Supplemental Material

This article contains the following supporting material:

• Movie 1 - Time-lapse fluorescence microscopy of an axon from a cortical neuron transfected with wild-type EGFP-calsyntenin-1. Cells were transfected at 3 DIV and subjected to live imaging at 5 DIV. Time intervals between frames were 2 s. Most vesicles moved continuously in anterograde direction (right to left). Only a minority of vesicles stopped within this axonal segment.

• Movie 2 - Time-lapse fluorescence microscopy of an axon from a cortical neuron transfected with EGFP-calsyntenin-1 WW mutant. Live imaging was performed under the same conditions as in Movie 1. Many vesicles stopped or turned during the time of observation. Only few vesicles moved in a continuous manner.

• Figure 1 - Characterization of affinity-purified rabbit anti-calsyntenin-1 antibodies R85 and R140. Antibody R85 was raised against the N-terminal cadherin domains, while R140 is a peptide antibody directed against the very C-terminus of calsyntenin-1. (A) Thirty micrograms of adult mouse brain homogenate (H) and a crude microsomal membrane pellet (P3) were subjected to Western blotting and probed with the indicated antibodies at a concentration of 1 μg/ml. Antibody R85 recognizes full-length calsyntenin-1 (150 kD) and the ectodomain (120 kD). Antibody R140 detects the full-length molecule and a 31 kD band that corresponds to the transmembrane stump after extracellular cleavage. Note the relative enrichment of the full-length form of calsyntenin-1 and the stump in the P3 fraction.

• Figure 2 - Calsyntenin-1 and KLC1 specifically co-immunoprecipitate from HeLa cell extracts. HeLa cells were transfected with either HA-KLC1 and EGFP-calsyntenin-1 or HA-KLC1 and an EGFP control plasmid. Both samples were subjected to immunoprecipitations using the indicated antibodies. Bound proteins were analyzed by immunoblotting using mouse anti-HA and mouse anti-GFP antibodies. Anti-calsyntenin-1 antibodies R85 and R140, as well as anti-GFP antibodies precipitated full-length EGFP-calsyntenin-1 and HA-KLC1. R140 pre-immune IgG did neither precipitate EGFP-calsyntenin-1 nor HA-KLC1. R85, R140 and anti-GFP antibodies did not precipitate HA-KLC1 from cells that were transfected with HA-KLC1 and EGFP. In control immunoprecipitations, anti-GFP antibody precipitated EGFP, but not HA-KLC1. Bands marked with a star indicate degradation products of EGFP-calsyntenin-1, as determined by peptide mass fingerprinting (data not shown).

• Figure 3 - Temporal and spatial co-segregation of calsyntenin-1 and Kinesin-1 in mouse brain. (A) Extracts of mouse brains from various developmental stages to adulthood were analyzed by Western blotting using the indicated antibodies. High expression levels of calsyntenin-1 were found during the first postnatal week, reaching a peak at postnatal day seven (P7). The expression peak of KLC and KHC coincided with the highest expression of calsyntenin-1. The expression of synaptophysin gradually increased over time to reach the highest level in the adult brain. FL: full-length; ED: ectodomain; ST: stump. (B) Brains of P7 mice were homogenized and subjected to differential centrifugation (see Supplemental Methods). Three membrane fractions (V0, V1, V2) and a soluble fraction (S2) were obtained and analyzed by Western blotting. KHC and KLC were found in all membrane fractions, with trace amounts left in S2. Full-length calsyntenin-1 was enriched in V1, while the calsyntenin-1 stump was present in both V0 and V1. The calsyntenin-1 ectodomain was accumulated in S2, but was also detected in membrane fractions. Synaptophysin was found in both V0 and V1 membrane pellets.