Intrakinetochore Stretch Is Associated with Changes in Kinetochore Phosphorylation and Spindle Assembly Checkpoint Activity

• Published in: The Journal of cell biology Vol. 184; no. 3; pp. 373 - 381
• Main Authors: Maresca, Thomas J., Salmon, Edward D.
• Format: Journal Article
• Language: English
• Published: United States Rockefeller University Press 09.02.2009; The Rockefeller University Press
• Subjects: Anaphase; Animals; Cell cycle; Cell Cycle Proteins - genetics; Cell Cycle Proteins - metabolism; Cell division; Cell Line; Cells; Centromere - metabolism; Centromere - ultrastructure; Centromeres; Chromatin; Chromosomes; Colchicine - metabolism; Double stranded RNA; Drosophila melanogaster; Drosophila Proteins - genetics; Drosophila Proteins - metabolism; Epitopes - metabolism; Imaging; Kinetochores; Kinetochores - metabolism; Kinetochores - ultrastructure; Metaphase; Microtubules; Mitosis; Mitotic spindle apparatus; Paclitaxel - metabolism; Phosphorylation; Proteins; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; RNA Interference; Spindle Apparatus - metabolism; Spindle Apparatus - ultrastructure; Stress, Mechanical; Tubulin Modulators - metabolism
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.200808130

Cells have evolved a signaling pathway called the spindle assembly checkpoint (SAC) to increase the fidelity of chromosome segregation by generating a "wait anaphase" signal until all chromosomes are properly aligned within the mitotic spindle. It has been proposed that tension generated by the stretch of the centromeric chromatin of bioriented chromosomes stabilizes kinetochore microtubule attachments and turns off SAC activity. Although biorientation clearly causes stretching of the centromeric chromatin, it is unclear whether the kinetochore is also stretched. To test whether intrakinetochore stretch occurs and is involved in SAC regulation, we developed a Drosophila melanogaster S2 cell line expressing centromere identifier-mCherry and Ndc80-green fluorescent protein to mark the inner and outer kinetochore domains, respectively. We observed stretching within kinetochores of bioriented chromosomes by monitoring both inter- and intrakinetochore distances in live cell assays. This intrakinetochore stretch is largely independent of a 30-fold variation in centromere stretch. Furthermore, loss of intrakinetochore stretch is associated with enhancement of 3F3/2 phosphorylation and SAC activation.