JIP1 mediates anterograde transport of Rab10 cargos during neuronal polarization

Abstract

Axon development and elongation require strictly controlled new membrane addition. Previously, we have shown the involvement of Rab10 in directional membrane insertion of plasmalemmal precursor vesicles (PPVs) during neuronal polarization and axonal growth. However, the mechanism responsible for PPV transportation remains unclear. Here we show that c-Jun N-terminal kinase-interacting protein 1 (JIP1) interacts with GTP-locked active form of Rab10 and directly connects Rab10 to kinesin-1 light chain (KLC). The kinesin-1/JIP1/Rab10 complex is required for anterograde transport of PPVs during axonal growth. Downregulation of JIP1 or KLC or disrupting the formation of this complex reduces anterograde transport of PPVs in developing axons and causes neuronal polarity defect. Furthermore, this complex plays an important role in neocortical neuronal polarization of rats in vivo. Thus, this study has demonstrated a mechanism underlying directional membrane trafficking involved in axon development.

Keywords: Jip1; Rab10; neuronal polarity; vesicle trafficking.

Figure 1.

KLC1 is required for anterograde transport of Rab10-PPVs. A, Cortical neurons were transfected with pSUPER vector or pSUPER-encoding siKLC1 or siScr together with KLC1^Res. Endogenous KLC (KLC1 and KLC2) and KLC1^Res levels were analyzed by IB with antibody against KLC. Actin and GAPDH were probed as controls. B, Representative kymographs of TD-Rab10 vesicles in cultured hippocampal neurons transfected with siScr or siKLC1, either alone or together with KLC1^Res. Scale bar, 10 μm. C, Quantification for the percentage of anterograde, retrograde, or immobile TD-Rab10 vesicles in neurons transfected with siScr, siKLC1, or siKLC1 plus KLC^Res. Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, Student's t test. D, Quantification for the mean velocity of TD-Rab10 vesicles in axons of cultured hippocampal neurons transfected with indicated plasmids. Data are presented as the mean ± SEM. *p < 0.05, Student's t test.

Figure 2.

Rab10 associates with KLC via JIP1. A–C, Association of Rab10, JIP1, and KLC in the rat brain. Homogenates of P0 rat brains were incubated with indicated antibodies. The same amount of control IgG (Ctrl IgG) was used as the control. Resulting immunocomplexes were subjected to IB with indicated antibodies. D, Purified His6-Rab10 (2 μg) was incubated with glutathione-Sepharose beads immobilized with the same amount of GST or GST-JIP1 (5 μg). Bound proteins were subject to IB with anti-His antibody. E, Purified His6-Rab10 (2 μg) preloaded with GTPγS or GDP was incubated with beads coupled with GST-JIP1 (5 μg). Bound proteins were subjected to IB with anti-His or GST antibody. F, G, Lysates of HEK293 cells transfected with Rab10 (WT or Q68L form) and Flag-JIP1 were subjected to IP and IB with indicated antibodies. H, Formation of the KLC1/JIP1/Rab10 complex in HEK293 cells transfected with indicated constructs. I, J, JIP1 enhances Rab10 association with KLC1 in HEK293 cells. Low and high exposures were used to display differences in band intensity, in the absence and presence of JIP1 expression. K, L, Cell lysates from HEK293 cells transfected with Flag-JIP1 and HA-Rabs were subjected to IP and then IB with anti-Flag or HA antibody. Note that only Rab10 is associated with JIP1.

Figure 3.

Mapping of the KLC1 and JIP1 domains mediating the formation of the KLC1/JIP1/Rab10 complex. A, Schematic structures of KLC1 full-length and various fragments. B, Lysates of HEK293 cells transfected with Flag-JIP1, together with Myc-tagged KLC1 fragments were subjected to IP with anti-Flag antibody, and then IB with anti-Myc or Flag antibody. C, Schematic structures of JIP1 full-length and various fragments. D, Lysates of HEK293 cells transfected with CFP-KLC1, together with control plasmid or that encoding indicated Myc-tagged JIP1 fragments were subjected to IP with antibody against GFP, and then IB with Myc or GFP antibody. E, Lysates of HEK293 cells transfected with HA-Rab10 and Myc-tagged JIP1 domains were subjected to IP with antibody against HA, and then IB with Myc or HA antibody. F, Summary of interactions between JIP1 fragments and KLC1 or Rab10. G, Effects of JIP1 fragments on KLC1 association with Rab10. HEK293 cells were cotransfected with CFP-KLC and HA-Rab10, together with vehicle vectors or that encoding various JIP1 fragments. Cell lysates were subjected to IP and then IB with indicated antibodies.

Figure 4.

JIP1 interaction with KLC regulates the localization of Rab10 in axonal tips. A, GFP-transfected hippocampal neurons at different stages were stained for Rab10 and JIP1. Scale bar, 20 μm. B, Relative immunofluorescence intensity of Rab10 and JIP1 along the axon of a hippocampal neuron at stage 3. C, Cortical neurons were transfected with siScr, siJIP1, or siJIP1 plus Myc-JIP1^Res, the siRNA-resistant form of JIP1. Cell lysates were blotted with anti-JIP1 antibody, with actin and GAPDH as loading controls. D, Cortical neurons were transfected with CFP-KLC and HA-Rab10, together with siScr or siJIP1 constructs. Cell lysates were subjected to IP with antibody against HA, followed by IB with indicated antibodies. Note the almost complete disruption of the KLC1-Rab10 association under the condition with JIP1 knockdown. E, DIV3 hippocampal neurons transfected with siScr or siJIP1 were stained with anti-Rab10 antibody. Shown are representative images of the Rab10 signals at distal regions of axons or the longest neurites. Scale bar, 10 μm. F, G, Distributions of Rab10 along axons or longest neurites in siScr (F) or siJIP1 (G) neurons. Shown are plots of relative intensity of Rab10 in the axon compared with that of the soma. Each color line represents an example neuron. H, Distributions of Rab10 in distal regions of axons or longest neurites of DIV3 hippocampal neurons transfected with indicated plasmids. Scale bar, 10 μm. I–L, Distributions of Rab10 along axons or longest neurites in DIV3 hippocampal neurons transfected with vehicle (I), KLC1-ΔN (J), KLC1-TPR1 (K), or JIP1-M (L). Data are presented as the ratio of the Rab10 intensity along the neurite to that of the soma with each color line representing an example cell. M, N, Quantification for relative intensities of Rab10 at distal regions of axons or the longest neurites (10 μm from the tip) of hippocampal neurons transfected with indicated constructs. Data are presented as the mean ± SEM. ***p < 0.001, Student's t test.

Figure 5.

JIP1 mediates anterograde transport of Rab10 vesicles. A, Hippocampal neurons were transfected with TD-Rab10, together with siScr, siJIP1, or siJIP1 plus JIP1^Res. At DIV3, transportation of individual TD-Rab10 vesicles was recorded with live-imaging microscopy. Shown are representative kymographs of TD-Rab10 in axons or the longest neurites. Scale bar, 10 μm. B, Quantification for the percentage of anterograde, retrograde, or immobile TD-Rab10 vesicles for axons or longest neurites, in transfected hippocampal neurons. Data are presented as the mean ± SEM. **p < 0.01, ***p < 0.001, Student's t test. C, Quantification for the mean velocity of TD-Rab10 vesicles in axons of hippocampal neurons of indicated groups. Data are presented as the mean ± SEM. *p < 0.05, Student's t test. D, Hippocampal neurons were transfected with TD-Rab10, together with vehicle plasmid or that encoding KLC1-ΔN or JIP1-M, followed by live imaging at DIV3. Shown are representative kymographs of TD-Rab10 vesicles in axons or longest neurites. Scale bar, 10 μm. E, Quantification for the percentage of anterograde, retrograde, and immobile TD-Rab10 vesicles. Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, Student's t test.

Figure 6.

KLC/JIP1/Rab10 complex is important for axon development. A, D, Hippocampal neurons were transfected with indicated plasmids, together with EGFP, and then stained with Smi312 antibody at DIV3. Shown are representative images for neurons in each group. Scale bar, 20 μm. B, E, Quantification for the percentage of cells with different morphology (0 axon, neurons without axon; 1 axon, neurons with a single axon positive for Smi312 and longer than 100 μm; ≥2 axons, neurons with >2 axons). Data are presented as the mean ± SEM. **p < 0.01, ***p < 0.001, Student's t test. C, F, Quantification for average length of axons in each group. *p < 0.05, **p < 0.01, ***p < 0.001, Student's t test.

