Waiting for Anaphase: Mad2 and the Spindle Assembly Checkpoint

Although all the pieces of the puzzle may seem to be in place, there are still several key processes that have been widely posited but remain experimentally unverified. What attracts Mad2 to the kinetochore and what causes its release? Is it Mad1, as has been hypothesized from its apparent loss (along with Mad2) from the kinetochore after microtubule attachment (Figure 3AFigure 3A)? A pair of reports reveal a fly in the ointment (3xBasto, R., Gomes, R., and Karess, R.E. Nat. Cell Biol. 2000; 2: 939–943Crossref | PubMed | Scopus (91)See all References, 5xChan, G.K.T., Jablonski, S.A., Starr, D.A., Goldberg, M.L., and Yen, T.J. Nat. Cell Biol. 2000; 2: 944–947Crossref | PubMed | Scopus (128)See all References). Specifically, they demonstrate that the zeste-white 10 protein (Zw10) and its binding partner, rough deal (Rod), both originally discovered in Drosophila and without homologs in yeast, are required for activation of the spindle checkpoint in human cells and flies. These proteins have been previously shown to recruit dynein and dynactin to the kinetochore and, upon microtubule attachment, to “stream” along kinetochore microtubules to the spindle poles—an attractive mechanism for turning the checkpoint off. So it comes as a big surprise that disruption of Zw10 or Rod function genetically (Basto et al. 2000xBasto, R., Gomes, R., and Karess, R.E. Nat. Cell Biol. 2000; 2: 939–943Crossref | PubMed | Scopus (91)See all ReferencesBasto et al. 2000) or with antibodies (Chan et al. 2000xChan, G.K.T., Jablonski, S.A., Starr, D.A., Goldberg, M.L., and Yen, T.J. Nat. Cell Biol. 2000; 2: 944–947Crossref | PubMed | Scopus (128)See all ReferencesChan et al. 2000) did not arrest cells in mitosis but instead prevented cells from activating the checkpoint in the presence of spindle damage. In human cells, antibody inhibition of Zw10 or Rod function leaves Mad2, the trusty wait signal, still present at the kinetochores of the unattached chromosomes even though the checkpoint is off (Chan et al. 2000xChan, G.K.T., Jablonski, S.A., Starr, D.A., Goldberg, M.L., and Yen, T.J. Nat. Cell Biol. 2000; 2: 944–947Crossref | PubMed | Scopus (128)See all ReferencesChan et al. 2000)!Figure 3Unresolved Questions in the Spindle CheckpointFeatures of checkpoint signaling yet to be experimentally verified include (A) what is Mad2*, (B) what deactivates it, and (C) how do the components upstream of Mad2 generate Mad2* at unattached kinetochores and how is such signaling silenced upon microtubule attachment?View Large Image | View Hi-Res Image | Download PowerPoint SlideHow could this be? One possible interpretation is that some proteins may be required to bring Mad2 to the kinetochore (e.g., Mad1), whereas others, Zw10 or Rod for example, may be responsible for releasing Mad2*. If this is the case, Zw10 and Rod must only be important for releasing Mad2* from unattached kinetochores because Mad2 is released from attached kinetochores despite the loss of Zw10 and Rod (Figure 3AFigure 3A). This hypothesis is now directly testable—does Mad2 fail to cycle at unattached kinetochores in Zw10 or Rod disrupted cells?Determining the components of the inhibitory signal (Figure 3BFigure 3B) will firmly establish the molecular link between the unattached kinetochore and the inhibition of anaphase. The bigger question, of course, is how these elements are coordinated upstream of Mad2 and downstream of spindle attachment (Figure 3CFigure 3C). Attachment of microtubules to the kinetochore is likely to be mediated by dynein and kinesin motors (e.g., CENP-E, XKCM1/MCAK; for review see Sharp, et al. 2000xSharp, D.J., Rogers, G.C., and Scholey, J.M. Nature. 2000; 407: 41–47Crossref | PubMed | Scopus (364)See all ReferencesSharp, et al. 2000), especially CENP-E, a kinetochore kinesin that can directly bridge between spindle microtubules and the checkpoint kinase BubR1 (4xChan, G.K., Jablonski, S.A., Sudakin, V., Hittle, J.C., and Yen, T.J. J. Cell Biol. 1999; 146: 941–954Crossref | PubMed | Scopus (269)See all References, 19xYao, X., Abrieu, A., Zheng, Y., Sullivan, K.F., and Cleveland, D.W. Nat. Cell Biol. 2000; 2: 484–491Crossref | PubMed | Scopus (254)See all References) and which can be essential for kinetochore signaling in vitro (Abrieu et al. 2000xAbrieu, A., Kahana, J.A., Wood, K.W., and Cleveland, D.W. Cell. 2000; 102: 817–826Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesAbrieu et al. 2000). Microtubule attachment leads to even more cascades that will undoubtedly involve mitotic kinases such as Mps1, MAP kinase, Bub1, and BubR1 and the little talked about phosphatases such as budding yeast Glc7/PP1. In addition, proteins such as Bub3, Mad3, Mad1, and Zw10/Rod all play an as yet undefined role in the generation, release, regulation, and/or composition of the wait signal. Stay tuned. Much more remains to be unraveled.‡To whom correspondence should be addressed (e-mail: dcleveland@ucsd.edu).