Anaphase onset in vertebrate somatic cells is controlled by a checkpoint that monitors sister kinetochore attachment to the spindle.

Abstract

Abstract. To test the popular but unproven assumption that the metaphase-anaphase transition in vertebrate somatic cells is subject to a checkpoint that monitors chromosome (i.e., kinetochore) attachment to the spindle, we filmed mitosis in 126 PtK t cells. We found that the time from nuclear envelope breakdown to anaphase onset is linearly related (r 2 = 0.85) to the duration the cell has unattached kinetochores, and that even a single unattached kinetochore delays anaphase onset. We also found that anaphase is initiated at a relatively constant 23-min average interval after the last kinetochore attaches, regardless of how long the cell possessed unattached kinetochores. From these results we conclude that vertebrate somatic cells possess a metaphase-anaphase checkpoint control that monitors sister kinetochore attachment to the spindle.We also found that some cells treated with 0.3-0.75 nM Taxol, after the last kinetochore attached to the spindle, entered anaphase and completed normal poleward chromosome motion (anaphase A) up to 3 h after the treatment-well beyond the 9-48-min range exhibited by untreated cells. The fact that spindle bipolarity and the metaphase alignment of kinetochores are maintained in these cells, and that the chromosomes move poleward during anaphase, suggests that the checkpoint monitors more than just the attachment of microtubules at sister kinetochores or the metaphase alignment of chromosomes. Our data are most consistent with the hypothesis that the checkpoint monitors an increase in tension between kinetochores and their associated microtubules as biorientation occurs.T HE transition from metaphase to anaphase is a key cell cycle event that commits the cell to exit mitosis and enter a new interphase (reviewed in Murray, 1992;Sluder and Rieder, 1993). Since the equal segregation of chromosomes at mitosis is predicated on each acquiring a bipolar attachment to the spindle before anaphase onset, the metaphase-anaphase transition must be tightly coordinated with proper completion of chromosome attachment. Ensuring the essential coordination of these events would be relatively straightforward if the mitotic portion of the cell cycle was fixed in duration, but sufficiently long so that all chromosomes had time to acquire a proper attachment before disjoining. However, the stochastic nature of spindle formation makes chromosome attachment an unpredictable and errorprone process. In vertebrate cells a chromosome first attaches when one of its kinetochores, usually the one closest to and facing a spindle pole at nuclear envelope breakdown

Mad2 and BubR1 Function in a Single Checkpoint Pathway that Responds to a Loss of Tension

“…This is in contrast to our PtK1 cells at 37°C, which spent an average of 21 min in metaphase (N ϭ 8), ranging from 8 to 32 min. This period is similar to previous findings that PtK1 cells at 37°C spend an average of 23 min between when the last chromosome begins congression and anaphase onset (Rieder et al, 1994). At the lower temperature, sister kinetochores exhibited poleward and antipoleward oscillations near the spindle equator during the extended metaphase, motility typical of metaphase kinetochores at 37°C but with slightly slower velocities, as previously reported (Wise et al, 1991).…”

SAC‐ing mitotic errors: How the spindle assembly checkpoint (SAC) plays defense against chromosome mis‐segregation

“…However, it is likely that an active mechanism is involved in relieving SAC signaling because anaphase begins within minutes after the last mis-aligned chromosome congresses to the metaphase plate [Rieder et al, 1994] or after laser ablation of the last unattached kinetochore [Rieder et al, 1995]. Such a mechanism may involve the active displacement of spindle checkpoint proteins from the kinetochore once a stable attachment to the spindle is established [Wojcik et al, 2001].…”

Centrobin regulates the assembly of functional mitotic spindles

“…However, a number of conditions can lead to SAC activation in metaphase cells including kinetochore malformation, defects in spindle dynamics and centrosome fragmentation as they prevent the formation of stable microtubule-kinetochore attachments (Lampson and Kapoor, 2005;Oshimori et al, 2006;Wong and Fang, 2006). The fidelity of the SAC is such that it can detect even a single unattached kinetochore (Rieder et al, 1994).…”