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Fig. 10. Kinetochore localization of spindle checkpoint proteins is differentially regulated by spindle assembly events. (A) The conditions underlying binding to unattached kinetochores are similar for all proteins and require phosphorylated kinetochores. (B) Once chromosomes become mono-oriented, the attached kinetochore retains little or no Mad2 and Bub1, generating an asymmetrical localization pattern. BubR1 and Bub3 levels are only slightly asymmetrical with the attached kinetochore exhibiting reduced staining because of some tension generated from anti-poleward forces (arrows). (C) Under reduced tension from taxol treatment mono-oriented chromosomes show symmetrical BubR1/Bub3 staining while Mad2/Bub1 labelling remains asymmetrical. (D) When bipolar attachment is achieved, Mad2/Bub1 no longer localize to kinetochores, 3F3/2 epitopes are dephosphorylated because of tension and BubR1 and Bub3 are released. (E) Disruption of tension with taxol leads to recruitment of BubR1 and Bub3 to kinetochores because 3F3/2 epitopes become rephosphorylated. However, Mad2 and Bub1 cannot accumulate at kinetochores because of microtubule occupancy.
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