Dynamics of centromere and kinetochore proteins; implications for checkpoint signaling and silencing.
BACKGROUND
The mitotic checkpoint prevents the onset of anaphase before all chromosomes are attached to spindle microtubules. The checkpoint is thought to act by the catalytic generation at unattached kinetochores of a diffusible "wait signal" that prevents anaphase. Mad2 and Cdc20, two candidate proteins for components of a diffusible wait signal, have previously been shown to be recruited to and rapidly released from unattached kinetochores.


RESULTS
Fluorescence recovery after photobleaching demonstrated that Mad1, Bub1, and a portion of Mad2, all essential mitotic-checkpoint components, are stably bound elements of unattached kinetochores (as are structural centromere components such as Centromere protein C [CENP-C]). After microtubule attachment, Mad1 and Mad2 are released from kinetochores and relocalize to spindle poles, whereas Bub1 remains at kinetochores.


CONCLUSIONS
A long residence time at kinetochores identifies Bub1, Mad1, and a portion of Mad2 as part of a catalytic platform that recruits, activates, and releases a diffusible wait signal that is partly composed of the rapidly exchanging portion of Mad2. The release of Mad1 and Mad2, but not Bub1, from kinetochores upon attachment separates the elements of this "catalytic platform" and thereby silences generation of the anaphase inhibitor despite continued rapid cycling of Mad2 at spindle poles.
Shah
Jagesh V
JV
Ludwig Institute for Cancer Research, Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA 92093 USA.
Botvinick
Elliot
E
Bonday
Zahid
Z
Furnari
Frank
F
Berns
Michael
M
Cleveland
Don W
DW
D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching; D005455:Fluorescent Antibody Technique; D018816:Genes, cdc / Q000502:physiology*; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000378:metabolism*; D064246:Mad2 Proteins; D008297:Male; D008394:Marsupialia; D009687:Nuclear Proteins; D010750:Phosphoproteins / Q000378:metabolism*; D011233:Precipitin Tests; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D012097:Repressor Proteins / Q000378:metabolism*; D012190:Retroviridae; D015398:Signal Transduction; D008941:Spindle Apparatus / Q000378:metabolism
D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D018076:DNA, Complementary; C501384:MAD1L1 protein, human; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D012097:Repressor Proteins; D011494:Protein Kinases; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases