Supplemental data
Supplemental Figures
Files in this Data Supplement:
- Supplemental Fig. 1 - Supplemental figure 1: Time-lapse movie of a growth cone on laminin. Adult dissociated DRG neurons were grown on laminin (5�g/ml). Digital still images were taken after 1 day in culture every minute and later compiled together to make the movie. The growth cone continually grows forward, and possesses multiple filopodia. The movie plays at 60x speed.
- Supplemental Fig. 2 - Supplemental figure 2: Time-lapse movie of a dystrophic growth cone on aggrecan/laminin. Adult dissociated DRG neurons were grown on spots made of aggrecan (0.7mg/ml) and laminin (5�g/ml) and imaged after 2 days in culture. Again, images were taken every minute. The growth cone is extremely active and continually sends out veils of membrane as it struggles but largely fails to grow forward. The movie plays at 60x speed.
- Supplemental Fig. 3 - Supplemental figure 3: High-speed time-lapse movie demonstrates that vesicles form at the leading edge of the dystrophic growth cone and move retrogradely. Adult DRG neurons were seeded onto the aggrecan/laminin spots and imaged after 3 days in culture. The vesicles appear to form at the peripheral edge of the active dystrophic ending and move towards the rear of the growth cone, where they eventually disappear, suggesting that the membrane may be locally recycled. The movie plays at 260x speed.
- Supplemental Fig. 4 - Supplemental figure 4: Cytoskeletal proteins are expressed throughout dystrophic growth cones in vitro. A-D, a growth cone grown on laminin-only double-stained for type III �-tubulin (b, green) and phalloidin Texas-Red to visualize F-actin (c, red). The phase-contrast image is shown in panel A. Tubulin and F-actin were fairly well separated in the growth cone. E-H, a double-stained dystrophic growth cone on aggrecan/laminin for -type III �-tubulin (f, green) and phalloidin-Texas Red (g, red). E, the phase-contrast image of the dystrophic growth cone. Both proteins were located at the tip of the growth cone (arrow in h), which was not seen in control growth cones. Scale bar: 10�m.
- Supplemental Fig. 5 - Supplemental figure 5: Cytoskeletal proteins are also expressed throughout dystrophic endings following spinal cord injury in vivo. A-C, confocal images of a dystrophic growth cone (arrows) 1 day following a dorsal column lesion. The growth cone was double-stained for �-tubulin III (a, green) and F-actin (b, red). The merged image is shown in c. There were areas that were positive for �-tubulin III and not for F-actin (arrowhead), suggesting that the overlap was not due to bleed-through of the fluorophore. D-F, confocal images of a dystrophic ending (arrows) 7 days after a dorsal column lesion. Again, �-tubulin III is depicted in green (d) and F-actin in red (e). The merged image is shown in f. There was expression of tubulin and F-actin throughout the endball in both examples, confirming the phenotype that was found in dystrophic endings in vitro as described above. Scale bars: 25�m.
- Supplemental Fig. 6 - Supplemental figure 6: Dystrophic growth cones ectopically express surface alpha 1 integrin. A-I, integrin staining in control and dystrophic growth cones was done without using a detergent. A-C, images of a growth cone growing on laminin-only stained for �-tubulin III (a,c, green) and alpha 1 (b,c, red). There was virtually no alpha 1 immunoreactivity on the growth cone. The only visible alpha 1 was associated with �-tubulin-negative satellite cells that were also present. D-F-, �-tubulin III (d,f, green) and alpha 1 (e,f, red) localization on a dystrophic growth cone on the aggrecan/laminin spot. Unlike growth cones on laminin, this growth cone expressed alpha 1 all over the surface. G-I, confocal microscopy further demonstrated that alpha 1 integrin (h,i, red) was located all over the surface (arrow) of a different �-tubulin-positive dystrophic growth cone (g,i, green) on aggrecan/laminin. J-L, images of a growth cone growing on laminin stained for �-tubulin III (j,l, green) and alpha 1 (k,l, red) using triton X-100. Membrane permeation allowed for �1 visualization in control growth cones. Insets are enlarged images of selected growth cones. Scale bars: 30�m.
- Supplemental Fig. 7 - Supplemental figure 7: alpha 1 integrin is expressed on the surface of dystrophic growth cones in vivo. 1 day following a spinal cord lesion, dystrophic endballs (arrows) near the lesion expressed alpha 1 (a, b, red). There was no visible alpha 1 expressed distal to the lesion (c). The surface expression of alpha 1 was no longer visible in axons near the lesion by 7 days post lesion (d). Scale bar: 20�m.
- Supplemental Fig. 8 - Supplemental figure 8: Myelin causes growth cone collapse. A, selected frames from a time-lapse microscopy movie of an adult DRG growth cone cocultured with oligodendrocytes. Although the growth cone (arrow) appeared collapsed and quiescent, it was able to recover briefly and send out lamellipodia before collapsing and again. B, C, selected frames from a time-lapse microscopy movie of an adult DRG growth cone before (b) and after (c) application of a purified myelin membrane. The growth cone collapsed after myelin membrane was added to the media. Note that in growth cones collapsed by either coculture with oligodendrocytes or purified myelin membrane, no large vesicles were seen. D-G, the same growth cone (b,c) was double-stained for �-tubulin III (e, g, green) and F-actin (f, g, red) that collapsed after myelin membrane was added to the media. The merged image is shown in g. The phase-contrast image is shown in d. Small filipodia (arrows) were rich with F-actin and had small amounts of tubulin. Furthermore, it appeared that tubulin does not fill the filopodia, suggesting that there was still some, although not complete, separation of cytoskeletal proteins in collapsed growth cones. Scale bars: 5�m.
