JCS02796 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. 1. Nucleotide sequence of the rat C6ST-1 cDNA and deduced amino acid sequence and hydropathy plot of the protein. (A) The coding sequence (GenBank™ accession number AF178689) is shown above the deduced amino acid sequence. The putative transmembrane domain (underlined) is towards the N-terminal. N-glycosylation sites are indicated in boxes. (B) The hydropathy plot was calculated according to the Kyte-Doolittle method with a window of 19 amino acids. (C) Amino acid sequence alignment of the rat C6ST-1 with those of mouse, human, and chick. Alignment was performed with the Clustal W programme. Black boxes indicate positions with conserved residues; shaded boxes indicate positions with homologous residues. The newly cloned rat C6ST-1 displays 93%, 87% and 74% identity to the mouse (AB008937), human (AB012192) and chick (D49915) sequences respectively. Dashes indicate gaps introduced for maximal alignment. Arrows bracket the putative PAPS binding sites (5¢PSB, 5¢-phosphosulphate binding; 3¢PB, 3¢-phosphate binding).

• Supplemental Figure 2 -

  Fig. 2. Emigration of cells from a DRG explant maintained in 10% FBS-DMEM-F12 supplemented with NGF (20 ng/ml) at 37°C (see also Movie 1). The migration front is towards the lower right and the explant tissue is beyond the upper left, outside the field of view. Schwann cells are distinguishable by phase-bright edges and their frequent variations in morphology as they migrate. The bar charts show profiles of cell mobility of a random selection of Schwann cells (as marked) at the migration front (950-1400 mm from the DRG centre) – (a) wide mobility-range cells (0 to 4.6 mm/minute, n=5), (b) intermediate mobility-range cells (0-2.9 mm/minute, n=6), and (c) narrow mobility-range cells (0-1.7 mm/minute, n=3). As they advance, they make transient contacts with axonal arbors and change axonal contacts along the way. In contrast, fibroblasts (asterisk) adopt flat morphologies that remain interdigitating with neighbouring fibroblasts. On the left side of the field of view, a fascicle of axons can be seen extending towards the frontier; Schwann cells (arrow) associated with the fascicle are much less mobile than those at the frontier. Bars, 100 mm.

• Supplemental Figure 3 -

  Fig. 3. Emigration of cells from a DRG explant during 3 hours in 10% FBS-DMEM-F12 supplemented with NGF (20 ng/ml) at 37°C following a 16-hour pre-treatment with chondroitinase ABC (see also Movie 2). The migration front is towards the right and the explant tissue is beyond the lower left, outside the field of view. The phase-contrast view shows cell distribution at the start of video recording. All cells mapped for mobility measurement fell into the wide mobility range (0 to 4.6 mm/minute). The bar charts show sub-classification of cell mobility of a random selection of Schwann cells (as marked) at the migration front (950-1400 mm from the DRG centre) – high-mobility-range cells (≥50% of steps exceeding 2.3 mm/minute, n=4), (b) intermediate-mobility-range cells (≥50% of steps of 1.5-2.3 mm/minute, n=6). None of the mapped cells fell into the low mobility range (≥50% of steps of £1.5 mm/minute). Bars, 100 mm.

• Movie 1 -

  Movie 1. Time-lapse video microscopy showing emigration of cells from a DRG explant maintained in 10% FBS-DMEM-F12 supplemented with NGF (20 ng/ml) at 37°C. The migration front is towards the lower right and the explant tissue is beyond the upper left, outside the field of view. Schwann cells are distinguishable by phase-bright edges and their frequent variations in morphology as they migrate.

• Movie 2 -

  Movie 2. Time-lapse video microscopy showing emigration of cells from a DRG explant during 3 hours in 10% FBS-DMEM-F12 supplemented with NGF (20 ng/ml) at 37°C following a 16-hour pre-treatment with chondroitinase ABC. The migration front is towards the right and the explant tissue is beyond the lower left, outside the field of view. The phase-contrast view in Fig. S2 shows cell distribution at the start of video recording.