Mammalian KIF3AC is classified as a heterotrimeric kinesin-2 that is best known for organelle transport in neurons, yet in vitro studies to characterize its single molecule behavior are lacking. The results presented show that a KIF3AC motor that includes the native helix α7 sequence for coiled-coil formation is highly processive with run lengths of ∼1.23 μm and matching those exhibited by conventional kinesin-1. This result was unexpected because KIF3AC exhibits the canonical kinesin-2 neck-linker sequence that has been reported to be responsible for shorter run lengths observed for another heterotrimeric kinesin-2, KIF3AB. However, KIF3AB with its native neck linker and helix α7 is also highly processive with run lengths of ∼1.62 μm and exceeding those of KIF3AC and kinesin-1. Loop L11, a component of the microtubule-motor interface and implicated in activating ADP release upon microtubule collision, is significantly extended in KIF3C as compared with other kinesins. A KIF3AC encoding a truncation in KIF3C loop L11 (KIF3ACΔL11) exhibited longer run lengths at ∼1.55 μm than wild-type KIF3AC and were more similar to KIF3AB run lengths, suggesting that L11 also contributes to tuning motor processivity. The steady-state ATPase results show that shortening L11 does not alter kcat, consistent with the observation that single molecule velocities are not affected by this truncation. However, shortening loop L11 of KIF3C significantly increases the microtubule affinity of KIF3ACΔL11, revealing another structural and mechanistic property that can modulate processivity. The results presented provide new, to our knowledge, insights to understand structure-function relationships governing processivity and a better understanding of the potential of KIF3AC for long-distance transport in neurons.

Propofol is the most widely used i.v. general anesthetic to induce and maintain anesthesia. It is now recognized that this small molecule influences ligand-gated channels, including the GABA A receptor and others. Specific propofol binding sites have been mapped using photoaffinity ligands and mutagenesis; however, their precise target interaction profiles fail to provide complete mechanistic underpinnings for the anesthetic state. These results suggest that propofol and other common anesthetics, such as etomidate and ketamine, may target additional protein networks of the CNS to contribute to the desired and undesired anesthesia end points. Some evidence for anesthetic interactions with the cytoskeleton exists, but the molecular motors have received no attention as anesthetic targets. We have recently discovered that propofol inhibits conventional kinesin-1 KIF5B and kinesin-2 KIF3AB and KIF3AC, causing a significant reduction in the distances that these processive kinesins can travel. These microtubule-based motors are highly expressed in the CNS and the major anterograde transporters of cargos, such as mitochondria, synaptic vesicle precursors, neurotransmitter receptors, cell signaling and adhesion molecules, and ciliary intraflagellar transport particles. The single-molecule results presented show that the kinesin processive stepping distance decreases 40-60% with EC 50 values <100 nM propofol without an effect on velocity. The lack of a velocity effect suggests that propofol is not binding at the ATP site or allosteric sites that modulate microtubule-activated ATP turnover. Rather, we propose that a transient propofol allosteric site forms when the motor head binds to the microtubule during stepping.anesthesia | allosteric inhibitor | microtubule | etomidate | ketamine

Microtubule-based molecular motors mediate transport of intracellular cargo to subdomains in neurons. Previous evidence has suggested that the anesthetic propofol decreases the average run-length potential of the major anterograde transporters kinesin-1 and kinesin-2 without altering their velocity. This effect on kinesin has not been observed with other inhibitors, stimulating considerable interest in the underlying mechanism. Here, we used a photoactive derivative of propofol, meta-azipropofol (AziPm), to search for potential propofol-binding sites in kinesin. Single-molecule motility assays confirmed that AziPm and propofol similarly inhibit kinesin-1 and kinesin-2. We then applied AziPm in semiquantitative radiolabeling and MS microsequencing assays to identify propofol-binding sites within microtubule–kinesin complexes. The radiolabeling experiments suggested preferential AziPm binding to the ATP-bound microtubule–kinesin complex. The photolabeled residues were contained within the kinesin motor domain rather than at the motor domain–β-tubulin interface. No residues within the P-loop of kinesin were photolabeled, indicating an inhibitory mechanism that does not directly affect ATPase activity and has an effect on run length without changing velocity. Our results also indicated that when the kinesin motor interacts with the microtubule during its processive run, a site forms in kinesin to which propofol can then bind and allosterically disrupt the kinesin–microtubule interaction, resulting in kinesin detachment and run termination. The discovery of the propofol-binding allosteric site in kinesin may improve our understanding of the strict coordination of the motor heads during the processive run. We hypothesize that propofol's potent effect on intracellular transport contributes to various components of its anesthetic action.

