Research in my laboratory is on motor proteins and the mechanisms by which they function in the spindle to ensure normal chromosome distribution during cell division. Molecular motors are force-generating proteins that drive movements of the spindle and chromosomes in dividing cells. We are currently trying to understand the mechanism of motor function – how motors use ATP to produce force and movement in cells – and the contributions by motors to spindle and chromosome dynamics during division.
The approach we use combines molecular genetics with structural biology and biophysical methods to determine the basis of motor function. We focus on the kinesin spindle motor, Ncd, discovered in my laboratory, and its ‘backwards’ or minus-end directed motility. Ncd is unusual in that it moves on microtubules in the opposite direction as kinesin-1, the founding member of the kinesin family.
By constructing chimeric Ncd-kinesin motor proteins, we identified residues required for the reversed movement of Ncd. We then mutated single amino acid residues and converted Ncd into a bidirectional motor that moves either towards the microtubule plus or minus end. Single-molecule laser-trap assays showed that the minus-end directionality of Ncd is due to a large conformational change that occurs when the motor binds to a microtubule.
We are using mutants to trap the motor in different structural states to visualize the conformational changes that occur in the motor as it hydrolyzes ATP and moves on microtubules. We reported a new crystal structure of Ncd that show a large rotation of the coiled-coil stalk – this was interpreted to represent a lever-like movement that amplifies smaller movements within the motor domain. Together with other changes in the motor domain, the stalk rotation is likely to explain the motor mechanism of force generation.
We are also studying the effects of Ncd and other motors on spindle and chromosome dynamics in live cells by transforming into Drosophila the motors fused to GFP, the green fluorescent protein. High-resolution imaging and dynamic studies, including FRAP (fluorescence recovery after photobleaching), give rate constants for motor binding to the spindle and diffusion coefficients in live cells.