Spindle checkpoint protein dynamics at kinetochores in living cells.
BACKGROUND
To test current models for how unattached and untense kinetochores prevent Cdc20 activation of the anaphase-promoting complex/cyclosome (APC/C) throughout the spindle and the cytoplasm, we used GFP fusions and live-cell imaging to quantify the abundance and dynamics of spindle checkpoint proteins Mad1, Mad2, Bub1, BubR1, Mps1, and Cdc20 at kinetochores during mitosis in living PtK2 cells.


RESULTS
Unattached kinetochores in prometaphase bound on average only a small fraction (estimated at 500-5000 molecules) of the total cellular pool of each spindle checkpoint protein. Measurements of fluorescence recovery after photobleaching (FRAP) showed that GFP-Cdc20 and GFP-BubR1 exhibit biphasic exponential kinetics at unattached kinetochores, with approximately 50% displaying very fast kinetics (t1/2 of approximately 1-3 s) and approximately 50% displaying slower kinetics similar to the single exponential kinetics of GFP-Mad2 and GFP-Bub3 (t1/2 of 21-23 s). The slower phase of GFP-Cdc20 likely represents complex formation with Mad2 since it was tension insensitive and, unlike the fast phase, it was absent at metaphase kinetochores that lack Mad2 but retain Cdc20 and was absent at unattached prometaphase kinetochores for the Cdc20 derivative GFP-Cdc20delta1-167, which lacks the major Mad2 binding domain but retains kinetochore localization. GFP-Mps1 exhibited single exponential kinetics at unattached kinetochores with a t1/2 of approximately 10 s, whereas most GFP-Mad1 and GFP-Bub1 were much more stable components.


CONCLUSIONS
Our data support catalytic models of checkpoint activation where Mad1 and Bub1 are mainly resident, Mad2 free of Mad1, BubR1 and Bub3 free of Bub1, Cdc20, and Mps1 dynamically exchange as part of the diffuse wait-anaphase signal; and Mad2 interacts with Cdc20 at unattached kinetochores.
Howell
Bonnie J
BJ
Department of Biology, CB#3280, 607 Fordham Hall, University of North Carolina, Chapel Hill, NC 27599 USA.
Moree
Ben
B
Farrar
Emily M
EM
Stewart
Scott
S
Fang
Guowei
G
Salmon
E D
ED
D064173:Anaphase-Promoting Complex-Cyclosome; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000378:metabolism; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism*; D003952:Diagnostic Imaging; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000378:metabolism*; D008164:Luminescent Proteins; D064246:Mad2 Proteins; D008667:Metalloproteins / Q000378:metabolism; D008938:Mitosis / Q000502:physiology*; D008954:Models, Biological; D009687:Nuclear Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D011233:Precipitin Tests; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D016601:RNA-Binding Proteins; D012097:Repressor Proteins / Q000378:metabolism; D012269:Ribosomal Proteins; D015398:Signal Transduction; D014162:Transfection; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism / Q000502:physiology*
D002135:Calcium-Binding Proteins; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; D008164:Luminescent Proteins; C501384:MAD1L1 protein, human; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D008667:Metalloproteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; C083686:RPS27 protein, human; D012097:Repressor Proteins; D012269:Ribosomal Proteins; D049452:Green Fluorescent Proteins; C577488:CDC20 protein, human; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D011494:Protein Kinases; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases