Recruitment of Mad2 to the Kinetochore Requires the Rod/Zw10 Complex

Abstract

Compromising the activity of the spindle checkpoint permits mitotic exit in the presence of unattached kinetochores and, consequently, greatly increases the rate of aneuploidy in the daughter cells. The metazoan checkpoint mechanism is more complex than in yeast in that it requires additional proteins and activities besides the classical Mads and Bubs. Among these are Rod, Zw10, and Zwilch, components of a 700 Kdal complex (Rod/Zw10) that is required for recruitment of dynein/dynactin to kinetochores but whose role in the checkpoint is poorly understood. The dynamics of Rod and Mad2, examined in different organisms, show intriguing similarities as well as apparent differences. Here we simultaneously follow GFP-Mad2 and RFP-Rod and find they are in fact closely associated throughout early mitosis. They accumulate simultaneously on kinetochores and are shed together along microtubule fibers after attachment. Their behavior and position within attached kinetochores is distinct from that of BubR1; Mad2 and Rod colocalize to the outermost kinetochore region (the corona), whereas BubR1 is slightly more interior. Moreover, Mad2, but not BubR1, Bub1, Bub3, or Mps1, requires Rod/Zw10 for its accumulation on unattached kinetochores. Rod/Zw10 thus contributes to checkpoint activation by promoting Mad2 recruitment and to checkpoint inactivation by recruiting dynein/dynactin that subsequently removes Mad2 from attached kinetochores.

In Vivo Dynamics ofDrosophilaNuclear Envelope Components

“…Larval neuroblasts were prepared and imaged as described previously (Buffin et al, 2005), using an Olympus IX-70 inverted microscope equipped with a focused xenon lamp and an OrcaER camera (Hammamatsu, Bridgewater, NJ), piloted by Cell-R hardware and software system (Olympus). Depending on the fly line, image acquisition times were 100 -400 ms for GFP and 250 -400 ms for red fluorescent protein (RFP).…”

“…In Drosophila neuroblasts, Mad2 is primarily in the nucleoplasm, but a fraction of it is associated with the NE (Buffin et al, 2005). To further explore Mad2's association with the NE, we directly compared the behavior of GFP-Mad2 with that of mRFP-Nup107 during mitotic entry and exit.…”

Systems Biology Modeling of Five Pathways for Regulation and Potent Inhibition of the Anaphase-Promoting Complex (APC/C): Pivotal Roles for MCC and BubR1

“…The formation of Bub3:BubR1:Cdc20 is accelerated in the presence of unattached chromosomes (Kulukian et al, 2009a) and it may be that MCC forms as an intermediate complex from which OMad2 rapidly dissociates (Kulukian et al, 2009a;Malureanu et al, 2009;Medema, 2009). Microtubule attachment depletes the template-complexes from the respective kinetochore, thereby silencing SAC-signaling locally (Buffin et al, 2005;Sivaram et al, 2009). Thus, after proper attachment of the last chromosome, SAC signaling ceases and passage to anaphase is granted.…”

The perpetual movements of anaphase

“…In another study, Savoian and colleagues used zw10 and rod Drosophila mutant spermatocytes to specifically perturb dynein localization at kinetochores [99,100] and showed that anaphase chromosome motion to the poles was severely affected in those mutants. However, some potential caveats with this experimental approach are related with the fact that zw10/rod are also required to recruit the SAC protein Mad2 to unattached kinetochores, thereby compromising SAC function [101][102][103]. Kinetochore dynein is also involved in the initial MT capture at the entry of mitosis, which could indirectly compromise chromosome motility if anaphase is triggered with normal kinetics [100].…”