In this HeLa cell, which is expressing both a kinetochore marker (EGFP-CENP-B) and a centrosome marker (Venus-centrin), one can visualize the dynamic nature of kinetochore movements during metaphase and anaphase. During metaphase, sister kinetochore pairs, seen as paired dots oriented parallel to the x-axis, are attached to opposite spindle poles. Poles are marked by the two pairs of venus centrin dots near the left and right edges of the movie and seen as single dots in this movie. Kinetochores in metaphase HeLa cells typically undergo oscillatory movements around the equator of the spindle. At the onset of anaphase, the sisters separate and move poleward. As seen in this movie, however, kinetochores can continue to switch between poleward and away from pole movements even after sister separation. Note that the centrin dots are approximately 12 microns apart at the start of the movie. This movie was part of a set that won first place in the 2007 ASCB Celldance contest.
This HeLa cell was analyzed by time-lapse microscopy 48 hours after DNA transfection and 30 hours after transfection with control siRNA (Ambion Negative Control #1). Prior to filming, the cell was switched to CO2-independent media (Life Technologies). The cell was imaged with a Deltavision RT system (Applied Precision) equipped with a CCD camera, 60X/1.42 NA lens (Olympus) and a 37C environmental chamber (Applied Precision). Z-stacks containing five focal planes with 0.5 microns spacing were acquired at intervals of 2 seconds. Images were deconvolved and projected as a 2-D image stack using Softworx software (Applied Precision). The movie was cropped, bleach corrected, and contrast adjusted using Image J. Display rate is 25 frames per second. Original resource created on August 20, 2007.Original resource: 284.8 Mb Deltavision image stack.
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