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Comparative Study
. 2005 Aug 17;25(33):7669-81.
doi: 10.1523/JNEUROSCI.2680-05.2005.

Src-dependent tyrosine phosphorylation at the tips of growth cone filopodia promotes extension

Estuardo Robles  1 , Stephanie Woo, Timothy M Gomez
Affiliations
Comparative Study

Src-dependent tyrosine phosphorylation at the tips of growth cone filopodia promotes extension

Estuardo Robles et al. J Neurosci. .

Abstract

Extracellular cues guide axon outgrowth by activating intracellular signaling cascades that control the growth cone cytoskeleton. However, the spatial and temporal coordination of signaling intermediates remains essentially unknown. Live imaging of tyrosine phosphorylation in growth cones revealed dynamic phospho-tyrosine (PY) signals in filopodia that directly correlate with filopodial behavior. Local PY signals are generated at distal tips of filopodia during extension and are lost during retraction. Active Src family kinases localize to the tips of filopodia, and Src activity regulates both filopodial dynamics and local PY signaling. Positive guidance cues stimulate filopodial motility by locally increasing tyrosine phosphorylation in a cell division cycle 42 (Cdc42)-dependent manner. Locally reduced Src activity on one side of the growth cone generates an asymmetry in filopodial motility and PY signaling that promotes repulsive turning, suggesting that local changes in filopodial PY levels may underlie growth cone pathfinding decisions. p21-activated kinase (PAK), a Cdc42 effector whose activity is regulated by Src phosphorylation, also localizes to the tips of extending filopodia and controls filopodial motility. Coordinated activation of cytoskeletal effector proteins by GTPase binding and Src-mediated tyrosine phosphorylation may function to produce specific growth cone behaviors in response to guidance cues.

