In vivo dynamics of the rough deal checkpoint protein during Drosophila mitosis.
Rough Deal (Rod) and Zw10 are components of a complex required for the metazoan metaphase checkpoint and for recruitment of dynein/dynactin to the kinetochore. The Rod complex, like most classical metaphase checkpoint components, forms part of the outer domain of unattached kinetochores. Here we analyze the dynamics of a GFP-Rod chimera in living syncytial Drosophila embryos. Uniquely among checkpoint proteins, GFP-Rod robustly streams from kinetochores along microtubules, from the time of chromosome attachment until anaphase onset. Prometaphase and metaphase kinetochores continuously recruit new Rod, thus feeding the current. Rod flux from kinetochores appears to require biorientation but not tension because it continues in the presence of taxol. As with Mad2, kinetochore- and spindle-associated Rod rapidly turns over with free cytosolic Rod, both during normal mitosis and after colchicine treatment, with a t1/2 of 25-45 s. GFP-Rod coimmunoprecipitates with dynein/dynactin, and in the absence of microtubules both Rod and dynactin accumulate on kinetochores. Nevertheless, Rod and dynein/dynactin behavior are distinguishable. We propose that the Rod complex is a major component of the fibrous corona and that the recruitment of Rod during metaphase is required to replenish kinetochore dynein after checkpoint conditions have been satisfied but before anaphase onset.
Basto
Renata
R
Centre de National de Récherche Scientifique, Centre de Génétique Moléculaire, Avenue de la Terasse, 91198, Gif-sur-Yvette, France.
Scaerou
Frédéric
F
Mische
Sarah
S
Wojcik
Edward
E
Lefebvre
Christophe
C
Gomes
Rui
R
Hays
Thomas
T
Karess
Roger
R
D000818:Animals; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000378:metabolism*; D003078:Colchicine; D004330:Drosophila; D029721:Drosophila Proteins / Q000378:metabolism; D004398:Dyneins / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D018386:Kinetochores / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008938:Mitosis / Q000502:physiology*; D017239:Paclitaxel; D011233:Precipitin Tests
D018797:Cell Cycle Proteins; D029721:Drosophila Proteins; D008164:Luminescent Proteins; D008869:Microtubule-Associated Proteins; C064993:ROD protein, Drosophila; D049452:Green Fluorescent Proteins; C076055:Zw10 protein, Drosophila; D004398:Dyneins; D017239:Paclitaxel; D003078:Colchicine