Those kinetochores that lacked an associated bundle of microtubules (K-fiber) had intense MAD2 staining, whereas in the presence of a K-fiber the MAD2 staining was reduced or absent (Fig. meiosis) is likely to affect the relative contributions of attachment and tension. We support the idea that MAD2 is usually attachment-sensitive and that tension stabilizes microtubule attachments. homologues of MAD2 bind to kinetochores that are not attached by microtubules (Chen et al., 1996; Li and Benezra, 1996). As soon as the chromosomes properly attach to the spindle, MAD2 staining is usually lost and is not visible at kinetochores again until the next cell cycle. A single unaligned chromosome is sufficient to activate the spindle checkpoint (Nicklas, 1997), and only unaligned chromosomes stain positive for Procyclidine HCl MAD2 (Chen et al., 1996; Waters et al., 1998). Apparently the availability of free microtubule binding sites or an absence of tension around the kinetochore causes MAD2 to be recruited to kinetochores where it activates the spindle checkpoint (Elledge, 1996). In a study of animal mitotic cells Procyclidine HCl designed to differentiate between these two alternatives, the disappearance of MAD2 staining appeared to be more dependent on microtubule attachment than tension (Waters et al., 1998). No studies have yet been published around the localization of MAD2 Procyclidine HCl in meiotic cells. Recent studies have provided the necessary link between MAD2 and the cell cycle regulatory proteins that initiate anaphase (Elledge, 1998). The link is usually Cdc20 (with homologues known as Sleepy, p55CDC, and Fizzy), a protein that Rabbit polyclonal to INMT imparts substrate specificity to the anaphase-promoting complex (APC1; Visitin et al., 1997). The APC is usually involved in the ubiquitination and degradation of proteins such as Pds1 that inhibit the onset of anaphase (King et al., 1996). Evidence from a variety of sources suggest that Mad2 delays anaphase because it not only interacts with (Fang et al., 1998; Hwang et al., 1998; Kallio et al., 1998; Kim et al., 1998; Wassmann and Benezra, 1998), but inhibits the action of Cdc20 (Kim et al., 1998). Unattached kinetochores may act as catalytic sites for the activation of MAD2, allowing the active MAD2 or CDC20/MAD2 to diffuse and inhibit APC activity throughout the cell (Gorbsky et Procyclidine HCl al., 1998; Kallio et al., 1998). Cytological evidence in animal systems suggests that protein phosphorylation, perhaps regulated by tension, plays a key role in the spindle checkpoint pathway (Campbell and Gorbsky, 1995; Nicklas, 1997). The Procyclidine HCl 3F3/2 antibody recognizes a phosphoepitope that is localized to prometaphase kinetochores until the chromosomes have aligned properly at the metaphase plate (Gorbsky and Ricketts, 1993; Nicklas et al., 1995). A strong correlation exists between 3F3/2 staining, tension at the kinetochore, and progression to anaphase. When tension is usually manually applied to a single unaligned chromosome, anaphase commences (Li and Nicklas, 1995) and 3F3/2 staining disappears (Li and Nicklas, 1997; Nicklas, 1997). Further, when the 3F3/2 antibody is usually injected into metaphase cells, anaphase onset is delayed (Campbell and Gorbsky, 1995). Although the 3F3/2 epitope appears to have an important checkpoint function, no information is usually yet available whether the epitope and its function are broadly conserved among eukaryotes. Here we describe the identification of a maize homologue of MAD2 and detailed immunolocalization studies designed to investigate its role in the spindle checkpoint. The data are interpreted in the context of apparent differences in both kinetochore morphology and spindle formation between plants and animals. Whereas animal kinetochores have a three-layered morphology (Earnshaw, 1994), herb kinetochores have a nondescript ball-shaped structure (e.g., Braselton and Bowen, 1971; Jensen, 1982). Animal mitotic spindles are initiated from centrosomes at the spindle poles, whereas herb spindles and animal meiotic spindles are initiated from the nuclear envelope or the chromosomes (Baskin and Cande, 1990; Smirnova and Bajer, 1992; Rieder et al., 1993; Waters and Salmon, 1997). Our data indicate that MAD2 localization patterns in mitosis are basically conserved among eukaryotes, but that at least in maize, the localization patterns in meiosis differ from those in mitosis. Based on MAD2 as well as 3F3/2 staining, we argue that microtubule attachment has a major role in the mitotic spindle checkpoint but the meiotic spindle checkpoint may rely more heavily on sensing the amount of tension at the kinetochore. Materials and Methods Generation of Recombinant Proteins and Antibodies The clones of two ESTs (expressed sequence tags) were gifts from Pioneer Hi-Bred. At.