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Figures

Figure 1.
Figure 1.
Imaging PY dynamics in living cells using GFP-dSH2. A, Structural domains of c-Src used in the construction of PY indicator (modified from Kirchner et al., 2003). M, Myristoylation sequence; U, unique region; R, regulatory domain. B, Schematic representation of the GFP-dSH2 construct. C, Live GFP-dSH2-expressing non-neuronal cell cultured from Xenopus neural tube. Note distinct focal adhesion labeling at cell periphery (arrowheads) and PY puncta at the tips of microspikes (arrows). D, The same cell fixed and triple labeled for PY (PY99; green), vinculin (red), and filamentous actin (blue). PY labeling in fixed cells is attributable entirely to immunofluorescent staining, because GFP-dSH2 fluorescence is lost during fixation. Note close correlation between live and fixed PY labeling (arrows) as well as colocalization with vinculin (arrowheads). Scale bar: C, D, 10 μm; E-H, 5 μm. E, Live growth cone on LN expressing GFP-dSH2. Note intense labeling in point contact structures within the central domain (arrowheads) and puncta in filopodial tips (arrows). F, Growth cone from E fixed and immunolabeled with anti-phospho-tyrosine primary antibody (PY99). G, Merged image of live GFP-dSH2 and fixed PY99 fluorescence. Note that bright GFP-dSH2 puncta at the periphery colocalize with immunolabeled PY (arrows and arrowheads), whereas diffuse GFP fluorescence in the central domain shows no colocalization. H, Merged images of a growth cone on LN expressing GFP-dSH2 (green) and labeled with TMR-D (red). I, Time-lapse images during application of 10 μm HA of boxed regions indicated in H at 2× magnification. Note that, before tyrosine kinase inhibition, many filopodia contain PY puncta at their tip (arrows) and point contacts near their base (arrowheads). At 60 s after HA treatment, PY is lost from the tips of filopodia (arrows) but remains in some point contacts (arrowheads). At 5 min after HA treatment, most PY clusters have dissolved, but several stable filopodia remain.
Figure 2.
Figure 2.
Tyrosine phosphorylation at the tips of filopodia correlates with filopodial protrusion. A-C, Time-lapse images at 30 s intervals of GFP-dSH2 fluorescence in a growth cone extending on LN. D, RGB three-time-point merge of this growth cone color-coded red, green, and blue at indicated times. PY puncta at the tips of extending filopodia appear as separate colors (arrows), whereas a stable PY point contact merges to white within the growth cone (arrowhead). E-G, Time-lapse images at 90 s intervals of GFP-dSH2 in a growth cone extending on PDL. Arrows indicate a new filopodium initiated during the image sequence (see H-J). H, RGB three-time-point merge of this growth cone at indicated times. Note lack of stable PY puncta (white label) in the central domain of the growth cone as well as the extension of many PY-positive filopodia. I, Differential interference contrast (DIC) image of growth cone in E-H. J, Kymographs of DIC and GFP-dSH2 images within boxed region in I, which corresponds to the newly generated filopodium indicated with arrows in E-H. The interval between images in kymograph is 5 s. Before filopodial initiation, there is a gradual accumulation of a discrete PY puncta that remains at the tip during extension. Scale bar: A-I, 5 μm; J, 3 μm. K, Incidence of tip PY during periods of filopodial extension, stabilization, and retraction on both LN and PDL. n > 60 filopodia in each condition. Data were obtained only from filopodia that exhibited all three behaviors. **p < 0.001.
Figure 3.
Figure 3.
Src family kinase activity mediates tip PY and is required for filopodial initiation and elongation. A, B, Immunofluorescent staining of a growth cone on PDL for P-Src and PY. C, Merge of P-Src (red), PY99 (blue), and F-actin (green). Arrows indicate a subset of PY-positive filopodia with strong enrichment of P-Src. Arrowhead denotes a PY-positive filopodia lacking obvious enrichment in P-Src. D, Immunoblot for P-Src from Xenopus neural tube lysates incubated in control media or media containing either a tyrosine kinase inhibitor (20 μm HA) or a tyrosine phosphatase inhibitor [100 μm phenylarsine oxide (PAO)] and then immunoblotted with anti-P-Src antibody. Note that the density of the band at 60 kDa, which represents one or more Src family kinases, was altered in response to pharmacological manipulations. This blot was reprobed with anti-β-tubulin (β-tub) antibody to control for protein loading. Assay was repeated four times with similar results. Quantified band intensities are normalized to values for the control lane. E, DIC and GFP-dSH2 fluorescence images of a growth cone on PDL before and after acute application of an Src-specific inhibitor (1 μm PP2). Immediately after Src inhibition, a subset of filopodia briefly lengthen during loss of PY (arrows), but all filopodia stop protruding after complete loss of PY. F, The effects of tyrosine kinase inhibitors [1 and 5 μm HA, 1 μm SU6656 (SU), 1 μm PP2, and 1 μm PP3, an inactive analog of PP2] on the number of PY-positive and protrusive filopodia, expressed as a percentage of the number before drug treatment. n > 8 growth cones for each treatment. G, Src inhibition reduces the rate of filopodial initiation on LN and PDL as measured during 5 min image sequences before and after drug application. n > 10 growth cones for each condition. Scale bar: A-C, 3 μm; E, 5 μm. **p < 0.001.
Figure 4.
Figure 4.
c-Src mutants alter filopodial dynamics and PY signaling. A, Fluorescent image of a growth cone on PDL expressing GFP alone. Note the short, regularly spaced filopodia extending from the lamellar leading edge of the growth cone. B, Growth cone expressing GFP and KD-Src. Note reduced number of filopodia and simplified morphology. C, Growth cone expressing GFP and CA-Src. Note complex growth cone morphology and many filopodial protrusions. D-F, KD-Src and CA-Src expression alters the percentage of PY99-positive filopodial tips (D), the percentage of motile filopodia (E), and the rate of filopodial initiation (F). n > 20 growth cones for each condition. Scale bar, 5 μm. *p < 0.05; **p < 0.001.
Figure 5.
Figure 5.
