The mechanochemistry of the kinesin-2 KIF3AC heterodimer is related to strain-dependent kinetic properties of KIF3A and KIF3C

Abstract

<jats:p>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 determine their transport potential under load, we assayed motor activity using interferometric scattering microscopy and optical trapping. The distribution of stepping durations of KIF3AC molecules is described by a rate (<jats:italic>k</jats:italic><jats:sub><jats:italic>1</jats:italic></jats:sub>= 11 s<jats:sup>−1</jats:sup>) without apparent kinetic asymmetry. Asymmetry was also not apparent under hindering or assisting mechanical loads in the optical trap. KIF3AC shows increased force sensitivity relative to KIF3AA yet is more capable of stepping against mechanical load than KIF3CC. Interestingly, the behavior of KIF3C mirrors prior studies of kinesins with increased interhead compliance. Microtubule gliding assays containing 1:1 mixtures of KIF3AA and KIF3CC result in speeds similar to KIF3AC, suggesting the homodimers mechanically impact each other’s motility to reproduce the behavior of the heterodimer. Our observations are consistent with a mechanism in which the stepping of KIF3C can be activated by KIF3A in a strain-dependent manner, similar to application of an assisting load. These results suggest that the mechanochemical properties of KIF3AC can be explained by the strain-dependent kinetics of KIF3A and KIF3C.</jats:p>

KIF3A accelerates KIF3C within the kinesin-2 heterodimer to generate symmetrical phosphate release rates for each processive step

“…These results are also in agreement with the work by Bensel et al. ( 22 ), where they found that fitting KIF3AC dwell time cumulative distribution data generated from interferometric scattering microscopy experiments could only be statistically justified with a single-exponential function rather than the sum of two exponentials. Moreover, their step trajectory analysis showed that alternating steps had similar step lifetimes.…”

“…The results presented here and those from Bensel et al. ( 22 ) indicate that KIF3AC is an unconventional processive kinesin, yet the question becomes how do these mechanochemical properties enable the physiological function of KIF3AC in neurons? Because neurons are highly polarized cells that depend on selective transport of specific vesicles to axons and dendrites ( 1 , 29 , 30 , 31 ), the first thought is that KIF3AC transports specific cargoes yet to be identified.…”

“…MT gliding assays revealed that an equal mixture of KIF3AA and KIF3CC achieve the same velocity of MT gliding as KIF3AC. The authors conclude that the stepping of KIF3C can be activated by KIF3A in a strain-dependent manner and that the mechanochemical properties of KIF3AC are due to the strain-dependent kinetics of KIF3A and KIF3C ( 22 ).…”

“…Recent results from Bensel et al. ( 22 ) evaluated the stepping kinetics of KIF3AC in the absence of load by interferometric scattering and under load by optical trapping. Their results indicate that the stepping durations of KIF3AC single molecules are best described by a single rate without apparent asymmetry.…”

CyLaKS: the Cytoskeleton Lattice-based Kinetic Simulator

“…Second, inspired by previous work on heterogeneity in the microtubule lattice [56–58], we show how motor motility changes if the lattice contains a random mixture of sites with different motor binding affinity. Third, building on previous work on heterodimeric kinesin motors [104–109], we create a toy model of a heterodimeric motor in which one head is immobile while bound while the other head can diffuse while singly bound. The model demonstrates the expected crossover from directed to diffusive motility as the diffusion coefficient of the second head increases.…”