Investigating the Interaction between Kinesin-13s and Microtubules by Single Molecule Fluorescence Polarization Microscopy

Abstract

Kinesins are motor proteins that translocate along microtubules (MTs) and assist in transportation of intracellular cargo by utilizing the energy generated from ATP hydrolysis. Interestingly, Kinesin-13s do not walk but depolymerize microtubules at their ends, an activity that is essential for regulating MT dynamics during cell-division. Prior research efforts have shown that Kinesin-13s target MT ends very quickly by the process of one-dimensional diffusion along the MT lattice. In an effort to elucidate the mechanism of such diffusive interactions and MT depolymerization activity, we have investigated the orientation and dynamics of BSR-labeled KLP10A (Drosophila m. Kinesin-13) constructs interacting with MTs in the presence of ATP analogues using fluorescence polarization microscopy (FPM). Unlike conventional kinesins, which show high mobility in nucleotide conditions that induce weak MT binding (presence of ADP), KLP10A shows relatively high fluorescence anisotropy in all nucleotide states investigated, including ADP. Such observations indicate that the nucleotide present has minimal effects on the binding configuration of the majority of KLP10A molecules bound to the MT lattice. However, FPM data acquired at the single-molecule levels suggest that the Kinesin-13 motor domain becomes very mobile (tumbling) when undergoing one-dimensional diffusion. Ongoing experiments with KLP10A constructs of different lengths and mutants will help in identifying the residues in the Kinesin-13 molecule that mediate one-dimensional lattice diffusion.