Mad2 and BubR1 function in a single checkpoint pathway that responds to a loss of tension.
The spindle checkpoint monitors microtubule attachment and tension at kinetochores to ensure proper chromosome segregation. Previously, PtK1 cells in hypothermic conditions (23 degrees C) were shown to have a pronounced mitotic delay, despite having normal numbers of kinetochore microtubules. At 23 degrees C, we found that PtK1 cells remained in metaphase for an average of 101 min, compared with 21 min for cells at 37 degrees C. The metaphase delay at 23 degrees 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 degrees C, and metaphase kinetochores at 23 degrees C exhibited higher levels of 3F3/2, Bub1, and BubR1 compared with 37 degrees C. Microinjection of anti-BubR1 antibody abolished the metaphase delay at 23 degrees 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.
Shannon
Katie B
KB
SPIRE fellow, Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599-3280, USA. ktphd@email.unc.edu
Canman
Julie C
JC
Salmon
E D
ED
D000818:Animals; D000906:Antibodies / Q000378:metabolism; D000972:Antineoplastic Agents, Phytogenic / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002875:Chromosomes / Q000378:metabolism; D000939:Epitopes / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching; D005456:Fluorescent Dyes / Q000378:metabolism; D018816:Genes, cdc; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000378:metabolism; D064246:Mad2 Proteins; D008845:Microinjections; D008870:Microtubules / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins; D008941:Spindle Apparatus / Q000378:metabolism*; D013314:Stress, Mechanical; D013696:Temperature; D014747:Vinblastine / Q000378:metabolism
D000906:Antibodies; D000972:Antineoplastic Agents, Phytogenic; D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D000939:Epitopes; D005456:Fluorescent Dyes; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014747:Vinblastine; D011494:Protein Kinases; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases