Timing and Checkpoints in the Regulation of Mitotic Progression

Abstract

Accurate chromosome segregation relies on the precise regulation of mitotic progression. Regulation involves control over the timing of mitosis and a spindle assembly checkpoint that links anaphase onset to the completion of chromosome-microtubule attachment. In this paper, we combine live-cell imaging of HeLa cells and protein depletion by RNA interference to examine the functions of the Mad, Bub, and kinetochore proteins in mitotic timing and checkpoint control. We show that the depletion of any one of these proteins abolishes the mitotic arrest provoked by depolymerizing microtubules or blocking chromosome-microtubule attachment with RNAi. However, the normal progress of mitosis is accelerated only when Mad2 or BubR1, but not other Mad and Bub proteins, are inactivated. Moreover, whereas checkpoint control requires kinetochores, the regulation of mitotic timing by Mad2 and BubR1 is kinetochore-independent in fashion. We propose that cytosolic Mad2-BubR1 is essential to restrain anaphase onset early in mitosis when kinetochores are still assembling.

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

“…Mutation of Drosophila bub1 [Basu et al, 1999] or deletion of mad2 in mice [Dobles et al, 2000] or C. elegans [Kitagawa and Rose 1999] promotes chromosome mis-segregation and early embryonic lethality. These results suggest that the spindle checkpoint regulates mitotic timing in every cell cycle, an idea that is supported by experiments demonstrating that impaired function of Mad2p or Mad3p/BubR1p accelerates mitotic progression Meraldi et al, 2004a].…”

“…Similarly, human (h) Bub1p and hMad2 mRNA abundance is increased in proliferative tissues [Ouyang et al, 1998;Pangilinan et al, 1997]. The up-regulation of SAC proteins in rapidly dividing tissues may be necessary because of the critical role they play in regulating mitotic timing Meraldi et al, 2004a;Taylor and McKeon 1997].…”

The Spindle Assembly Checkpoint: Clock or Domino?

“…Later studies further indicated that the remaining checkpoint kinase Mps1, participated as Mad2 and BubR1 in the timing mechanism (Tighe et al 2008). Interestingly, the dual functions of Mad2 and BubR1 correlated with different protein pools, localized at kinetochores (checkpoint) or in the cytoplasm (timer), respectively (Meraldi et al 2004).…”

“…This indicated that the checkpoint proteins control the timing of cell division Gorbsky et al 1998;Shannon et al 2002;Taylor and McKeon 1997). A more detailed study based on RNAi depletions of all the human Mad and Bub proteins revealed the existence of two different, separable phenotypes: while all the Mad and Bub proteins are necessary for delaying anaphase in the presence of unattached kinetochores, only Mad2 and BubR1 depletion also resulted in a significant acceleration of mitosis when compared to control-depleted cells (Meraldi et al 2004). This suggested that Mad2 and BubR1, in addition to their checkpoint function, also act as mitotic "timers", while the other proteins, Bub1, Bub3, and Mad1, have a pure checkpoint role with regard to mitotic timing.…”

“…However, to prevent such a premature activation of APC/C towards Cyclin B and securin a cytosolic pool of Mad2 and Mad3/BubR1 is present that inhibits APC/C Cdc20 and thus functions as a mitotic timer, by inhibiting anaphase onset. As kinetochores rapidly recruit all their components, they will activate the spindle assembly checkpoint machinery in the absence of microtubule attachment, resulting in a sustained APC/C Cdc20 inhibition until all kinetochores are attached and the cell is ready to segregate their sister chromatids accurately (Meraldi et al 2004). An alternative hypothesis is that such a timing mechanisms represent a very basic form to prevent premature anaphase onset, without direct necessary input from the kinetochores.…”