Rieder's story began with a 1988 call from Leland Hartwell, shortly before Hartwell had put forward the idea of cell cycle checkpoints (Hartwell and Weinert, 1989). Hartwell asked whether anyone had definitively shown that cells delayed anaphase until all chromosomes were hooked up to the spindle. Rieder noted that there was one obscure abstract concluding that newt cells never started anaphase in the presence of a monooriented chromosome (Zirkle, 1970). And Sluder, as a graduate student, had found that inhibiting spindle assembly delayed anaphase onset in sea urchin eggs (Sluder, 1979). But although there were some anecdotal reports of a chromosome attachment or spindle assembly checkpoint in other cell types, a proper study had not been done.
Schultz, Rieder, and Sluder find that unattached kinetochores tell mitotic cells to wait before dividing.
Now, the two road-trippers thought, was the perfect time to test the idea in mammalian somatic cells before someone else did. Rieder already had an undergraduate, Adriene Schultz, collecting video data on 126 individual PtK1 cells, which remain flat through mitosis and have 12 easy-to-follow chromosomes. She was measuring the time from nuclear envelope breakdown to anaphase onset and how long the cell had unattached kinetochores. The data lined up on a near-perfect linear regression (Rieder et al., 1994).
In addition, the group determined that a single unattached kinetochore was enough to delay anaphase. But once the last kinetochore attached to the spindle, anaphase would always proceed ∼20 min later. Sluder suggested using the drug Taxol to artificially delay microtubule attachment for up to three hours. These cells also entered anaphase when the last kinetochore joined up.
“The unattached kinetochore was doing something that shuts down the whole spindle system,” says Sluder. According to Rieder: “The next question was, what does the cell monitor? Either it was monitoring bipolar attachment [of each chromosome] or the unattached kinetochore is screaming ‘Wait!’ through negative feedback.”
To test what the checkpoint monitored, the group relied on a handy laser set-up. Rieder had installed it to do chromosome microsurgery for aster ejection force studies. But he now found that ablating the centromere of the last unattached chromosome—to destroy either the entire centromeric region or just the unattached kinetochore—could override the checkpoint (Rieder et al., 1995). Blasting off a kinetochore on one of the already attached chromosomes to create a new monooriented chromosome did not delay the cell cycle, however. This indicated that the inhibitory signal was coming from the unattached kinetochore itself.
Sluder and Rieder went on to show that in cells with multipolar spindles any unattached chromosome pair would delay the whole system (Sluder et al., 1997), although in fused cells with two spindles the inhibitory signal was specific to the spindle with the unattached kinetochore (Rieder et al., 1997). By then, multiple spindle assembly checkpoint proteins, such as Mad2, had been identified, and they began to show up at the kinetochore (Chen et al., 1996). A literature battle waged for a while about whether the checkpoint was relieved by tension on the bioriented kinetochore or microtubule occupancy of the kinetochore. Occupancy won out when Mad2 binding was shown to be dependent on accumulated microtubules, although tension probably plays an important upstream role (Nicklas et al., 2001).