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First published online August 9, 2006
doi: 10.1242/10.1242/jcs.03084

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RhoA-kinase coordinates F-actin organization and myosin II activity during semaphorin-3A-induced axon retraction

Drexel University College of Medicine, Department of Neurobiology and Anatomy, 2900 Queen Lane, Philadelphia, PA 19129, USA

View larger version (100K): [in a new window]   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.

View larger version (48K): [in a new window]   Fig. 2. Semaphorin 3A induces axonal F-actin bundles. (A) Control (CNT) axons exhibit F-actin staining restricted to the distal axon (arrows in color-inverted inset). Semaphorin 3A (SemaIIIA) treatment increased phalloidin staining relative to control axons and induced F-actin bundles throughout the axon (arrows in inset). Bar,10 µm. (B) Four panels showing details of semaphorin-3A-induced axonal F-actin bundles (images were color inverted). Arrowheads denote what appear to be continuous F-actin bundles that cross from one side of the axon to the other. Bar, 2 µm. (C) Examples of axons double labeled with phalloidin and myosin-II-isoform-specific antibodies. Axons were treated for 20 minutes with either semaphorin 3A (Sema) or BSA as a control (CNT) prior to fixation. Note colocalization of myosin IIA aggregates with areas of axonal buckling (yellow arrows) and at the tip of the retracting axon (yellow arrowhead). I did not observe a similar organization of myosin IIB in semaphorin-3A-treated axons, in which myosin IIB distribution appeared relatively uniform. (D) Quantification of the percentage of axons that exhibit F-actin bundles as a function of distance from the tip of the axon in 5 µm bins. Control axons (CNT) reliably (>50%) exhibited bundles only in the distal-most 20 µm. Semaphorin 3A treatment for 30 minutes (IIIA) induced axon F-actin bundles up to 100 µm behind the growth cone. Blebbistatin (Bleb) did not alter semaphorin 3A induction of bundles. y-27632 at 10 µM (Y) partially decreased semaphorin-3A-induced formation of axon bundles. Thirty axons were measured per group. (E) Increase in axonal F-actin content relative to controls not treated with semaphorin 3A. Controls were treated with DMSO or transfected with BSA, for y-27632 (10 µM) and C3 transfection, respectively. Numbers indicate n of axons measured. (F) Examples of axons transfected with BSA or C3 and then treated with semaphorin 3A and stained with phalloidin to reveal F-actin. Note that the C3-transfected axon retrained the growth cone and did not develop prominent axonal F-actin bundles following treatment with semaphorin 3A (IIIA+C3, right panel).

View larger version (35K): [in a new window]   Fig. 3. Constitutively active RhoA-induced RhoA-kinase-dependent axonal F-actin bundles. (A,B) Chariot-peptide-mediated transfection of axons with constitutively active RhoA (L63RhoA) induced axon bundles in axons that (A) underwent retractions and also in axons that (B) did not retract but exhibited partially collapsed growth cones with distal accumulation of F-actin. Insets show magnified and color-inverted regions of the axons as denoted by the white lines. Bar, 10 µm. (C) Quantification (in percent) of axon segments exhibiting F-actin bundles (as shown in Fig. 2) revealed that treatment with y-27632 at the time of L63RhoA transfection completely inhibited the formation of F-actin bundles. (D) L63RhoA decreased the percentage of time that axon spent extending relative to BSA-treated controls. Data are normalized to BSA-transfected axons. (E) Inhibition of ROCK minimized L63RhoA-induced axon retraction. The percentage of axons simultaneously transfected with L63RhoA and treated with y-27632 that exhibit retraction during imaging was decreased by 69% relative to axons transfected with L63RhoA.

View larger version (26K): [in a new window]   Fig. 4. Semaphorin 3A activates myosin II. (A) Neurons were loaded with 2.5 µM of the volumetric fluorescent reporter CellTracker prior to treatment with semaphorin 3A or BSA (control) for 10 minutes. Cultures were then fixed and stained with an antibody against phosphorylated regulatory myosin light chains (rMLC-p). Treatment with semaphorin 3A increased the levels of rMLC-p in axons relative to controls. (B) Quantification of the ratio of rMLC-p staining to CellTracker staining (sample size is given within bars). Semaphorin 3A increased rMLC-p staining in the distal 20 µm of the axon by 69% (P<0.0001, Welch t-test). The mean and s.e.m. are shown in all presentations of quantitative data. (C) Pretreatment with 10 µM y-27632 (30 minutes) prevents the semaphorin-3A-induced increase in the ratio of rMLCp to CellTracker staining (P>0.7, Welch t-test; sample site is givin within bars). Data are shown normalized to treatment with y-27632 alone.

