Strain-Dependent Kinetic Properties of KIF3A and KIF3C Tune the Mechanochemistry of the KIF3AC Heterodimer

Abstract

KIF3AC is a mammalian neuron-specific kinesin-2 implicated in intracellular cargo transport. It is a heterodimer of KIF3A and KIF3C motor polypeptides which have distinct biochemical and motile properties as engineered homodimers. Single-molecule motility assays show that KIF3AC moves processively along microtubules at a rate faster than expected given the motility rates of the KIF3AA and much slower KIF3CC homodimers. To resolve the stepping kinetics of KIF3A and KIF3C motors in homo-and heterodimeric constructs, and to determine their transport potential under mechanical load, we assayed motor activity using interferometric scattering (iSCAT) microscopy and optical trapping. The distribution of stepping durations of KIF3AC molecules is described by a rate (k1 = 11 s-1) without apparent kinetic asymmetry in stepping. Asymmetry was also not apparent under hindering or assisting mechanical loads of 1 pN in the optical trap. KIF3AC shows increased force sensitivity relative to KIF3AA, yet is more capable of stepping against mechanical load than KIF3CC. Microtubule gliding assays containing 1:1 mixtures of KIF3AA and KIF3CC result in speeds similar to KIF3AC, indicating the homodimers mechanically impact each others motility to reproduce the behavior of the heterodimer. We conclude that the stepping of KIF3C can be activated by KIF3A in a strain-dependent manner which is similar to application of an assisting load, and the behavior of KIF3C mirrors prior studies of kinesins with increased interhead compliance. These results suggest that KIF3AC-based cargo transport likely requires multiple motors, and its mechanochemical properties arise due to the strain-dependences of KIF3A and KIF3C.\n\nSignificance StatementKinesins are important long-range intracellular transporters in neurons required by the extended length of the axon and dendrites and selective cargo transport to each. The mammalian kinesin-2, KIF3AC, is a neuronal heterodimer of fast and slow motor polypeptides. Our results show that KIF3AC has a single observed stepping rate in the presence and absence of load and detaches from the microtubule rapidly under load. Interestingly, both KIF3A and assisting loads accelerate the kinetics of KIF3C. These results suggest that KIF3AC is an unconventional cargo transporter and its motile properties do not represent a combination of alternating fast and slow step kinetics. We demonstrate that the motile properties of KIF3AC represent a mechanochemistry that is specific to KIF3AC and may provide functional advantages in neurons.