Application on advanced optical tweezers to the study of mitotic motor proteins

The complex mechanics of mitosis occur by the coordinated action of many diverse motor proteins. Microtubule motor proteins localize to the chromosomes in two distinct areas: (1) the kinetochore, which is the site of principal attachment to microtubules of the mitotic spindle, contains motor and motor-like proteins (dynein, CENP-E, etc.), and (2) the chromosome arms, where chromokinesins such as Xkid have been discovered. This study uses advanced optical tweezers to probe the motor proteins of mitotic chromosomes in vitro. In these assays, an individual microtubule is suspended between two optically-trapped microspheres in a sample chamber. The optical tweezers are used to bring the microtubule into contact with a mitotic chromosome isolated from prometaphase-arrested CHO cells and adhered to the upper surface of the chamber. In the presence of ATP, the chromosomal motor proteins bind to and act upon the microtubule, causing changes in the motion of the trapped microspheres. A back focal plane interferometry detection system tracks the microsphere movement at 7 kHz; this system can detect nm-level displacements and pN-level forces. We present the preliminary results from these assays.