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. 2001 Oct 29;155(3):427-38.
doi: 10.1083/jcb.200107063. Epub 2001 Oct 22.

Transmission of growth cone traction force through apCAM-cytoskeletal linkages is regulated by Src family tyrosine kinase activity

D M Suter  1 , P Forscher
Affiliations

Transmission of growth cone traction force through apCAM-cytoskeletal linkages is regulated by Src family tyrosine kinase activity

D M Suter et al. J Cell Biol. .

Abstract

Tyrosine kinase activity is known to be important in neuronal growth cone guidance. However, underlying cellular mechanisms are largely unclear. Here, we report how Src family tyrosine kinase activity controls apCAM-mediated growth cone steering by regulating the transmission of traction forces through receptor-cytoskeletal linkages. Increased levels of tyrosine phosphorylation were detected at sites where beads coated with apCAM ligands were physically restrained to induce growth cone steering, but not at unrestrained bead binding sites. Interestingly, the rate and level of phosphotyrosine buildup near restrained beads were decreased by the myosin inhibitor 2,3-butanedione-2-monoxime, suggesting that tension promotes tyrosine kinase activation. While not affecting retrograde F-actin flow rates, genistein and the Src family selective tyrosine kinase inhibitors PP1 and PP2 strongly reduced the growth cone's ability to apply traction forces through apCAM-cytoskeletal linkages, assessed using the restrained bead interaction assay. Furthermore, increased levels of an activated Src family kinase were detected at restrained bead sites during growth cone steering events. Our results suggest a mechanism by which growth cones select pathways by sampling both the molecular nature of the substrate and its ability to withstand the application of traction forces.

