Rapid microtubule-independent dynamics of Cdc20 at kinetochores and centrosomes in mammalian cells.
Cdc20 is a substrate adaptor and activator of the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase whose activity is required for anaphase onset and exit from mitosis. A green fluorescent protein derivative, Cdc20-GFP, bound to centrosomes throughout the cell cycle and to kinetochores from late prophase to late telophase. We mapped distinct domains of Cdc20 that are required for association with kinetochores and centrosomes. FRAP measurements revealed extremely rapid dynamics at the kinetochores (t1/2 = 5.1 s) and spindle poles (t1/2 = 4.7 s). This rapid turnover is independent of microtubules. Rapid transit of Cdc20 through kinetochores may ensure that spindle checkpoint signaling at unattached/relaxed kinetochores can continuously inhibit APC/CCdc20 targeting of anaphase inhibitors (securins) throughout the cell until all the chromosomes are properly attached to the mitotic spindle.
Kallio
Marko J
MJ
University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA. marko-kallio@ouhsc.edu
Beardmore
Victoria A
VA
Weinstein
Jasminder
J
Gorbsky
Gary J
GJ
D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism*; D018385:Centrosome / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000378:metabolism*; D018374:LLC-PK1 Cells; D008025:Ligases / Q000378:metabolism; D064246:Mad2 Proteins; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism; D008938:Mitosis; D008954:Models, Biological; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D013997:Time Factors; D043743:Ubiquitin-Protein Ligase Complexes
C577495:CDC20 protein, S cerevisiae; D002135:Calcium-Binding Proteins; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D011494:Protein Kinases; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases; D008025:Ligases