Supplemental Material

This article contains the following supporting material:

• Supplemental Video 1 - Kidins220/ARMS is actively transported in PC12 cells. Cells were injected with EGFP-Kidins220/ARMS and imaged as described in Materials and Methods. The cell body is located on the left. Frames were taken every 2 s and correspond to a field of 32.8 μm x 25 μm. The video consists of 150 frames played at 10 frames/s.
• Supplemental Video 2 - mRFP-KIM expression impairs the trafficking of Kidins220/ARMS. Cells were coinjected with EGFP-Kidins220/ARMS and mRFP-KIM and imaged as described in Materials and Methods. In the presence of mRFP-KIM, EGFP-Kidins220/ARMS-positive structures are mainly stationary or undergo short range movements. Frames were taken every 2 s and correspond to a field of 13 μm x 21.7 μm. The video consists of 100 frames played at 10 frames/s.
• Supplemental Video 3 - The expression of mRFP-KIM(Y24A) does not alter the trafficking of Kidins220/ARMS. Cells were coinjected with EGFP-Kidins220/ARMS and mRFP-KIM(Y24A) and imaged as described in Materials and Methods. The trafficking of EGFP-Kidins220/ARMS-positive carriers is unaffected by the expression of mRFP-KIM(Y24A). Frames were taken every 2 s and correspond to a field of 51.09 μm x 31.2 μm. The video consists of 100 frames played at 10 frames/s.
• Supplemental Figure 1 - Pairwise testing of interactions by yeast two-hybrid analysis. Relative strength of interactions between different Kidins220/ARMS and kinesin-1 constructs, together with positive and negative controls, was measured by colony growth on high stringency medium (A) and by quantifying β-galactosidase activity associated with the resulting colonies (B). (A) Growth of yeast cotransformed with KC and KLC1 encoding plasmids was in the range of that of SyD/JIP-3 and KLC1 (set to 100%). A reduced number of colonies was observed for yeast cotransformed with KC and the TPR regions of KLC1 (KLC1-TPR, corresponding to KLC1-F in Figure 3), and no colonies were observed for the cotransformation with Pp5 (Pp5-TPR), indicating that TPR motifs are not sufficient to bind Kidins220/ARMS. The addition of a portion of the HR repeats (KLC1-ΔN, corresponding to the construct KLC1-E in Figure 3) partially rescued the interaction (see also B), suggesting that Kidins220/ARMS interacts with KLC1 via a novel docking site, which includes both HR and TPR regions. The mutation Y1379A, which corresponds to Y24 in KIM(Y24A), strongly impairs the interaction between Kidins220/ARMS and KLC1, as shown by both yeast growth (A) and β-galactosidase activity (B), and is in agreement with the GST-pull down results shown in Figure 4D. (B) Quantitative β-galactosidase assay on single yeast colonies (see Materials and Methods) confirmed that the TPR domain alone (KLC1-TPR) is unable to bind Kidins220/ARMS, and that a portion of the HR repeats (KLC1-ΔN) is required. The β-galactosidase activity of the yeast cotransformed with KC and KLC1 encoding plasmids was set to 100%, whereas 0% corresponds to the activity of yeast transformed with KC alone. n=3 colonies were measured for each couple. Error bars represent s.e.m.
• Supplemental Figure 2 - Kidins220/ARMS and kinesin-1 partially colocalise in primary MNs. Differentiating rat spinal cord MNs (1 d in culture) were stained with Kidins220/ARMS antibodies (b, f), and anti-KLC (c) or anti-KHC (g) antibodies, and analysed by confocal microscopy. Structures containing both Kidins220/ARMS and KLC (d) or KHC (h) appear in yellow (arrowheads). Scale bars=5 μm.
• Supplemental Figure 3 - Quantification of GST and GST-fusion proteins used in the biochemical analysis of the Kidins220/ARMS - KLC interaction. (A) (a) Coomassie staining of the SDS-PAGE gel corresponding to the GST-pull down in Figure 1B. The GST : GST-KC molar ratio is 3. (b) Ponceau staining of the nitrocellulose membranes corresponding to the GST-pull down in Figure 1C. The GST : GST-KC molar ratio is 3.4 for the experiment done with PC12 cell extract, and 2.3 for the GST-pull down from rat brain. (B) Coomassie staining of the SDS-PAGE gels corresponding to the GST-pull down in Figure 3B. The GST : GST-KC molar ratio is 2.7 ± 0.4. The amount of GST-KC loaded in different samples is comparable. (C) (a) Coomassie staining of the SDS-PAGE gel corresponding to the GST-pull down in Figure 4C. The molar ratios of GST versus the different GST-fusion proteins are the following: 7.9 for KC-A, 3.0 for KC-E, 3.3 for KC-F, 1.5 for KC-K and 8.8 for KC-L. (b) Ponceau staining of the nitrocellulose membrane corresponding to the GST-pull down in Figure 4D. The molar ratios of GST versus the different GST-fusion proteins are the following: 3.7 for KC-A, 1.1 for KC-K, 2.1 for KC-L, 2.2 for KIM(Y24A). Molar ratios were calculated as described in Materials and Methods.
• Supplemental Tables 1 and 2