JIP3 Activates Kinesin-1 Motility to Promote Axon Elongation

Abstract

Kinesin-1 is a molecular motor responsible for cargo transport along microtubules and plays critical roles in polarized cells, such as neurons. Kinesin-1 can function as a dimer of two kinesin heavy chains (KHC), which harbor the motor domain, or as a tetramer in combination with two accessory light chains (KLC). To ensure proper cargo distribution, kinesin-1 activity is precisely regulated. Both KLC and KHC subunits bind cargoes or regulatory proteins to engage the motor for movement along microtubules. We previously showed that the scaffolding protein JIP3 interacts directly with KHC in addition to its interaction with KLC and positively regulates dimeric KHC motility. Here we determined the stoichiometry of JIP3-KHC complexes and observed approximately four JIP3 molecules binding per KHC dimer. We then determined whether JIP3 activates tetrameric kinesin-1 motility. Using an in vitro motility assay, we show that JIP3 binding to KLC engages kinesin-1 with microtubules and that JIP3 binding to KHC promotes kinesin-1 motility along microtubules. We tested the in vivo relevance of these findings using axon elongation as a model for kinesin-1-dependent cellular function. We demonstrate that JIP3 binding to KHC, but not KLC, is essential for axon elongation in hippocampal neurons as well as axon regeneration in sensory neurons. These findings reveal that JIP3 regulation of kinesin-1 motility is critical for axon elongation and regeneration.

Keywords: JIP3; KHC; axon; kinesin; molecular motor; motility; neurite outgrowth; regeneration.

FIGURE 1.

JIP3 interacts with dimeric KHC in at least a 2:1 ratio to promote KHC motility. A, schematic representation of the TIRF-based dimeric KHC motility assay. Rhodamine- and biotin-labeled microtubules (red line) are adhered to coverslips (black line) with streptavidin. A motility mix with GFP-JIP3 and FLAG-KHC protein lysates is added to the chamber. B, schematic representation of JIP3 mutants used in this study. JIP3ΔKLC removes amino acids 429–459, corresponding to the KLC binding domain. JIP3ΔKHC removes amino acids 5–81 corresponding to the KHC binding domain. JIP3ΔΔ removes both the KLC and KHC binding domains. KBD, KHC binding domain; JBD, JNK binding domain; LZ, leucine zipper (KLC binding domain). C, representative kymographs of motile FLAG-KHC events in the presence of the indicated JIP3 construct. Motile events are less frequent and shorter when JIP3 cannot bind KHC, as with JIP3ΔKHC. D, velocity of motile events. Activation of the KHC dimer by WT GFP-JIP3 in this replication was 0.25 μm/s, compared with JIP3ΔKHC, which cannot bind KHC and significantly reduces run velocity. #, p < 0.001; Student's t test. E, run length of motile events. No significant difference was detected in run length of motile events; p = 0.07, Student's t test. F, motile fraction was calculated as described by Sun et al. (5). Inability to bind KHC significantly reduces motile fraction compared with WT JIP3. #, p < 0.001, Student's t test. G, representative trace of the fluorescence intensity over time of a GFP-JIP3 punctum. Fluorescence intensity decreases in distinct steps (red lines), representing photobleaching of single GFP molecules. The total number of GFP-JIP3 molecules can then be calculated by dividing the overall drop in fluorescence intensity (blue line) by the intensity of a single GFP molecule. This example shows a punctum that is calculated to contain four GFP molecules. H, histogram of the number of GFP molecules in GFP-JIP3 bound to FLAG-KHC. Counts below 4 were rarely observed, suggesting that JIP3 associates with KHC with at least 2:1 stoichiometry. AU, arbitrary units; error bars, S.E.

FIGURE 2.

