The cytoplasmic segment of calsyntenin-1 binds directly to the light chain of Kinesin-1. (A) Schematic diagram illustrating the calsyntenin-1 bait construct (aa 878-924) used for the yeast two-hybrid screen. (B) Domain structure of KLC1. The regions encoding the 16 KLC1 cDNA clones isolated from the screen are depicted. (C) Recombinant KLC1 (15 μg) was incubated with beads coated with 5 μg GST or GST-calsyntenin fusion proteins (GST-Cst1, GST-Cst2, GST-Cst3). Bound proteins were analyzed by immunoblotting using an antibody against KLC1 (L15; top panel) and Ponceau-S staining of the membrane (bottom panel). All three calsyntenins interact with KLC1. (D) Estimation of the apparent binding affinity of calsyntenin-1 to KLC1. Increasing concentrations of KLC1 (0.2–5 μM) were incubated with 46 nM GST-Cst1. Bound and free KLC1 were measured, and the K_d was determined by curve-fitting as described in Materials and Methods (R² = 0.95, n = 3). (E) Immobilized GST-Cst1 or GST (50 μg) was incubated with mouse brain extract. Bound proteins were analyzed by immunoblotting using the indicated antibodies. GST-Cst1 pulled down both KLC1 and KHC. (F1 and F2) HeLa cells were cotransfected with EGFP-calsyntenin-1 and HA-KLC1 (F1) or the KLC1 deletion construct HA-L176 (F2) followed by immunoprecipitations using the indicated antibodies. Immunoprecipitates were analyzed by immunoblotting with the indicated antibodies. EGFP-calsyntenin-1 coimmunoprecipitated with HA-KLC1, but not with HA-L176.

Calsyntenin-1 and Kinesin-1 interact in live cells. (A) HeLa cells were triple-transfected with EGFP-calsyntenin-1, HA-KLC1, and myc-KHC. Cell lysates were subjected to immunoprecipitation using the indicated antibodies. The tripartite complex was isolated with R85, anti-HA, and anti-c-myc antibodies, but not with nonspecific IgG. (B) Immunoprecipitation of calsyntenin-1 from the V1 membrane fraction of P7 mouse brains (see Supplementary Methods). Antibody R85 and R140 immunoprecipitates contained KLC1, KLC2, and KHC, but not JIP-1, SNAP-25, synaptophysin, and GAP-43. Nonspecific IgG did not precipitate any of these proteins. Note that longer exposure times were required to detect KLC and KHC in R140 immunoprecipitates (insets).

Calsyntenin-1 colocalizes with Kinesin-1 in growth cones and is enriched in axonal growth cone vesicles. (A) Immunocytochemistry of cultured cortical neurons (14 DIV) using antibodies R85 or R140. Dendrites and axons (arrowheads) were discriminated by costaining with MAP2. Anti-calsyntenin-1 antibodies labeled tubulovesicular structures in the soma, in dendrites, and in the axon. Note the abundance of calsyntenin-1 immunoreactivity in the perinuclear region and in the axon. Scale bar, 30 μm. (B–D) Double stainings of axonal growth cones (4 DIV) with antibody R85 (B1–D1) and either tubulin (B2), KHC (C2), or synaptophysin (SyPh; D2). Note the alignment of calsyntenin-1–positive vesicles along microtubules (B3 and B4) and the partial colocalization with KHC (C3 and C4), but not with synaptophysin (D3 and D4). B4 to D4 show higher magnifications of the boxed areas in B3 to D3. Scale bar, 10 μm in D1 and 1 μm in D4. (E) Axonal growth cones were isolated from P7 mouse brains by subcellular fractionation and analyzed by Western blotting using the indicated antibodies. Full-length (FL) calsyntenin-1 and the calsyntenin-1 stump (ST) were enriched in the GCV fraction, with trace amounts present in the GCM fraction. Note the presence of KLC and KHC in the GCV fraction. ED, ectodomain; PNS, postnuclear supernatant. (A) Growth cone particle-enriched fraction; (B) synaptic elements; (C) axonal shafts. GCP, pelleted growth cone particles; GCV, growth cone vesicles; GCM, growth cone membranes.

KBS1 and KBS2 are conserved among calsyntenins and show sequence similarity to the vaccinia virus protein A36R. Alignment of KBS1 and KBS2 core motives of all calsyntenin members with an internal sequence (aa 95-101) of the vaccinia virus protein A36R. Note the sequence similarity at positions 1, 3, 4, and 5 in KLC-binding motives between calsyntenins and A36R. Underlined amino acid residues highlight important positions for the interaction.

Calsyntenin-1 contains two KLC1-binding segments. (A) Deletion mutants of the cytoplasmic domain of calsyntenin-1 were tested for KLC1 binding in GST pulldown assays as described in Figure 1C. Two KLC1-binding segments, KBS1 (aa 897-906) and KBS2 (aa 966-979), were found. (B) Schematic diagram summarizing the mapping analysis. Binding constructs are shown in green and nonbinding mutants in red. (C) Sequence alignment of the cytoplasmic domain of calsyntenins. Note the conservation of the WDDS core motif in KBS1 and KBS2. (D) Calsyntenin-1 point mutants in the KBS1 and KBS2 were generated by replacing the conserved tryptophan residues with alanine in either of the two binding sites (W1 and W2) or in both (WW) and tested in GST pulldowns as described in Figure 1C. (E) Analysis of KLC1-binding of calsyntenin-1 point mutants by quantitative GST pulldown assay. The apparent binding affinities of W mutant proteins are expressed as amount of bound KLC1 relative to the amount bound by wild-type GST-Cst1. Mutation in one binding segment (W1 and W2) caused a significant reduction of bound KLC1 to <30%. Mutation of both tryptophans (WW) reduced KLC1-binding to 3% (***p < 0.0001; n = 3). (F) HeLa cells were cotransfected with wild-type or mutant EGFP-calsyntenin-1 constructs and HA-KLC1 followed by immunoprecipitation using antibody R85. Note the reduction of bound HA-KLC1 and the relative abundance of unbound HA-KLC1 in WW mutant precipitates. (G) Effect of several KBS1 point mutations on KLC1-binding. Immobilized GST-Cst1 (897-911) and the corresponding point mutants were incubated with KLC1. Bound proteins were analyzed by immunoblotting. The MDWDDS core motif of KBS1 is boxed, and the mutated amino acid residues are depicted in bold. (H) Effect of phenylalanine F896 in the KBS of calsyntenin-3 on KLC1 binding. Immobilized GST-calsyntenin-3 (aa 869-956), GST-Δcalsyntenin-3 (891-956), and the corresponding F896/D mutant were incubated with KLC1. Bound proteins were analyzed by immunoblotting. mm, Mus musculus; hs, human; dm, D. melanogaster; ce, C. elegans. Error bars in E, SEM.

Calsyntenin-1 is enriched in developing nerve fiber tracts and is associated with tubulovesicular organelles. (A and B) Immunohistochemistry of P6 and adult mouse brain using anti-calsyntenin-1 antibody R113. (A) At P6 all major fiber tracts were strongly labeled, including the anterior commissure (small arrowhead) and the corpus callosum (large arrowhead). (B) In adult mice, only faint immunoreactivity was observed in these tracts. (C–F) Immunoelectron micrograph of calsyntenin-1 in the corpus callosum of P8 mice using antibody R113. Immunogold particles labeled the membranes of tubulovesicular organelles. Scale bar, 2 mm in A and B, 0.2 μm in C–F.

Axonal transport of calsyntenin-1–containing vesicles depends on the binding between the cytoplasmic segment of calsyntenin-1 and Kinesin-1. (A) Three consecutive frames of a time-lapse sequence (2-s interval) showing one moving vesicle (A1, arrowheads) and one tubular organelle (A2, ∗) containing EGFP-calsyntenin-1 moving anterogradely within the same axonal segment. (B) Velocity distribution of vesicles (B1) and tubules (B2) carrying wt, W1, W2, and WW EGFP-calsyntenin-1. (C) Kymographs illustrating the movements of wt (top) and WW (bottom) transport organelles. (D) Schematic representation of different types of trajectories obtained by manual organelle tracking (see Materials and Methods). (E) Quantitative analysis of organelle motility. (E1) At least 50% of wt, W1, and W2 transport organelles moved continuously, compared with 30% of WW organelles. WW organelles differed significantly from wt, W1, and W2 organelles (wt, n = 221; W1, n = 217; W2, n = 127; WW, n = 317). (E2) Ratio of stop-to-turn events for wt and WW mutant organelles. After pausing, WW organelles initiated retrograde movement significantly more often than wt organelles. (F1 and G1) Displacement-distribution (run-lengths) of wt, W1, W2, and WW vesicles (F1) and tubules (G1). (F2) The displacement of WW vesicles was significantly reduced compared with wt, W1, and W2 vesicles. (wt, n = 96; W1, n = 42; W2, n = 31; WW, n = 36). (G2) WW tubules were not significantly different from wt, W1, and W2 tubules (wt, n = 32; W1, n = 23; W2, n = 42; WW, n = 37). Error bars, SEM. Scale bar in A, 1 μm.