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*CIL – Cell Image Library accession number. Please use this to reference an image.

CIL:8053*  Cite 
Description

This HeLa cell is expressing two fluorescent proteins, including EGFP-CENP-B, a fluorescent protein that marks kinetochores (seen as paired dots oriented parallel to the x-axis and attached to opposite spindle poles), and Venus-centrin, which marks the centrosomes (seen as single dots near the left and right edges of the movie) and was depleted of the mitotic kinesin, Kif18A. In control cells at this stage in mitosis, sister kinetochore pairs typically undergo oscillatory movements around the equator of the spindle. In this cell depleted of Kif18A, however, kinetochores are still bioriented and undergo oscillatory movements but fail to align at the spindle equator. Depletion of Kif18A leads to a two-fold increase in oscillation amplitude (from 1.2 microns to 2.3 microns). The increased oscillation amplitude can be explained by both a decrease in the rate of directional switches and an increase in the velocity of movement. As kinetochore movements are highly coordinated with the dynamics of kinetochore microtubules, we hypothesize that Kif18A may control kinetochore movements by regulating kinetochore microtubule dynamics. Note that the centrin dots are approximately 19 microns apart at the start of the movie. This movie is part of a group that captured first place at the 2007 ASCB Celldance contest.

Technical Details

This HeLa cell was analyzed by time-lapse microscopy 48 hours after DNA transfection and 30 hours after transfection with Kif18A siRNA (GCCAAUUCUUCGUAGUUUU). Prior to filming the cell was switched to CO2-independent media (Life Technologies). The cell were 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 created on August 18, 2007.Original resource: 54.6 Mb Deltavision image stack.

Biological Sources
NCBI Organism Classification
Homo sapiens
Cell Type
cervical cancer
epithelial cell
Cell Line
HeLa
Cellular Component
kinetochore
spindle pole centrosome
Attribution
Names
Jason Stumpff
George von Dassow
Michael Wagenbach
Charles Asbury
Linda Wordeman
Published
Dev Cell. Feb;14(2):252-62. (2008)
Pubmed
18267093
Citation
Digital Object Identifier (DOI)
doi:10.7295/W9CIL8053
Archival Resource Key (ARK)
ark:/b7295/w9cil8053
Grouping This image is part of a group.
Imaging
Image Type
recorded image
Image Mode
time lapse microscopy
widefield illumination
fluorescence microscopy
Parameters Imaged
fluorescence emission
Source of Contrast
distribution of a specific protein
Visualization Methods
VenusFP
EGFP
Processing History
3D-deconvolution
Data Qualifiers
processed data
Sample Preparation
Methods
living tissue
Relation To Intact Cell
dispersed cells in vitro
Dimensions
Spatial Axis Image Size Pixel Size
X 328px ——
Y 278px ——
Time 6 seconds 150