JIP3 regulates distinct aspects of kinesin-1 motility via differential binding to KLC and KHC subunits. A, schematic representation of the tetrameric kinesin-1 motility assay. FLAG-KHC and KLC-mCit are used with FLAG-JIP3 mutants as described in the legend to Fig. 1B. Because KLC has no intrinsic motility along microtubules, mCit puncta move along microtubules only when kinesin-1 is functioning as a heterotetramer. B, representative Western blot of KLC-mCit and FLAG-KHC lysates. FLAG-KHC and KLC-mCit were both present in their expected size ranges (n = 3). C, representative Western blot of FLAG-JIP3 lysates, including a control lysate lacking JIP3 and FLAG-KHC for comparison. 1 μl of each of the JIP3 mutants and control lysate and 3 μl of the FLAG-KHC were loaded in the gel. All JIP3 constructs were expressed at much greater levels than FLAG-KHC (n = 3). D, representative kymographs from TIRF experiments. Each y axis represents 100 s of imaging time. Scale bar, 2 μm. E, binding frequency was measured as the number of motile and nonmotile binding events per micron of microtubule. WT JIP3 and JIP3ΔKHC significantly increased the microtubule binding frequency of kinesin-1 compared with untransfected controls, but JIP3ΔKLC did not. F and G, n = 74 (control), 93 (WT JIP3), 85 (JIP3ΔKLC), and 92 (JIP3ΔKHC) microtubules analyzed in four replicates (ANOVA, p = 1.65 × 10⁻⁵; #, p ≪ 0.001 compared with control; Tukey's HSD). F, motile efficiency was measured as the number of motile events per 10 μm of microtubule. All JIP3 mutants increased motile efficiency compared with control, but JIP3ΔKHC produced a significantly lower motile efficiency than WT JIP3 (ANOVA, p = 0.01; *, p < 0.05; ***, p < 0.001; n.s., not significant; Tukey's HSD). G, velocity of motile events. WT JIP3 and JIP3ΔKLC significantly increase run speed compared with control, but JIP3ΔKHC did not. G–J, n = 41 (control), 108 (WT JIP3), 80 (JIP3ΔKLC), or 86 (JIP3ΔKHC) motile events in four replicates (ANOVA, p = 9.65 × 10⁻⁵; **, p < 0.01; ***, p < 0.001 compared with control; Tukey's HSD). H, run length of motile events. WT JIP3, JIP3ΔKLC, and JIP3ΔKHC all significantly increase run length compared with controls, but JIPΔKHC does not increase run length to the extent that WT JIP3 does (Kruskal-Wallis, p = 4.2 × 10⁻⁵; *, p < 0.05; **, p < 0.01; #, p ≪ 0.001; Mann-Whitney U test). I, histogram of velocity of motile events. J, histogram of run length of motile events. Error bars, S.E.

FIGURE 3.

JIP3 is required for axon elongation. A, representative Western blots from JIP3 WT (+/+), heterozygous (+/−), and KO (−/−) littermate embryonic whole brain lysates. B, quantification of A. JIP3 heterozygosity results in an ∼50% reduction of JIP3 expression compared with WT. n = 5 pups in each group, litter-matched. C, Tau (axons) and MAP2 (dendrites) staining of littermate WT, heterozygous, and KO embryonic hippocampal neurons at DIV5. Most neurons produce one Tau-positive axon and multiple MAP2-enriched dendrites. Scale bar, 50 μm. D, quantification of axon number from C. Axons were defined as Tau-positive neurites. No difference in axon number was detected between any of the groups (Kruskal-Wallis, p = 0.22). D–H, n = 100 neurons/group over two replicates. E, quantification of total axon length from C. Heterozygous and KO axons are significantly shorter than WT (ANOVA, p = 0.018; *, p < 0.05; **, p < 0.01 compared with WT; Tukey's HSD). F, quantification of axon branching from C by Sholl analysis. No differences in axon branching were detected in any of the groups (ANOVA, p = 0.23). G, total dendrite length quantification of C. No differences in dendrite length were detected in any group (ANOVA, p = 0.81). H, dendrite branching quantification of C by Sholl analysis. No difference in dendrite branching was detected in any group (ANOVA, p = 0.87). n.s., not significant; Error bars, S.E.

FIGURE 4.

JIP3 binding to KHC is required to promote axon elongation. A, rescue of axon length defects in JIP3 KO mouse hippocampal neurons by JIP3 WT and mutants shown in Fig. 1B. Top, Tau (axons) and MAP2 (dendrites) staining of mouse JIP3 KO hippocampal neurons. Bottom, GFP signal produced by the GFP-JIP3 fusion constructs. As in Fig. 2C, most neurons extend a single axon. Arrowheads, cell bodies; arrows, tip of the longest axon. Scale bar, 50 μm. B, quantification of axon number from A. No differences in axon number were detected between groups (p = 0.15, Kruskal-Wallis). C, quantification of total axon length from A. WT JIP3 and JIP3ΔKLC rescued axon length defects of the JIP3 KO, but JIP3ΔKHC and JIP3ΔΔ did not. B–F, n = 59 (vector), 66 (WT JIP3), 71 (JIP3ΔKLC), 67 (JIP3ΔKHC), or 53 (JIP3ΔΔ) neurons from three replicates (ANOVA, p = 0.065; *, p < 0.05; **, p < 0.01 compared with vector; Tukey's HSD). D, quantification of axon branching from A by Sholl analysis. No changes in axon branching was detected among any of the groups (ANOVA, p = 0.35). E, total dendrite length quantification from A. No change in dendrite length was detected between any of the groups (ANOVA, p = 0.25). F, dendrite branching from A analyzed by Sholl analysis. No change in dendrite branching was measured between any groups (ANOVA, p = 0.34). n.s., not significant; error bars, S.E.

