Rough Deal (Rod) and Zw10 are components of a complex required for the metazoan metaphase checkpoint [1, 2] 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.
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We thank A. Royou, S. Brown, C. Talbot and A. Sossick for confocal help, M. Serr for immunoprecipitations, J. Minden for rhodamine-histone, and J. Raff for fly stocks, and advice. Work at the CGM, UPR2167 was supported by CNRS, ARC (France) for RK; the FCT (Portugal) grants BD/11488/97 and P/BIA/111055/1998 for RB; the MNESR (France) for FS. Grants to TH and EW were NIH GM44757, and GM19123, respectively, and the American Heart Association (96002200). The Gif confocal facility was supported by IFR87 “La plante et son environment” and Conseil Général de l'Essonne (Programme ASTRE).