Mechanical Characterization of the Yeast Centrosome

Abstract

Reflection Fluorescence Microscopy system measured an Mtw1p-Nuf2p fluorescence colocalization of 34 5 2.2%. We performed a similar experiment, immobilizing the kinetochores onto microtubules. We measured a colocalization on microtubules of 8156.4% 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 5 3.4% Mtw1p-Bub1p colocalization when the particles were immobilized onto a coverslip and 50 5 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.