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Metaphase 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 shows that sister kinetochore pairs oscillate between the two spindle poles as their respective kinetochore fibers lengthen and shorten. The top kinetochore of the first sister pair on the right exhibits abnormal attachment to spindle microtubules, as it is bound to microtubules coming from both spindle poles, rather than just one (merotelic kinetochore orientation). As a consequence of this mis-attachment, the merotelically oriented kinetochore remains relatively stationary while its normally oriented sister oscillates between poleward and anti-poleward movements.
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 20-60 seconds. The movie is played at 10 frames per second. Elapsed time is shown in min:sec. Corresponds to video 1 in Curr Biol. 2004 Dec 14;14(23):2149-55.
Lisa A Cameron
Edward D Salmon
Curr Biol. 2004 Dec 14;14(23):2149-55.
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