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 (k1= 11 s−1) 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.

250 max) 249 words 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 homoand 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 other's 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.Key Words: kinesin  molecular motors single-molecule motility high-speed optical trapping iSCAT microscopy  neuron  microtubule  intracellular transport  cytoskeleton Significance Statement -(120 max) 120 words Kinesins 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.

ability) completely abolishes the effects of retigabine. This finding suggests that retigabine interacts with KCNQ channels via a hydrogen bond with the Trp indole group, a rarely demonstrated mode of drug:target interaction. Supporting this model, substitution with fluorinated Trp analogs can strengthen retigabine potency by increasing the indole N-H bond polarity. Lastly, the potency of numerous retigabine analogs for KCNQ3 channels was found to correlate with the negative electrostatic surface potential of a conserved carbonyl oxygen atom. These findings demonstrate the detailed chemical interactions and functional groups that likely underlie the effects of retigabine and other KCNQ activators, highlighting a rarely observed mode of drug interaction with multifunctional amphipathic Trp side chains. These stringent constraints for models of retigabine interactions with KCNQ channels may guide the rational development of improved retigabine derivatives. Kinesin-5 slides antiparallel microtubules apart during mitosis and is necessary for bipolar spindle formation. Besides its unique homotetrameric configuration, determined by the coiled-coil domain, kinesin-5 motor domains also possess specific properties optimal for their spindle organizing function. To study properties intrinsic to the kinesin-5 motor domain, we generated functional kinesin-5 dimers by fusing the kinesin-5 head and 18-residue neck linker to the coiled-coil rod of kinesin-1. In ATP this kinesin-5 dimer decorated plusends of taxol-stabilized microtubules and slowed depolymerization of GMPCPP microtubules. On dynamic microtubules, kinesin-5 dimer increased the microtubule growth rate by more than a factor of two and reduced the catastrophe frequency three-fold. These findings are consistent with kinesin-5 acting as a microtubule polymerase. To understand this polymerase mechanism, TIRF microscopy was used to visualize individual GFP-labeled kinesin-5 dimers on immobilized microtubules. Motors walked to the ends of microtubules and remained bound there for 7 seconds, much longer than the 0.1 s motor step time. We hypothesize that kinesin-5 promotes microtubule polymerization by stabilizing longitudinal interactions between tubulin subunits on a single protofilament. Consistent with this protofilament stabilization hypothesis, fluorescence analysis suggests that microtubule plus-ends are more tapered in the presence of kinesin-5. Furthermore, curved and looped ends were observed, which occasionally resolved into straight microtubules, consistent with kinesin-5 stabilizing long protofilament bundles. In cells, these endbinding and polymerase activities should enhance the ability of kinesin-5 to establish and maintain spindle pole separation during mitosis. 101-PlatWhy are Kinesin-2 KIF3AB and KIF3AC so Processive? Mammalian KIF3AC is a heterotrimeric kinesin-2 that is best known for roles in organelle transport and remodeling of the microtubule cytoskeleton in neuronal tissue, yet in vitro studies to characterize its single molecule behavior are lack...

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Cytoplasmic dynein is involved in a diverse set of cellular functions including cargo transport and microtubule organization. Dynein's activity is controlled by regulatory proteins, such as Lis1 and Ndel1. In yeast, Lis1 facilitates plus-end tip localization of dynein. In vitro studies showed that Lis1 inhibits the motility of yeast cytoplasmic dynein, but the molecular mechanism of this regulation is not well understood. In this study, we studied the motility and force generation of Lis1-bound dynein at the single molecule level. We observed that Lis1 stably binds to dynein as it walks along a microtubule. One Lis1 dimer is recruited per motor domain of dynein. Dynein velocity decreases with the increasing stoichiometry of Lis1. Using an optical trap, we showed that Lis1 binding does not change the stall force of dynein, but it increases the stall duration. Lis1 binding also reduces the asymmetry in force-induced detachment of dynein from the microtubules regardless of its interaction with dynein's coiled-coil stalk.