Research
Whole genome functional analysis identifies novel components required for mitotic spindle integrity in human cells
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* Corresponding authors: Sumit K Chanda schanda@burnham.org - Jeremy S Caldwell jcaldwell@gnf.org
1 Genomics Institute of Novartis Research Foundation, John Jay Hopkins Drive, San Diego, California 92121, USA
2 Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Marris Park Avenue, Bronx, New York 10461, USA
3 Novartis Institute for Biomedical Research Inc., Mass Avenue, Cambridge, Massachusetts 02139, USA
4 Novartis Pharma AG, Postfach, CH-4002 Basel, Switzerland
5 Infectious & Inflammatory Disease Center, Burnham Institute for Medical Research, North Torrey Pines Road, La Jolla, California 92037, USA
Genome Biology 2008, 9:R44 doi:10.1186/gb-2008-9-2-r44
Published: 26 February 2008Additional files
Additional data file 1:
Presented is a data file showing the complete list of candidate genes isolated from our initial mitotic index thresholding and OPI clustering results.
Format: XLS Size: 68KB Download file
This file can be viewed with: Microsoft Excel Viewer
Additional data file 2:
(A) Loss of spindle surveillance mechanisms or defects in cytokinesis may result in multinucleated cells. Thus, we also identified genes involved in checkpoint-independent spindle functions by analyzing changes in the cell populations based on their multinucleation status. We acquired an additional 308,736 images using a far-red cytoplasmic dye (DDAO-SE) to determine the number of discrete nuclei per cell (A, inset). For our analysis, images were segmented into ROIs based on each cell's cytoplasmic intensity. Image analysis of the ROIs then determined the number of discrete nuclei per cell. Using this approach we uncovered a number of siRNAs targeting known chromosomal passenger genes, such as INCENP, CDCA8, BIRC5, and AuroraB. Cytokinesis genes were also identified as an additional benefit of this approach, because failing to complete cell division can result in multiple nuclei per cell. Thus, we isolated MKLP-1, MgcRacGAP, and CIT along with other known cytokinesis members. (B) Shown is the nuclei DNA organization after the activation of apoptotic pathways resulting in noncircular shaped and fragmented nuclei patterns. (C) The approach that was used to fit nuclei and cell morphology pattern analysis. Those objects that have a poor fit are given a lower score. (D) Typical image of cytoplasmic and DNA channels of cells that are mostly rounded. (E) Graph illustrates the time dependent change in nuclear shape. Cells treated with PLK1 siRNAs undergo apoptotic events relatively soon after treatment and show a high degree of fragmentation. (F) Watershed analysis (cytoplasmic masking) and signal intensity measurement approach for spindle and microtubule intensity on a cell by cell basis. (G) Image segmentation used to identify individual nuclei for proliferation comparisons.
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Additional data file 3:
The data table demonstrates all of the OPI clusters along, with calculated values listed.
Format: XLS Size: 171KB Download file
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Additional data file 4:
The data table lists the statistically significant clusters of genes associated with the mitotic/spindle processes.
Format: XLS Size: 28KB Download file
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Additional data file 5:
Enlarged version of the interaction map shown in Figure 3a, with protein labels added for reader's clarity.
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Provided is a sample live-cell movie of a mitotic cell transfected with an siRNA against SKA1 and expressing GFP-tubA1. The movie demonstrates the formation of a metaphase-like spindle before falling apart prior to the onset of anaphase.
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Provided is a sample live-cell movie of a HeLa cell transfected with the open reading frame for GFP-SKA1 driven by a cytomegalovirus promoter.
Format: MPG Size: 14.2MB Download file
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