The spindle checkpoint monitors microtubule attachment and tension at kinetochores to ensure proper chromosome segregation. Previously, PtK1 cells in hypothermic conditions (23°C) were shown to have a pronounced mitotic delay, despite having normal numbers of kinetochore microtubules. At 23°C, we found that PtK1 cells remained in metaphase for an average of 101 min, compared with 21 min for cells at 37°C. The metaphase delay at 23°C was abrogated by injection of Mad2 inhibitors, showing that Mad2 and the spindle checkpoint were responsible for the prolonged metaphase. Live cell imaging showed that kinetochore Mad2 became undetectable soon after chromosome congression. Measurements of the stretch between sister kinetochores at metaphase found a 24% decrease in tension at 23°C, and metaphase kinetochores at 23°C exhibited higher levels of 3F3/2, Bub1, and BubR1 compared with 37°C. Microinjection of anti-BubR1 antibody abolished the metaphase delay at 23°C, indicating that the higher kinetochore levels of BubR1 may contribute to the delay. Disrupting both Mad2 and BubR1 function induced anaphase with the same timing as single inhibitions, suggesting that these checkpoint genes function in the same pathway. We conclude that reduced tension at kinetochores with a full complement of kinetochore microtubules induces a checkpoint dependent metaphase delay associated with elevated amounts of kinetochore 3F3/2, Bub1, and BubR1 labeling.


Biological Sciences

Keywords and Phrases

Bub1 Related Protein; Protein; Protein Mad2; Unclassified Drug; Vinblastine; Antibody; Antineoplastic Agent; Bub1 Spindle Checkpoint Protein; Calcium Binding Protein; Cell Cycle Protein; Epitope; Fluorescent Dye; Hybrid Protein; MAD2L1 Protein, Human; Protein Kinase; Repressor Protein; Centromere; Chromosome; Fluorescence Microscopy; HeLa Cell; Human; Human Cell; Metaphase; Mitosis; Priority Journal; Animal; Cell Cycle; Cell Line; Fluorescence Recovery After Photobleaching; Gene; Genetics; Mechanical Stress; Metabolism; Microinjection; Microtubule; Mitosis Spindle; Physiology; Temperature; Animals; Antibodies; Antineoplastic Agents, Phytogenic; Calcium-Binding Proteins; Cell Cycle; Cell Cycle Proteins; Cell Line; Chromosomes; Epitopes; Fluorescence Recovery After Photobleaching; Fluorescent Dyes; Genes, Cdc; Hela Cells; Humans; Kinetochores; Microinjections; Microtubules; Mitotic Spindle Apparatus; Protein Kinases; Recombinant Fusion Proteins; Repressor Proteins; Stress, Mechanical; Temperature; Vinblastine

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© 2002 American Society for Cell Biology, All rights reserved.

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