Although extremely rare, errors in chromosome segregation lead to aneuploidy, which has been linked to cancer and birth defects. Dividing cells rely on a surveillance mechanism called the spindle assembly checkpoint (SAC), which ensures that chromosomes are properly segregated during mitosis. The SAC, comprising several proteins on the mitotic kinetochore emits a diffusible ‘wait’ signal upon detection of improper and/or unattached kinetochores, delaying mitosis. Proper attachment and biorientation silences the ‘wait’ signal allowing progress through mitosis. ‘Wait’ signal silencing is thought of in two possible ways: First, the SAC responds to kinetochore-microtubule tension. Second, the SAC responds to attachment of microtubules to kinetochores, independent of tension. This attachment-tension debate is still unresolved due to the challenge of independently controlling attachment states and levels of tension on kinetochores in vivo. To help shed more light, we purified mitotic kinetochores from budding yeast with core kinetochore components, Mtw1p and Nuf2p, fluorescently labeled. We immobilized purified kinetochores onto a coverslip in our in vitro assay, and using a very sensitive Total Internal Reflection Fluorescence Microscopy system measured an Mtw1p-Nuf2p fluorescence colocalization of 34 ± 2.2%. We performed a similar experiment, immobilizing the kinetochores onto microtubules. We measured a colocalization on microtubules of 81±6.4% indicating that Nuf2p remained bound to the kinetochore upon kinetochore-microtubule attachment in vitro, as expected. We repeated this experiment for kinetochores with fluorescently labeled Mtw1p and Bub1p, a SAC component. We measured a 12 ± 3.4% Mtw1p-Bub1p colocalization when the particles were immobilized onto a coverslip and 50 ± 1.9% on microtubules. These experiments indicate that kinetochore-microtubule attachment in yeast may not be sufficient to release Bub1p. Current efforts are focused on Mad1p, a checkpoint protein that is believed to be released from kinetochores upon microtubule attachment in vivo.
