place, consistent with a general picture that the degradation of cyclin B1 correlates the transition from metaphase to anaphase.
Background: Cyclin B1 is the major mitotic regulator and controls the progression of mitosis by modulating activity of the cyclin-dependent protein kinase Cdc2 (CDK1). The active complex of cdc2-cyclin B1 initiates many mitotic events in both the cytoplasm and the nucleus. The amount of cyclin B1 increases during interphase and is rapidly degraded during mitosis. The destruction of cyclin B1 during mitosis is essential for cells to exit from mitosis. Live images of cyclin B1-GFP clearly show the spatial and temporal change of localization of cyclin B1 in a cell cycle. Regulation of cyclin B1 localization may be important to control the progression of mitosis.
1. Molecular Biology of the Cell, Third edition, Garland Publishing, Inc., Bruce Albert et. al.
Methods: Transfection; Ca phosphate method. Microscopy; The DeltaVision (Applied Precision Inc. Seattle, USA) fluorescence microscope system. Microscope equipments; an Olympus inverted microscope IX70, UApo 40x (NA=1.35), a Peltier-cooled CCD camera (photometrics Ltd., Tucson, USA), a 2 m optical fiber for illumination, a UNIX workstation Indigo2 (Silicon Graphics, USA), a temperature controlled room and a microinjector (Eppendorf model 5171 and 5242). The experiment was carried out at 37 C and the time-lapse image data were collected every 1 min at an exposure of 1 sec for cyclinB1-GFP and 0.5 sec for Hoechst33342 up to 133 min except that the first three images were taken every 15 min.