Regulation and single-molecule mechanics of microtubule-based motors in living Chlamydomonas

Abstract

Whether motors of different directionality are functionally coordinated in cells or operate in a semi-random “tug of war” is unclear. We tested the hypothesis that the microtubule-based motion of the transmembrane protein FMG-1 in the flagella of Chlamydomonas is functionally coordinated for unidirectional transport. A laser trap was used to position microspheres on the plasma membrane of paralyzed Chlamydomonas flagella. The anterograde and retrograde movements of the microsphere were measured with nanometer resolution as microtubule-based motors moved FMG-1. Based on stall forces, we find that an average of 10 motors act to move the microsphere in either direction, with mean step sizes of 4 and 8 nm. Reverse steps were uncommon, and quiescent periods separated every transport event, suggesting the exclusive activation of motors of one direction. Temperature-sensitive mutants of kinesin-2 showed exclusively retrograde steps after jumps to the non-permissive temperature. These data suggest that molecular motors in living cells can be reciprocally coordinated to engage in large numbers and for transport in a single direction, even when motors of mixed directionality are present. The predominance of 4 nm steps suggests sub-steps or other novel behavior of these motors in the cytoplasm. This novel technique should prove beneficial for studying the mechanics, regulation and bidirectional coordination of molecular motors in vivo.