The Journal of Neuroscience, February 1, 2006, ():
Rac1 and RhoA Promote Neurite Outgrowth through Formation and Stabilization of Growth Cone Point Contacts
J. Neurosci. Woo and Gomez
26: 1418
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplemental Figure 1. Dose-dependent collapse of RGC growth cones, but not spinal neuron growth cones, in response to Sema3A. Explants of spinal cord or retinal tissue cultured on LN were treated with control media (black) or 400 ng/ml (red), 1 µg/ml (magenta), or 2 µg/ml (pink) Sema3A for 15 min then fixed prior to F-actin labeling with Alexa 488-conjugated phalloidin. Growth cones lacking lamellipodia were scored as collapsed. Sema3A induces a dose-dependent increase in the percentage of collapsed retinal, but not spinal neuron, growth cones.
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Supplemental Figure 2. A sub-collapsing concentration of Sema3A subtly alters protrusion dynamics. (A-B) Two representative RGC growth cones expressing paxillin-GFP were imaged at 10 s intervals for 5-15 min before and 15 min after addition of 400 ng/ml Sema3A. (A’-B’) Kymographs were generated along the red lines in A, B. Red arrowheads indicate the time points of images in A, B. White arrows indicate time point of Sema3A addition. Sema3A can cause noticeable changes in membrane protrusion (B’), but the effect is often more subtle (A’). (C) Quantification of protrusion persistence, distance, velocity, frequency and retraction distance, as well as rate of outgrowth (n=13 growth cones). Low-dose Sema3A causes only a modest decrease in protrusion persistence and an increase in the distance of protrusion retraction, which together may explain the reduced rate of neurite outgrowth. Scale bars 5 µm or as indicated.
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Movie 1. Fluorescence image sequence of a Xenopus spinal neuron growth cone expressing paxillin-GFP migrating on LN. Arrows point to representative point contacts. Note the presence of both transient point contacts with symmetrical, punctate morphologies and stable point contacts that elongate into streak-like structures. Stable point contacts tend to elongate proximally (see Fig. 4 B-C). Frames were acquired at 10 s intervals and movie playback is at 10 frames/s. All frames of this movie were processed with the smooth function in ImageJ.
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Movie 2. Fluorescence image sequence of a Xenopus spinal neuron growth cone expressing paxillin-GFP on PDL acutely stimulated with LN. 25 µg/ml soluble LN was perfused in at 10 min. Arrows point to representative point contacts. Note that the first point contact appears approximately 7 min after LN addition accompanied by an increase in the rate of neurite outgrowth (see Fig. 5). Frames were acquired at 10 s intervals and movie playback is at 10 frames/s.
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Movie 3. Fluorescence image sequence of a Xenopus spinal neuron growth cone growth cone on LN coexpressing paxillin-GFP and myc-CA Rac1. Arrows point to representative point contacts. Note that point contacts are relatively transient and retain a symmetrical, punctate morphology (see Fig. 8, Movie1). Frames were acquired at 10 s intervals and movie playback is at 10 frames/s.
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Movie 4. Fluorescence image sequence of a Xenopus RGC growth cone expressing paxillin-GFP while growing upon LN. 400 ng/ml Sema3A was added at 5 min. Note that pre-existing point contacts dissolve upon Sema3A addition and that this growth cone changes shape, but does not collapse or retract. In the presence of Sema3a, this growth cone continues to form transient point contacts, but at a reduced frequency. Frames were acquired at 10 s intervals and movie playback is at 10 frames/s.
