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Mitotic PtK1 cell microinjected with X-rhodamine-labeled tubulin and Alexa 488-labeled CENP-F antibodies to fluorescently label kinetochore fibers (red) and kinetochores/spindle poles (green), respectively. The movie starts in metaphase and shows sister kinetochore pairs aligned at the metaphase and oscillating between the two spindle poles as their respective kinetochore fibers lengthen and shorten. As the cell enters anaphase and the sister chromatids separate, sister kinetochores move away from each other as their kinetochore fibers shorten (anaphase A). Almost simultaneously, the two spindle poles move away from each other leading to elongation of the mitotic spindle (anaphase B). The compacted DNA of the chromosome arms excludes the background fluorescence so that chromosome arms appear as dark rod-like structures and the movement of the sister chromatid arms away from each other during anaphase can also be followed.
Digital images were collected with an Orca ER cooled CCD camera (Hamamatsu Photonics) coupled to a Yokogawa spinning disk confocal CS10 unit (Perkin Elmer Life Sciences Wallac), which was attached to a Nikon TE300 inverted microscope. Illumination was achieved via an Argon/Krypton laser and 488 nm and 568 nm filters in an excitation filter wheel (Sutter Instruments) through a 60 x 1.4 NA Plan-Apochromatic DIC objective lens. Nearly simultaneous fluorescence images were acquired at 488 and 568 nm at a single focal plane every 60 seconds. The movie is played at 10 frames per second. Elapsed time is shown in min:sec.
Lisa A Cameron
Edward D Salmon
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