Article Figures & Data
Figures
- Figure 1.
Aggrecan-laminin spot gradient causes the formation of dystrophic endballs. A, A low magnification image of the aggrecan-laminin spot showing the distribution of the aggrecan as visualized via CS56 staining (red). The proteoglycan concentration is lower in the center of the spot and higher in the rim (area between both arrowheads). B, The laminin, however, has the opposite pattern (green), and its deposition is greatest in the center of the spot and least at the periphery. C, β-tubulin III immunohistochemistry of a high-density dissociated adult DRG culture on the aggrecan-laminin spot. There are virtually no neurites in the outermost part of the inhibitory rim, but many that struggle into the inner part of the rim (arrow). D, A lower-density culture of DRG neurons (in green via β-tubulin staining) on the aggrecan-laminin spot (aggrecan in red via CS56 staining). There are fewer neurons growing in the center of the spot (arrowhead), and although there are some in the inner portion of the rim, no neurites are in the outermost rim (arrow). E, The appearance of a club ending on a neurite growing in the inner portion of the rim of the aggrecan-laminin spot (β-tubulin in green; CS56 in red). F, G, β-tubulin III staining of DRGs on control spots of either BSA and laminin (F) or fibronectin and laminin (G). Neurites are able to grow well and cross the rims of both spots, demarcated by the dashed lines, into the laminin surround. Scale bars: A, B, F, G, 50 μm; D, 30 μm; E, 20 μm.
- Figure 2.
Increasing laminin concentration modifies the aggrecan gradient and allows neurite crossing of the inhibitory rim. A, A merged image of an aggrecan-laminin spot double stained for CS56 (red) and laminin (green), again demonstrating their inverse gradients. The main area of interest, the rim of the spot, is 70 μm wide. B, Quantification of the number of neurites crossing the rims of spots containing 0.7 mg/ml aggrecan and 5, 10, or 25 μg/ml laminin. Increasing the laminin concentration allowed for a significant increase in the number of neurites that were able to successfully cross the inhibitory rim (n = 8 spots per group; p < 0.01). C-E, Quantification of average pixel intensity of CS56 and anti-laminin stainings in the 70-μm-wide rim region of spots containing 0.7 mg/ml aggrecan and 5 (C), 10 (D), or 25 (E) μg/ml laminin. Measurements were taken every 10 μm. The effect of the various concentrations on neurite outgrowth in the spot is shown in F-H, respectively. Despite the increase in initial laminin concentrations, the slopes of the increase in CS56 pixel intensity and decrease in anti-laminin pixel intensity remained similar. Also, as more laminin was used, less aggrecan bound to the substrate. This resulted in more outgrowth in the central portion, with some crossing of the spot when 10 μg/ml laminin was used, and many fibers crossing when 25 μg/ml was used. Scale bars: A, 50 μm; F-H, 30 μm.
- Figure 3.
Dystrophic growth cones are dynamic. A, Selected frame from a time-lapse microscopy movie of an adult DRG growth cone on laminin only. The growth cone looked stereotypical of those that grow rapidly in that they contain multiple filopodia. B, Quantification of the rate of neurite outgrowth on either laminin only or aggrecan-laminin. Net advancement of growth cones on laminin (n = 8) and aggrecan-laminin (n = 8) was quantified and compared. Although growth cones on laminin displayed a net outgrowth forward, there was a net retraction of growth cones on aggrecan-laminin, although they were highly active (p < 0.0001). C, Drawings by Ramón y Cajal (1928) (reprinted with permission) of dystrophic endings near a lesion site in vivo look like those in the rim in vitro. D, E, Scanning electron microscopy of a control growth cone on laminin (D) and a dystrophic one on aggrecan-laminin (E). The growth cone on laminin was flattened and sent out long thin filopodia (D, arrow). The dystrophic endings contained many membrane ruffles (E, arrow) and looked entirely different from growth cones on laminin. F-H, Quantification of the dynamism displayed by growth cones (I-K, respectively) on aggrecan-laminin. Time-lapse movies were made of three different endings on the aggrecan-laminin spots. The distance from the distal-most tip of the growth cone from the initial point was measured every 5 min for a total elapsed period of 2 hr. Moreover, the appearance (asterisk) or disappearance (open square) of a membrane veil was also noted. Note that although all three growth cones were able to extend past their initial points, there was a net retraction. I-K, Examples of the multiple shapes displayed by the three different growth cones. Even within short time frames, the same growth cone looked completely different, demonstrating that they are extremely dynamic. Scale bars: A, 10 μm; D, E, 1 μm; I-K, 5 μm.
- Figure 5.
Uptake of dextran by dystrophic endings demonstrates activity in vitro. After 5 d in culture, cultures were exposed to dextran-Texas Red for 30 min followed by 1 hr wash. A, A β-tubulin III-positive (green) control growth cone growing on laminin does not endocytose much dextran (red). B, A dystrophic growth cone grown on aggrecan-laminin for 5 d that was then incubated with dextran-Texas Red for 30 min at 4°C. No dextran was taken up under these conditions. A β-tubulin-rich filopodia is highlighted by the arrowhead (see Discussion). C, An example of a dystrophic growth cone that had ingested dextran (arrow). Inset is a high-magnification confocal image of the same growth cone. Because no dextran was taken up by dystrophic endings at 4°C, this suggests that the dextran seen in C is from an active uptake process and not caused by dextran passively attaching to the surface of the growth cone. D, The numbers of dextran particles in individual growth cones show obvious differences in uptake between control and dystrophic endings. The averages are depicted by the bars (*p < 0.01). Scale bar, 10 μm.
- Figure 4.
Vesicle formation occurs at the leading edge of the dystrophic growth cone. A single vesicle (highlighted in red) was tracked in individual image files taken from a time-lapse movie of a growth cone on aggrecan-laminin from its initial appearance at the front of the growth cone over a period of 12 min, when the vesicle could no longer be readily identified. During that time, the vesicle gradually moved toward the rear of the growth cone. Scale bar, 5 μm.
- Figure 6.
Dextran uptake by dystrophic endings demonstrates activity in vivo well after injury. A, B, Confocal images of dystrophic endings of axons (arrows) in spinal cord that were able to take up dextran-Texas Red 1 d after a lesion. C, D, Confocal images of dystrophic endings (arrows) of spinal cord axons that took up dextran-Texas Red 7 d after lesion. Some of these active endings contain large vacuoles (A, C). Scale bar, 10 μm.
Additional Files
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.