View larger version (86K): [in a new window]   Fig. 5. Semaphorin 3A blocks the formation of axonal F-actin patches that serve as precursors to filopodial and lamellipodial extension. (A) Example of filopodial protrusion from a spontaneously formed axonal F-actin patch in an eYFP-actin expression axon. Numbers in panels reflect seconds. At 15 seconds, a patch forms (arrow) that subsequently gives rise to a filopodium (sec 30-45). By 60 seconds, the filopodium has retracted and the patch has disappeared. As described by Loudon et al., the majority of axonal protrusive events are preceded by patch formation (Loudon et al., 2006), although only approximately 6-7% of F-actin patches give rise to filopodial or lamellipodial protrusion. (B) Determination of the frequency of F-actin patch formation, during 6-minute sampling periods (6-second interframe intervals) in eYFP-actin-transfected axons revealed that semaphorin 3A inhibited the formation of patches. Inhibition of patches occurred with a similar time course to that of growth cone collapse (i.e. during the first 10 minutes of treatment). The effects of semaphorin 3A were blocked by 10 µM y-27632 (P values shown for comparison of semaphorin 3A treatment and controls within time points, Welch t-test, n=6-7 axons per group; y-27632 together with semaphorin 3A frequencies were not different from controls at either time points). (C) Example of axonal F-actin patch formation in a control axon, as previously described by Loudon et al. (Loudon et al., 2006). Patches form spontaneously and increase in size and fluorescence intensity. Double-magnification insets of the patches are shown in the bottom of each panel. Numbers in panels reflect seconds in the time-lapse sequence. Arrowhead in middle panel indicates a briefly detectable eYFP-actin patch. (D) Example of minimal patch-formation, and lack of patch development, in an axon treated with semaphorin 3A for 13 minutes. A small, but detectable, patch of eYFP-actin formed (middle panel) but the patch disappeared at 6 seconds. The rapid disappearance of patches in the semaphorin-3A-treated axon stands in contrast to the much longer live-span of patches observed in control axons (compare seconds elapsed in C and D). Arrowheads in middle panel indicate a briefly detectable eYFP-actin patch. Double-magnification insets of the patches are shown in the bottom of each panel; arrow in 0-second-panel magnification indicates the collapsed growth cone. Numbers in panels reflect seconds in the time-lapse sequence. (E) Growth cone pretreated with y-27632 followed by a 13-minute treatment with semaphorin 3A. Notice that the growth cone is not collapsed (as in D), and continues to undergo morphologic remodeling. Numbers in panels reflect minutes. (F) Example of an eYFP-actin patch (arrowhead) forming and developing in an axon pretreated with y-27632 followed by treatment with semaphorin 3A for 13 minutes. Notice the similarity to patch formation in control axons (in C). Number in panels reflect seconds. (G) Time-lapse sequence from an eYFP-actin-transfected axon treated with semaphorin 3A for 13 minutes (t=0); 72 seconds later, bundle-like eYFP-actin is apparent (arrowheads) and becomes more pronounced at 240 seconds. Image was inverted to increase the contrast of the eYFP-actin signal.

View larger version (15K): [in a new window]   Fig. 6. Model of the proposed role of RhoA-kinase (ROCK) in the coordinated regulation of the axonal F-actin cytoskeleton and myosin II activity in response to semaphorin 3A signaling. (A) Under normal conditions, RhoA-kinase (ROCK) activity is low. However, as previously reported (Loudon et al., 2006), endogenous baseline-ROCK-activity contributes to the negative regulation of protrusion, and promotes myosin II activity and the formation of a population of F-actin bundles in growth cones. (B) Following activation of RhoA by semaphorin 3A, ROCK activity is elevated, resulting in the suppression of protrusive activity, which contributes to growth cone collapse. In concert, elevated ROCK activity promotes the activation of myosin II and the formation of non-protrusive intra-axonal F-actin bundles that serve as a substratum for myosin II to generate the contractile forces required to drive axon retraction. Since inhibition of ROCK only partially blocked the formation of axonal bundles, additional pathways not elucidated in this report probably contribute to bundle formation. This diagram shows the proposed map of ROCK-myosin-II functions in growth cone collapse and axon retraction induced by semaphorin 3A. Since growth cone collapse occurs before axon retraction, the time course of the functions of ROCK-myosin-II in these two processes is not directly shown here.

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