FIGURE 5.

JIP3 mutants unable to bind KHC are dominant negative. Analysis of JIP3 deletion mutants shown in Fig. 1B in WT rat hippocampal neurons. A, WT rat hippocampal neurons electroporated with the indicated constructs were fixed and stained for Tau/MAP3 (axons/dendrites) at DIV5 (top). Arrowheads, cell bodies; arrows, tip of the longest axon. Scale bar, 50 μm. B, axon number was not significantly different between groups (p = 0.09, Kruskal-Wallis). C, total axon length quantification of A. Expression of JIP3ΔKHC or JIP3ΔΔ, but not WT JIP3 or JIP3ΔKLC, significantly reduced axon length compared with vector controls. B–F, n = 70 (vector), 68 (WT JIP3), 72 (JIP3ΔKLC), 73 (JIP3ΔKHC), or 66 (JIP3ΔΔ) neurons from five replicates (ANOVA, p ≪0.001; #, p ≪ 0.001 compared with vector; Tukey's HSD). D, axon branching quantification by Sholl analysis of A. Expression of JIP3ΔKHC and JIP3ΔΔ reduced axon branching, but expression of WT JIP3 or JIP3ΔKLC did not (ANOVA, p ≪ 0.001; #, p ≪ 0.001 compared with vector; Tukey's HSD). E, total dendrite length quantification of A. No difference in dendrite length was measured between any groups (ANOVA, p = 0.42). F, dendrite branching of A by Sholl analysis. Expression of JIP3ΔKHC and JIP3ΔΔ significantly reduced the number of dendrite branches but to a much smaller extent than axon branches (see Fig. 5C) (ANOVA, p = 2.0 × 10⁻⁴; **, p < 0.01; Tukey's HSD). n.s., not significant; error bars, S.E.

FIGURE 6.

JIP3 binding to KHC is not essential for axon outgrowth in DRG neurons. A, representative Western blot of spot cultures infected with lentiviral vector, GFP-tagged WT JIP3, JIP3ΔKLC, JIP3ΔKHC, or JIP3ΔΔ. GFP was used to probe for transgenic JIP3 expression. Note that the gel used did not allow for resolution of molecular weight changes between mutants and WT. B, axon growth of DRG neurons infected at DIV1 with mutant JIP3 lentivirus and assessed for axon growth at DIV3. Dotted lines indicate the boundary of the cell body spot and the point from which axon length was assessed. Expression of JIP3 mutants did not affect growth of DRG neurons. The gridlike overlay is an artifact of stitching images together to visualize the entire spot field. C, quantification of B. No significant differences in maximum axon projection from the spot culture boundary were detected between different conditions. n = 17 (vector), 19 (WT JIP3), 24 (JIP3ΔKLC), 21 (JIP3ΔKHC), or 17 (JIP3ΔΔ) fields of view analyzed from three replicates (4 spots/group/replicate); ANOVA, p = 0.30. n.s., not significant; error bars, S.E.

FIGURE 7.

JIP3 binding to KHC is required for axon regeneration in DRG neurons. A, representative images of infected spot cultures 12 h after axon injury, stained for the regenerating axon marker SCG10. Vector-, WT JIP3-, and JIP3ΔKLC-infected neurons undergo robust regeneration after injury, which is markedly reduced with expression of JIP3ΔKHC or JIP3ΔΔ. Dashed lines, site of injury. Scale bar, 100 μm. B, quantification of A. WT JIP3 and JIP3ΔKLC slightly, but not significantly, reduce axon regeneration compared with vector controls (p = 0.077 and 0.088, respectively). In contrast, JIP3ΔKHC and JIP3ΔΔ attenuate axon regeneration after injury. n = 15 (vector), 18 (WT JIP3), 23 (JIP3ΔKLC), 30 (JIP3ΔKHC), or 31 (JIP3ΔΔ) fields of view analyzed from four replicates (4 spots/group/replicate); ANOVA, p = 0.002; **, p < 0.01; ***, p < 0.001; Tukey's HSD). C, staining of the regeneration marker SCG10 compared with total tubulin. Note that SCG10 is present in regenerating tips in all groups, indicating that transport of SCG10 was not affected by JIP3 expression. However large tubulin-positive, SCG10-negative axon debris was noted in all groups (arrowheads), indicating that SCG10 is a suitable marker for axon regeneration. Scale bar, 50 μm. Dotted line, axotomy. D, top two rows, staining of activated phosphorylated c-Jun (p-cJun) after axotomy in JIP3 mutant-expressing neurons. All groups display strong nuclear staining of phospho-c-Jun, as indicated, with the DAPI counterstain. Bottom row, uninjured controls display no phospho-c-Jun staining in DAPI-positive cell bodies. Error bars, S.E.