Figure 7.

The Role of JIP1 in neocortical development in vivo. A, Representative images of E18.5 neocortical slices from rat embryos, which were electroporated at E15.5 with plasmids encoding siScr or siJIP1, together with pCAG-EYFP. Scale bar, 200 μm. Enlarged areas show YFP-positive neurons in the IZ of siScr- or siJIP1-electroporated cortical sections. Scale bar, 10 μm. B, Representative reconstructed images of YFP-positive cells in the IZ regions of electroporated cortical sections. Scale bar, 20 μm. C, Quantification for the percentage of monopolar/bipolar and multipolar YFP-positive neurons in the SVZ and IZ regions of electroporated cortical sections. Data are presented as the mean ± SEM. ***p < 0.001, Student's t test. D, F, Cortical sections from rat embryos electroporated at E15.5 with EYFP together with indicated plasmids were stained with Ki67 antibody (D) or Tbr2 antibody (F). Scale bars: D, 20 μm; F, 10 μm. E, G, Quantification for the percentage of Ki67-positive (Ki67+; E) or Tbr2-positive (Tbr2+; G) cells among YFP-positive cells in VZ/SVZ/IZ regions of cortical sections. n.s., Not significant, Student's t test. H, Representative images of rat neocortical slices from P0, P3, P7, or P14, after electroporation at E15.5. Scale bar, 100 μm. I, Quantification for the percentages of YFP-positive neurons in each cortical zone of rat neocortical slices at different stages. Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, Student's t test. J, P14 neocortical slices from rat embryos, which were electroporated at E15.5 with plasmids encoding siJIP1, together with pCAG-EYFP. Scale bar, 200 μm. Enlarged areas show YFP-positive neurons in layers IV–VI of siJIP1-electroporated cortical sections with various morphologies. Scale bar, 50 μm. K, P14 neocortical slices from rat embryos electroporated at E15.5 with plasmids encoding siScr or siJIP1, together with pCAG-EYFP, were stained with GM130 antibody. Scale bar, 20 μm. L, Representative tracings of YFP-positive neurons in layers IV–VI of siJIP1-electroporated cortical sections. Scale bar, 50 μm.

Figure 8.

The KLC/JIP1/Rab10 complex is required for neuronal polarization and axon growth in vivo. A, Representative images of YFP-positive neurons in the IZ of E17.5 cortical sections from rat embryos, which were electroporated at E15.5 with vehicle or JIP1-M plasmids, together with pCAG-EYFP. Scale bar, 20 μm. B, Representative tracings of YFP-positive cells in IZ regions of electroporated cortical sections. Scale bar, 20 μm. C, Quantification for the percentage of monopolar/bipolar and multipolar YFP-positive neurons in the SVZ and IZ of cortical sections with electroporation. Data are presented as the mean ± SEM. ***p < 0.001, Student's t test. D, F, Neocortical slices from rat embryos electroporated at E15.5 with EYFP together with indicated plasmids were stained with Ki67 antibody (D) or Tbr2 antibody (F). Scale bars: D, 20 μm; F, 10 μm. E, G, Quantification for the percentage of Ki67-positive (Ki67+; E) or Tbr2-positive (Tbr2+; G) cells among YFP-positive cells in VZ/SVZ/IZ regions of cortical sections. n.s., Not significant, Student's t test. H,Representative images for P3 neocortical slices from rat embryos electroporated at E15.5 with vehicle or JIP1-M plasmids, together with pCAG-EYFP. Enlarged areas show contralateral corpus callosum axon bundles. Scale bar, 500 μm. I, Intensity distribution of YFP expressed in contralateral axon bundles against the distance from the midline. The YFP intensity at the midline of the control group was normalized as 1.0. J, Quantification for the intensity of YFP in the midline. Data are presented as the mean ± SEM. *p < 0.05, Student's t test. K, Quantification of the length of axons measured from the midline to the end of axonal tracts. The value of the control group was normalized as 1.0. Data are presented as the mean ± SEM. *p < 0.05, Student's t test. L, Proposed model for anterograde transport of Rab10-PPVs along the axon. Rab10-PPVs are loaded to KIF5 by JIP1, which binds to KLC directly, and transported along microtubules during axon development.