spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

Right arrow Help viewing high resolution images
Right arrow Return to article
(Downloading may take up to 30 seconds.
If the slide opens in your browser, select File -> Save As to save it.)
Click on image to view larger version.


Figure 1


Fig. 1. Myosin II blocks semaphoring-3A-induced axon retraction but not growth cone collapse. (A) Still images from time-lapse sequences of the responses of axons to treatment with semaphorin 3A (SemaIIIA) or semaphorin 3A with blebbistatin pretreatment (50 µM, 1 hour). Controls were treated with DMSO, the vehicle for blebbistatin. Images of the same axons before (top panels) and 30 minutes after (bottom panels) treatment with semaphorin 3A. Treatment with semaphorin 3A caused growth cone collapse and axon retraction (arrows denote extent of retraction). Insets in blebbistatin-treated culture show a higher magnification view of the adjacent growth cone. Note that, in the presence of blebbistatin and after semaphorin 3A treatment, growth cones collapse but axons do not retract. Bar, 20 µm. (B) Quantification of the mean extension-retraction rates of axons pretreated with either 50 µM blebbistatin (Bleb), 10 µM y-27632 (Y) or transfected with 1 µg C3 (C3) by using Chariot with or without semaphorin 3A (IIIA) treatment. The control group for blebbistatin treatment alone (CNT) was treated with BSA. Relative to the CNT group, semaphorin 3A treatment induced strong axon retraction (P<0.0001). Blebbistatin inhibited semaphorin-3A-induced retraction (P<0.003, Bleb+IIIA versus IIIA). Pretreatment with Y or C3 also blocked retraction (P<0.0001 for both comparisons, Y+ IIIA and C3+IIIA versus IIIA). Neither Y nor C3 treatment altered axon extension rate relative to CNT (P>0.7 for both comparisons). Blebbistatin decreased axon extension rate relative to CNT (P<0.03). Numbers above or below bars represent the number of axons measured (n). (C) DRG explants were treated for 30 minutes with semaphorin 3A following pretreatment with Blebbistatin, y-27632 or C3. Fixed cultures were stained with Rhodamine-phalloidin and the percentage of collapsed growth cones determined blind of treatment. No statistically significant differences were observed in comparisons of blebbistatin with CNT, or IIIA relative to bleb+IIIA. Neither Y nor C3 treatment altered growth cone collapse relative to CNT or treatment with Chariot and BSA (data not shown). Both Y and C3 treatment decreased the percentage of growth cones that collapsed after treatment with semaphorin 3A (P<0.001 for both comparisons). However, in both Y- and C3-treated groups, semaphorin 3A slightly increased the percentage of collapsed growth cones relative to Y or C3 treatment alone (P<0.02 or P<0.03, respectively). Means were obtained from 10-15 cultures per group with 80-100 growth cones scored per culture. Welch t-test was used for comparisons. (D) Responses of axons pretreated with either C3 or y-27632 to treatment with semaphorin 3A. Images are shown before (pre) and 30 minutes after (post) treatment with semaphorin 3A. Ch, chariot. Arrows denote axon extension (upwards arrow) or retraction (downwards arrow). Bar, 10 µm.





Right arrow Return to article