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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).
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