LOCALIZATION OF MAD2 TO KINETOCHORES DEPENDS ON MICROTUBULE ATTACHMENT, NOT TENSION

A single unattached kinetochore can delay anaphase onset in mitotic ti ssue culture cells (Rieder, C.L., A. Schultz, R. Cole, G. Sluder. 1994 . J. Cell Biol. 127:1301-1310). Kinetochores in vertebrate cells conta in multiple binding sites, and tension is generated at kinetochores af ter attachment to the plus ends of spindle microtubules. Checkpoint co mponent Mad2 localizes selectively to unattached kinetochores (Chen, R .-H., J.C. Waters, E.D. Salmon, and A.W.Murray. 1996. Science. 274:242 -246; Li, Y., and R. Benezra. Science. 274: 246-248) and disappears fr om kinetochores by late metaphase, when chromosomes are properly attac hed to the spindle. Here we show that Mad2 is lost from PtK1 cell kine tochores as they accumulate microtubules and re-binds previously attac hed kinetochores after microtubules are depolymerized with nocodazole. We also show that when kinetochore microtubules in metaphase cells ar e stabilized with taxol, tension at kinetochores is lost. The phosphoe pitope 3f3/2, which has been shown to become dephosphorylated in respo nse to tension at the kinetochore (Nicklas, R.B., S.C. Ward, and G.J. Gorbsky. 1995. J. Cell Biol. 130:929-939), is phosphorylated on all 22 kinetochores after tension is reduced with taxol, In contrast, Mad2 o nly localized to an average of 2.6 out of the 22 kinetochores in taxol -treated PtK1 cells. Therefore, loss of tension at kinetochores occupi ed by microtubules is insufficient to induce Mad2 to accumulate on kin etochores, whereas unattached kinetochores consistently bind Mad2. We also found that microinjecting antibodies against Mad2 caused cells ar rested with taxol to exit mitosis after similar to 12 min, while uninj ected cells remained in mitosis for at least 6 h, demonstrating that M ad2 is necessary for maintenance of the tax ol-induced mitotic arrest. We conclude that kinetochore microtubule attachment stops the Mad2 in teractions at kinetochores which are important for inhibiting anaphase onset.