Modulation of filopodial tip PY by BDNF and netrin-1. A-D, Time-lapse DIC and GFP-dSH2 fluorescence images of a growth cone 2 min before and 8 min after BDNF application (100 ng/ml). Note that many filopodia lack enriched GFP-dSH2 at their tip before BDNF application but subsequently accumulate GFP-dSH2 after BDNF stimulation. E, F, Time-lapse DIC and GFP-dSH2 fluorescence images of boxed region indicated in A and C 30-120 s after BDNF application. Time between images is 10 s. Arrows point to one preexisting filopodia that acquires tip PY 80 s after BDNF stimulation and three newly extended, PY-containing filopodia. G, H, Kinetics of increased filopodial initiation and GFP-dSH2 accumulation in response to BDNF in growth cone in A-D. I, Relationship between GFP-dSH2 accumulation and the initiation of motility in 39 filopodia (on 3 different growth cones) that were immotile and did not contain tip PY before BDNF application. The black line represents theoretical condition in which no delay separates PY accumulation and increased motility. Data points show actual delay, with the gray line representing the linear best fit for all data points. Note that the delay to motility initiation is disproportionately increased when the delay to PY accumulation is longer than 5 min. J, K, Application of netrin-1 (200 ng/ml) to growth cones with low baseline PY on PDL also increases filopodial tip PY. L, Percentage of PY-positive filopodia per growth cone before and after stimulation by guidance cues. BDNF-induced PY accumulation does not occur in response to denatured BDNF or during simultaneous Src inhibition with 1 μm PP2 or 1 μm SU6656 (SU). Netrin effects are similarly blocked with 1 μm PP2. n > 8 growth cones for each data set. Scale bar: A-D, J, K, 5 μm; E, F, 3 μm. *p < 0.05; **p < 0.001.
Figure 6.
Figure 6.
BDNF increases Src activity in growth cones. A-F, Single-channel fluorescence (B, D, F) and merged images (A, C, E) of P-Src and phalloidin staining in control (A, B), BDNF-treated (C, D), and BDNF plus PP2-treated (E, F) growth cones. Note increased P-Src immunofluorescence at the tips of filopodia in response to BDNF and complete loss of this enrichment by simultaneous Src inhibition with PP2. G, BDNF increases P-Src immunofluorescence at the tips of filopodia and within the growth cone central domain. Both increases are completely blocked by simultaneous application of PP2. H, Src activity is required for promotion of neurite growth rate by BDNF. Scale bar, 5 μm. *p < 0.05; **p < 0.001.
Figure 7.
Figure 7.
Local Src inhibition stimulates repulsive growth cone turning. A, B, Single-channel and merged images of PY99 immunofluorescence and phalloidin staining of a growth cone exposed to a gradient of PP2 for 5 min. Note that the number of PY-positive filopodia (small arrows) is reduced on the side of the growth cone closest to the source of PP2 (indicated by large arrow in top right corner). C, D, Time-lapse images of a control growth cone exposed to pulsatile local application of culture medium. E, F, The same growth cone as in C and D exposed to local application of media containing the Src inhibitor PP2 (50 μm in the pipette) now exhibits repulsive turning away from the pipette. G, Cumulative distribution of neurite turning angles in response to culture medium (black) or Src inhibitor (red). Average turning angles for both conditions are indicated at bottom of graph. H, Quantification of filopodial initiation rates on each side of the growth cone (proximal and distal to pipette tip) in response to local application of control media or PP2-containing media. Note that, during local application of Src inhibitor, filopodia are disproportionately generated on the distal side of the growth cone. Scale bar: A, B, 5 μm; C-F, 15 μm. **p < 0.001.
Figure 8.
Figure 8.
Cdc42 localizes to filopodia and regulates tip PY. A, B, Immunofluorescent staining for Cdc42 and PY. C, Merge of channels in A and B with F-actin staining in green. Note high degree of Cdc42 and PY colocalization (magenta) at the tips of filopodia (arrows). D, Quantification of P-Src and Cdc42 colocalization to PY-positive and -negative filopodia. P-Src accumulations were not observed in PY-negative filopodia. E, The percentage of PY-positive filopodia per growth cone determined by PY99 immunolabeling shows that PY at filopodial tips is regulated by Cdc42 activity. Inhibition of all Rho GTPases with toxin B (ToxB) or Cdc42 selectively by expression of dominant-negative Cdc42 reduces the percentage of PY-positive tips. Expression of a constitutively active mutant Cdc42 does not significantly alter filopodial tip PY. WT, Wild type. F, Direct inhibition of Cdc42 by application of CRIB peptide reduces tip PY in dSH2-expressing growth cones with high initial baseline PY. Pretreatment with CRIB peptide also blocks BDNF-induced increases in tip PY. n > 8 growth cones for each data set. Scale bar, 5 μm. *p < 0.05; **p < 0.001.
Figure 9.
Figure 9.
PAK colocalizes with PY at the tips of extending filopodia. A, B, Fluorescence images of a growth cone expressing DsRed-PAK1 and GFP. Note robust enrichment of PAK at the tips of filopodia (arrows in B). C, Incidence of DsRed-PAK accumulation at filopodial tips during periods of extension (Ext), stabilization (Stab), and retraction (Retr) in growth cones on PDL. n > 60 filopodia in each condition. D-F, Individual fluorescence channels and merged image (F) of a growth cone coexpressing DsRed-PAK and GFP-dSH2. Note the high degree of colocalization at the tips of filopodia (arrows in D and E). G, Time series at 5 s intervals of DsRed-PAK and GFP-dSH2 fluorescence within boxed region in F at 2× magnification. Note the close colocalization of PAK and PY during filopodial extension (t = 15-45 s) and delocalization of both labels during retraction (t = 75-105 s). H, I, Merged image of a growth cone expressing DsRed-PAK and GFP before and after Src inhibition (1 μm PP2). Note the DsRed-PAK accumulation at several filopodia before inhibition and subsequent loss of all filopodial enrichment after Src inhibition (arrows). J, Immunoblot for phospho-PAK1 from Xenopus neural tube lysates incubated in control media or media containing the Src-specific inhibitor SU66056 (100 μm; SU) or the N-WASP CRIB peptide (250 μg/ml). A single band is detected at ∼62 kDa, and the intensity of this band was reduced in response to Src or Cdc42 inhibition. The blot was reprobed for β-tubulin (β-tub) to control for protein loading, and the assay was repeated three times with similar results. Separate trials demonstrated similar results for the Src inhibitor PP2. Quantified P-PAK band intensities for all experiments were normalized to control lane (Ctl). Scale bar: A, B, D-F, H, I, 5 μm; G, 2.5 μm. *p < 0.05; **p < 0.001.
Figure 10.
Figure 10.
PAK activity regulates filopodial motility. A, B, Fluorescence images of growth cones on PDL coexpressing DN-PAK or CA-PAK together with GFP. C, Stimulation of filopodial motility by BDNF is blocked by expression of either DN-Cdc42 or DN-PAK. n > 12 growth cones for each condition. D-F, Effects of DN-PAK or CA-PAK expression on the percentage of PY-positive tips (D), the rate of filopodial initiation (E), and the rate of filopodial extension (F). n > 20 growth cones for each condition. G, Examples of extending filopodia from growth cones with varying levels of PAK activity. The rate of extension is increased in a CA-PAK-expressing filopodium compared with wild type (WT). Scale bar: A, B, 5 μm; G, 2 μm. *p < 0.05; **p < 0.001.
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