The spindle checkpoint was characterized in meiosis of budding yeast. In the absence of the checkpoint, the frequency of meiosis I missegregation increased with increasing chromosome length, reaching 19% for the longest chromosome. Meiosis I nondisjunction in spindle checkpoint mutants could be prevented by delaying the onset of anaphase. In a recombination-defective mutant (spo11Delta), the checkpoint delays the biochemical events of anaphase I, suggesting that chromosomes that are attached to microtubules but are not under tension can activate the spindle checkpoint. Spindle checkpoint mutants reduce the accuracy of chromosome segregation in meiosis I much more than that in meiosis II, suggesting that checkpoint defects may contribute to Down syndrome.

We established a light microscopy-based assay that reconstitutes the binding of phagosomes purified from mouse macrophages to preassembled F-actin in vitro. Both endogenous myosin Va from mouse macrophages and exogenous myosin Va from chicken brain stimulated the phagosome-F-actin interaction. Myosin Va association with phagosomes correlated with their ability to bind F-actin in an ATP-regulated manner and antibodies to myosin Va specifically blocked the ATP-sensitive phagosome binding to F-actin. The uptake and retrograde transport of phagosomes from the periphery to the center of cells in bone marrow macrophages was observed in both normal mice and mice homozygous for the dilute-lethal spontaneous mutation (myosin Va null). However, in dilute-lethal macrophages the accumulation of phagosomes in the perinuclear region occurred twofold faster than in normal macrophages. Motion analysis revealed saltatory phagosome movement with temporarily reversed direction in normal macrophages, whereas almost no reversals in direction were observed in dilute-lethal macrophages. These observations demonstrate that myosin Va mediates phagosome binding to F-actin, resulting in a delay in microtubuledependent retrograde phagosome movement toward the cell center. We propose an "antagonistic/cooperative mechanism" to explain the saltatory phagosome movement toward the cell center in normal macrophages.

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Cell cycle checkpoints sense defects in chromosome metabolism, halt the cell cycle, and activate pathways that repair the defects. The spindle checkpoint arrests the cell cycle in response to defects in the interaction between microtubules and kinetochores (the proteinaceous complex assembled on centromeric DNA), but no repair function has been demonstrated for this checkpoint. We show that the roles of two spindle checkpoint components, Mad2 and Mad3, differ in meiosis I. In the absence of Mad2, meiosis I nondisjunction occurs at a high frequency and can be corrected by delaying the onset of anaphase. The absence of Mad3 does not induce nondisjunction, even though mad3Delta cells cannot arrest the cell cycle in response to kinetochores that lack either microtubules or tension on the linkage between chromosomes and microtubules. The two proteins have different roles in chromosome alignment. Compared to wild type and mad3Delta cells, mad2Delta mutants are slower to attach homologous chromosomes to opposite poles of the spindle. This observation suggests that Mad2 plays a role in reorienting chromosomes that are incorrectly attached to the spindle as well as delaying the cell cycle, whereas Mad3 is needed for the cell cycle delay, but not for chromosome reorientation.