2000), and in mammalian cells and cytoplasmic extracts, electroporation or microinjection of anti-Mad2 antibodies allows cells that have not aligned all of their chromosomes on the spindle to enter anaphase (Chen et al. from activating the APC. To test this model, we examined Mad2 dynamics in living PtK1 cells that were either injected with fluorescently labeled Alexa 488-XMad2 or transfected with GFP-hMAD2. Real-time, digital imaging revealed fluorescent Mad2 localized to unattached kinetochores, spindle poles, and spindle fibers depending on the stage PF429242 dihydrochloride of mitosis. FRAP measurements showed that Mad2 is a transient component of unattached kinetochores, as predicted by the catalytic model, with a Mad2 (XMad2) shares a 41 and 81% identity with the yeast (scMad2) and PF429242 dihydrochloride human (hMad2) homologues, respectively (Chen et al. 1996; Li and Benezra 1996). Further studies have shown Mad2 to have important roles in the spindle checkpoint in these organisms (for review see Hardwick 1998). For instance, in yeast and mice, the absence of Mad2 results in increased frequency of spontaneous chromosome loss (Li and Murray 1991; Amon, 1998; Hardwick 1998; Dobles et al. 2000), and in mammalian cells and cytoplasmic extracts, electroporation or microinjection of anti-Mad2 antibodies allows cells that have not aligned all of their chromosomes on the spindle to enter anaphase (Chen et al. 1996, Chen et al. 1998; Li and Benezra 1996; Gorbsky et al. 1998; Waters et al. 1998; Canman et al. 2000). Furthermore, Mad2-null mouse cells do not arrest in response to spindle damage, show widespread chromosome missegregation, and undergo apoptosis during initiation of gastrulation (Dobles et al. 2000). Mad2 and several other checkpoint components have been shown to immunolocalize to kinetochores that lack bound microtubules or have only a small fraction of their microtubule binding sites occupied, and to disappear from kinetochores as they become fully occupied with kinetochore microtubules during chromosome alignment at the spindle equator in prometaphase (for reviews see Hardwick 1998; Rieder and Salmon 1998; Amon 1999). Mad1 has been shown to be required PF429242 dihydrochloride for Mad2 localization to unattached kinetochores (Chen et al. 1998). A variety of evidence suggests the defects that activate the spindle checkpoint occur at kinetochores (Rieder and Salmon 1998). In 1995, Rieder et al. demonstrated that destruction of the last unattached kinetochore by laser ablation FANCD1 causes cells to enter anaphase. Furthermore, using micromanipulation of meiotic insect spermatocytes to move a chromosome far enough away from the metaphase plate to interfere with microtubule attachment inhibited anaphase onset (Li and Nicklas 1995; Zhang and Nicklas 1996). How does the cell know that a single kinetochore is improperly attached to the spindle? Recent studies have identified a link between Mad2 and the cell cycle proteins that regulate anaphase onset (for review see Elledge 1998). Initial studies in fission yeast identified a genetic interaction between Mad2 and the anaphase-promoting complex (APC; He et al. 1997). APC is involved in the ubiquitination and degradation of cyclin B1 (Holloway et al. 1993) and anaphase inhibitory proteins, such as Pds1/Cut2 (King et al. 1996), and its activation during mitosis requires an interaction with Cdc20 (Visitin et al. 1997; Fang et al. PF429242 dihydrochloride 1998; Hwang et al. 1998). Cdc20 has been shown to localize to kinetochores from late prophase to telophase, and partially to spindle microtubules and spindle poles (Kallio et al. 1998). Genetic analysis in fission and budding yeasts and biochemical experiments in frog egg extracts have shown that Cdc20 is the target that the spindle checkpoint inhibits; mutations in Cdc20 that block the binding of Mad2 destroy the checkpoint, and a PF429242 dihydrochloride tetrameric form of Mad2 binds Cdc20 and interferes with its ability to activate the APC in in vitro assays (Fang et al. 1998; Hwang et al. 1998; Kim et al. 1998). Therefore, the current model proposes that unattached kinetochores serve as catalytic sites for assembling Mad2CCdc20 complexes,.