The motility of biological molecular motors has typically been analyzed by in vitro reconstitution systems using motors isolated and purified from organs or expressed in cultured cells. The behavior of biomolecular motors within cells has frequently been reported to be inconsistent with that observed in reconstituted systems in vitro. Although this discrepancy has been attributed to differences in ionic strength and intracellular crowding, understanding how such parameters affect the motility of motors remains challenging. In this report, we investigated the impact of intracellular crowding in vitro on the mechanical properties of kinesin under a high ionic strength that is comparable to the cytoplasm. Initially, we characterized viscosity in a cell by using a kinesin motor lacking the cargo-binding domain. We then used polyethylene glycol to create a viscous environment in vitro comparable to the intracellular environment. Our results showed that kinesin frequently dissociated from microtubules under high ionic strength conditions. However, under conditions of both high ionic strength and crowding with polymers, the processive movement of kinesin persisted and increased in frequency. This setting reproduces the significant variations in the mechanical properties of motors measured in the intracellular environment and suggests a mechanism whereby kinesin maintains motility under the high ionic strengths found in cells.

Impact of physiological ionic strength and crowding on kinesin-1 motility