Dimerization of mammalian kinesin-3 motors results in superprocessive motion

Abstract

The kinesin-3 family is one of the largest among the kinesin superfamily and its members play important roles in a wide range of cellular transport activities, yet the molecular mechanisms of kinesin-3 regulation and cargo transport are largely unknown. We performed a comprehensive analysis of mammalian kinesin-3 motors from three different subfamilies (KIF1, KIF13, and KIF16). Using Forster resonance energy transfer microscopy in live cells, we show for the first time to our knowledge that KIF16B motors undergo cargo-mediated dimerization. The molecular mechanisms that regulate the monomer-to-dimer transition center around the neck coil (NC) segment and its ability to undergo intramolecular interactions in the monomer state versus intermolecular interactions in the dimer state. Regulation of NC dimerization is unique to the kinesin-3 family and in the case of KIF13A and KIF13B requires the release of a proline-induced kink between the NC and subsequent coiled-coil 1 segments. We show that dimerization of kinesin-3 motors results in superprocessive motion, with average run lengths of ∼10 μm, and that this property is intrinsic to the dimeric kinesin-3 motor domain. This finding opens up studies on the mechanistic basis of motor processivity. Such high processivity has not been observed for any other motor protein and suggests that kinesin-3 motors are evolutionarily adapted to serve as the marathon runners of the cellular world.

Keywords: helical plot; intracellular transport; microtubule; molecular motor.

Fig. 1.

Full-length KIF16B motors dimerize on the cargo surface to drive transport. (A–E) Live cell FRET microscopy. COS-7 cells coexpressing (A) wild-type KIF16B tagged with FRET donor (mCFP) and acceptor (mCit) fluorescent proteins or (B) the cargo-binding mutant LF/AA tagged with mCFP and mCit were imaged by FRET microscopy. For tail-to-tail FRET (A and B, Left), FPs were fused to the C termini of the motors, whereas for motor-to-motor FRET (A and B, Right), FPs were fused to the N termini of the motors. (C–E) FRET controls. COS-7 cells coexpressing (C) donor and acceptor FPs targeted to the cargo surface via the monomeric KIF16B PX domain, (D) the N-terminally tagged KIF16B NC mutant, or (E) donor and acceptor FPs extended 30 nm from the cargo surface by a SAH between the FP and PX domains were imaged by FRET microscopy. Representative images of the calculated average FRET efficiency (E_AVE) are shown. Yellow dotted lines indicate the outline of the cells. (Scale bars, 10 μm.) (F) Quantification of the FRET efficiencies in live COS-7 cells. For a negative control, the FRET efficiency between mCFP and mCit expressed as separate proteins was measured. For a positive control, the FRET efficiency between linked mCFP and mCit proteins (mCit-16aa-mCFP) was measured. n = 25–40 cells each over three independent experiments. The data are presented as mean ± SD. P values were calculated using the two-tailed t test. (G) Comparison of the FRET efficiency (E_AVE) to protein concentration (measured as fluorescence intensity) on a cell-by-cell basis.

Fig. 2.

The NC is a key regulatory element for kinesin-3 motor dimerization and processive motility. (A) Domain organization of kinesin-3 motors analyzed in this study. Truncations are indicated by black tick marks with the amino acid number. Truncations in green text indicate processive motors, whereas truncations in red text indicate nonprocessive motors based on analysis of motor behavior in CAD cells (Fig. S6) and/or single molecule assays (Fig. 3). CC, coiled-coil; FHA, forkhead-associated; MD, motor domain; NC, neck coil; PH, pleckstrin homology; and PX, Phox homology. (B–E) Heptad net plots of the NC–CC1 regions (gray shaded rectangles in A) of the kinesin-3 motors. The helix has been cut and opened flat to give a 2D representation of the amino acid residues and their potential interactions in the helix. The N terminus is at the top right. Acidic residues are shown in red, basic in blue, and all others in black. Preferred ionic interactions are shown as solid lines and alternative interactions are shown as dotted lines. Every seventh residue is repeated on the right of the plot (in brackets) so that all interactions can be shown. The violet dotted line defines the orientation of the α-helix axis. The light gray circles indicate ‘‘a’’ positions and the dark gray circles indicate ‘‘d’’ positions of the hydrophobic seam in the CC. Residues circled in green indicate truncations that yield processive motors and residues circled in red indicate truncations that yield nonprocessive motors.

Fig. 3.

Truncated kinesin-3 motors are superprocessive. Truncated kinesin-3 motors tagged with three tandem mCit FPs (3xmCit) at their C termini were expressed in COS-7 cells. Cell lysates were used for single-molecule motility assays. Histograms of the velocities (Upper) and run lengths (Lower) were plotted for each population of motors and fit to a single Gaussian. The peak represents the average velocity and run length of (A) KIF1A(1–393), (B) KIF1A(1–393)-LZ, (C) KIF13A(1–411∆P), (D) KIF13B(1–412∆P), (E) KIF16B(1–400), and (F) KHC(1–560), a well-characterized truncated kinesin-1 motor used as a motility control. Data are the averages from at least two independent experiments. The mean ± SEM and N values for each motor are indicated in the top right corners.

Fig. 4.

Dimerization of full-length kinesin-3 motors results in superprocessive motion. The oligomeric state and motility properties of full-length kinesin-3 mutants KIF1A(L478Q/M481Q), KIF1A(V483N), KIF13A(∆P390), and KIF13B(∆P391) were compared with that of an active dimeric kinesin-1 [KHC(1–560)] motor. (A) CAD cell assay to measure motor processivity in cells. Representative images of differentiated CAD cells expressing C-terminal mCit-tagged full-length mutant kinesin-3 motors or KHC(1–560). Expressing cells are outlined with a yellow dotted line. Arrowheads indicate neurite tips. Asterisks indicate nuclei of expressing cells. (Scale bars, 20 μm.) (B) TIRF single-molecule photobleaching assay to measure oligomeric state. The fluorescence intensity over time was measured by TIRF microscopy for individual 3xmCit-tagged motors walking along the microtubule in COS-7 cell lysates. The number of bleaching events per molecule was plotted in a histogram for the population. (C and D) TIRF single-molecule motility assays; 3xmCit-tagged motors in COS-7 cell lysates were added to polymerized microtubules in a flow chamber and observed by TIRF microscopy. The measured (C) velocities and (D) run lengths of each population were plotted in a histogram. The averages of each population are from at least two independent experiments. The mean ± SEM and N values for each motor are indicated in the top right corners.

Fig. 5.

Molecular mechanisms of kinesin-3 motor regulation. Kinesin-3 motors use a unique mechanism of regulation in which dimerization and processive motility are tied to cargo binding. Maintaining the monomeric state of cytosolic motors is due to intramolecular interactions between the NC and CC1 segments. (A) For KIF1A, the NC–CC1 region is proposed to form a parallel coiled-coil based on structural studies of CeUNC-104 (35). (B) For KIF16B, the shorter linker between the NC and CC1 helices results in the formation of an anti-parallel coiled-coil. (C and D) For KIF13A and KIF13B, the hinge between NC and CC1 is absent; however, the two segments maintain an anti-parallel coiled-coil conformation owing to the presence of a proline residue at the NC–CC1 junction. (E) Cargo binding increases the effective concentration of the motor such that intermolecular NC–NC interactions are favored, resulting in dimerization and highly processive motility. CG, Cap-Gly; PH, Pleckstrin homology; and PX, phox homology.