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Figures

Figure 1.
Figure 1.
Intense PY labeling at the leading edge, tips of filopodia, and in ruffles of growth cones. PY immunocytochemistry using the 4G10 antibody in Aplysia growth cones. (A) Low power magnification view of bag cell neuron; cell body position is marked by star and arrow points to increased labeling at contact site. (B–E and G–I) High power magnification views of growth cones. (B and C) Same growth cone is shown with PY labeling (B) and in DIC optics (C). Peripheral domain (P), transition zone (T), and central domain (C) are indicated. Intense PY labeling was detected along the leading edge (open arrows; 170% increase in PY intensity compared with the peripheral domain, see F), at tips of filopodia (arrowheads; 155% increase), and in ruffles (arrows). (E) PY levels are reduced in growth cones treated with 100 μM genistein for 25 min. (F) Quantification revealed that genistein treatment decreased PY intensity at the leading edge by 55.4% (P < 0.001; t test), at filopodia tips (arrowhead in E) by 54.8% (P < 0.001), and in the peripheral domain by 41.7% (P < 0.01). Data represents average fluorescence intensity values ± SEM; n = 7 (control, 46 growth cones); n = 3 (genistein, 36 growth cones). (G–I) PY proteins (green) partially colocalize with F-actin structures (red) in ruffles (arrow in G), filopodia (arrowheads in H), and hot spots after cytochalasin B treatment (arrow in I). Treatment with 5 μM cytochalasin B for 1 min results in opening of an F-actin–free gap (double-headed arrow) that has reduced levels of PY proteins. Bars: (A) 50 μm; (B–E and G–I) 10 μm.
Figure 2.
Figure 2.
Concentration of PY labeling at growth cone–growth cone contact sites and at Con A RBI sites. (A) Increased PY labeling at growth cone–growth cone contact sites. Double lines indicate corridor through which average fluorescence intensity has been measured. (B) Plot of relative PY fluorescence intensity through the contact site indicated in A. (C and D) DIC images at start (C) and end (D, 4 min after start) of the central domain extension phase during a Con A RBI. (E and F) Conventional (E) and confocal fluorescence microscopy (F) reveal concentration of PY labeling around the bead (arrow). Confocal z-sections in 1-μm steps suggest that the growth cone membrane wraps around the bead. Generally, PY labeling was more intense at interaction sites using Con A beads when compared with apCAM beads: average PY accumulation factor of 7.0 ± 0.3 for Con A versus 4.1 ± 0.7 for apCAM beads, respectively. n = 3, P < 0.01. See also Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200107063/DC1. Bars, 10 μm.
Figure 3.
Figure 3.
Correlation between tension and PY build up. (A–D) PY build up with increasing restraining time. (A–C) Con A beads were either unrestrained and moved with retrograde flow for 1 min (A) or restrained for the times indicated before RBI was completed (B and C, arrows point to bead positions). We used Con A beads in these experiments since their RBI latency times are less variable and generally shorter than those observed using apCAM beads (Suter et al., 1998). PY labeling at bead sites increased by 25% after 1 min of restraint (B) and by 90% after 2 min of restraint (C). (D) apCAM beads were either unrestrained (dotted arrow) or restrained for 1 (dashed arrow) or 2 min (solid arrow). Inset shows higher focus level to illustrate PY labeling around restrained beads. (E and F) BDM decreases PY buildup and increases RBI latency times. (E) Quantification of PY intensity at unrestrained Con A beads as well at Con A RBI sites. PY protein levels near restrained Con A beads were reduced in 5 mM BDM compared with control interactions. Asterisk indicates P < 0.05, t test, n = 3 (unrestrained); n = 3 (control RBI); n = 4 (BDM RBI). (F) Latency times of Con A RBIs under control conditions (n = 6) and in 5 mM BDM (n = 4, asterisk indicates P < 0.05). Bars, 10 μm.
Figure 4.
Figure 4.
Tyrosine kinase inhibition uncouples apCAM-mediated RBIs. (A–C) ApCAM RBI capability using 4E8 beads was tested on the same growth cone shown in control (A; 0.5% DMSO), after treatment with 100 μM genistein for 20 min (B), and after drug washout (C; 0.5% DMSO). Top, growth cone at the start of central domain extension (boundary marked by dashed yellow line) or, in the case of genistein, when central domain extension would have been expected. Bottom, completed RBI when central domain reached the bead or, in the case of the genistein, after a total restraining time of 16 min, which accounts for more than average control latency plus interaction times. RBI fully recovered after drug washout (C). (D and E) Cytoskeletal reorganizations typical of control RBIs (D) are absent after treatment with 100 μM genistein (E). Green signal around bead in E (yellow in D because of F-actin accumulation in control RBIs) results from the detection of the 4E8 antibody on the bead by the secondary antibody used for tubulin immunofluorescence. (F and G) F-actin and microtubule distribution in controls (F) and growth cones treated with 100 μM genistein for 25 min (G) without any bead interactions. Bars, 10 μm.
Figure 5.
Figure 5.
PTK inhibition uncouples RBIs without affecting retrograde F-actin flow. (A) Quantification of retrograde F-actin flow effects by treatment with 100 μM genistein for 25 min on growth cones without RBIs. Average values ± SEM for control (n = 6), drug (n = 6), and wash out (n = 3) flow rates are: 6.11 ± 0.56, 5.18 ± 0.56, 5.69 ± 0.46 μm/min, respectively; P > 0.1; 50–90 beads analyzed per condition. (B and C) PTK inhibition does not affect flow during uncoupled RBIs. All images refer to the same growth cone. Flow marker beads were placed with a laser tweezer both within the interaction corridor (on-axis) and on adjacent areas (off-axis) during an 4E8 RBI under control conditions (B) and after pretreatment with 50 μM genistein for 20 min (C). Panels on the right show simultaneous DIC time sequences of on- and off-axis bead movements in areas of interests marked on the left. Flow rates for the displayed on- and off-axis beads are 0.31 versus 5.64 μm/min in control and 4.47 versus 5.14 μm/min in genistein. Central domain extension in control is indicated by dashed line and leading edge growth by a white arrow. Laser beam focus is marked with an arrowhead in C. Bars, 5 μm.
Figure 7.
Figure 7.
Quantification of RBI inhibition by PTK inhibitors. RBI probability is indicated as the percentage of successfully completed RBIs per total number of experiments (n). PTK inhibitor experiments are shown as black bars, control RBIs as gray bars. Only PTK inhibitor RBI experiments that were preceded with a successfully completed control RBI on the same growth cone were taken into these statistics.
Figure 6.
Figure 6.
The Src family selective PTK inhibitor PP1 uncouples apCAM-mediated RBIs. (A–D) PP1 has minimal effects on growth cone morphology and cytoskeletal distribution. (A and B) High power magnification DIC images of the same growth cone in control condition (A) and after 20 min treatment with 10 μM PP1 (B). Note, no effect on ruffling and very little effect on filopodia extension (arrows). (C and D) F-actin and microtubule organization in control condition (C) and in 10 μM PP1 (D). (E) RBI with a 4E8-coated bead in control condition at start (top) and when the central domain reached the bead (bottom; boundary marked with yellow line). (F) The same growth cone after treatment with 25 μM PP1 for 20 min did not respond to the restrained apCAM substrate, even after a total restraining time of 30 min (G) RBI capability of this growth cone was recovered after drug washout. See also Videos 2–4, available at http://www.jcb.org/cgi/content/full/jcb.200107063/DC1. Bars, 10 μm.
Figure 9.
Figure 9.
Increased levels of activated Src family PTK at RBI sites. (A–C) Control RBI with a 4E8 bead at start (A) and end (B) of interaction. (C) Increased Src PY418 signal at RBI site compared with unrestrained beads (arrow) or control beads on the substrate (arrowhead). Lower panel shows Src PY418 intensity line scan through interaction site indicated with dashed lines. (D–F) RBI experiment with a 4E8 bead which was blocked by 25 μM PP2 at start (D) and after 12 min of bead restraint (E). (F) The levels of activated Src family PTK labeling at bead site was reduced by 78% compared with the control condition shown in C. Bar, 10 μm.
Figure 8.
Figure 8.
Immunolocalization of an activated Src family PTK in growth cones. (A) Western blot characterization of Src PY418 antibody using Aplysia CNS proteins extracted under different tyrosine kinase and phosphatase inhibitor conditions as indicated and separated on a 10% SDS-PAGE. A control lysate from chicken embryonic fibroblasts was loaded in lane 4. Reprobing for β-tubulin was done to normalize for equal protein loading. The 60-kD band in Aplysia CNS extracts was reduced by 41% using 20 μM PP1 in the experiment shown (lane 2). An average reduction of 71 ± 3% was found using 50 μM PP1 (n = 2). Molecular weights are indicated. (B–D) Immunocytochemistry of growth cones (B and D) and branching neurites (C) in culture using the Src PY418 antibody. (D) Double-staining for total PY (green) and activated Src family PTK (red). Bar, 10 μm.
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References

    1. Akiyama, T., J. Ishida, S. Nakagawa, H. Ogawara, S. Watanabe, N. Itoh, M. Shibuya, and Y. Fukami. 1987. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J. Biol. Chem. 262:5592–5595. - PubMed
    1. Atashi, J.R., S.G. Klinz, C.A. Ingraham, W.T. Matten, M. Schachner, and P.F. Maness. 1992. Neural cell adhesion molecules modulate tyrosine phosphorylation of tubulin in nerve growth cone membranes. Neuron. 8:831–842. - PubMed
    1. Beggs, H.E., P. Soriano, and P.F. Maness. 1994. NCAM-dependent neurite outgrowth is inhibited in neurons from Fyn-minus mice. J. Cell Biol. 127:825–833. - PMC - PubMed
    1. Beggs, H.E., S.C. Baragona, J.J. Hemperly, and P.F. Maness. 1997. NCAM140 interacts with the focal adhesion kinase p125(fak) and the SRC-related tyrosine kinase p59(fyn). J. Biol. Chem. 272:8310–8319. - PubMed
    1. Bray, D. 1984. Axonal growth in response to experimentally applied mechanical tension. Dev. Biol. 102:379–389. - PubMed

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