Mechanism of Kinesin-13 Binding to Microtubules as Revealed by Single Molecule Fluorescence Polarization Microscopy

Abstract

The kinesin superfamily of motor proteins are involved in diverse cellular processes, including intracellular organelle transport, cell division and cytoskeletal dynamics. The widely studied conventional kinesin (or kinesin-1) translocates along the microtubule (MT) by utilizing the energy generated from ATP hydrolysis. In contrast, members of the Kinesin-13 family do not walk along the MT lattice, but use their catalytic core to rapidly target microtubule ends by the process of one-dimensional diffusion (ODD) and promote depolymerization upon reaching there. However, the reason for such a significant difference in the behavior of the structurally conserved motor domain is not clearly understood. In order to reveal the mechanistic details of the kinesin-13 action, fluorescence polarization microscopy (FPM) has been employed to probe the configuration and mobility of BSR-labeled KLP10A (Drosophila m. Kinesin-13) molecules interacting with microtubules in the presence of different nucleotides. Experiments are being performed with KLP10A constructs of variable lengths to identify the potent mediators of diffusive motility. Preliminary results emerging from single-molecule FPM measurements have suggested that the motor core itself can undergo ODD without the assistance from the positively-charged neck domain. In addition, data acquired at both ensemble and single-molecule levels have revealed that the orientation of the KLP10A molecule relative to the MT filament is altered by mutating the crucial residues in the tubulin-binding sites on the motor domain. The structural and functional information extracted from analyses of our experimental findings will be discussed in details.