This HeLa cell, which is expressing both a kinetochore marker (EGFP-CENP-B) and a centrosome marker (Venus-centrin), has been depleted of the spindle assembly checkpoint protein Mad2. In the absence of Mad2, the cell rapidly progresses from early prometaphase through anaphase and cytokinesis. The spindle assembly checkpoint functions to generate a WAIT signal until all sister kinetochore pairs become properly attached to the mitotic spindle. Loss of checkpoint function leads to anaphase initiation before kinetochores are completely aligned at the spindle equator. Despite the rapid nature of these divisions, the majority of the kinetochores appear to separate properly in Mad2-depleted cells. However, at least a few lagging chromosomes are frequently observed in these cells, demonstrating the importance of checkpoint function. Note that the frame width is approximately 30 microns. This movie is part of a group that captured first place at the 2007 ASCB Celldance contest.
This HeLa cell was analyzed by time-lapse microscopy 48 hours after DNA transfection and 36 hours after transfection with 120 nM of Mad2-specific siRNAs (GGAUGACAUGAGGAAAAUA and GCGUGGCAUAUAUCCAUCU). 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 um spacing were acquired at intervals of 5 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 20 frames per second. Original resource created on February 18, 2007.Original resource: Deltavision Image stack
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