The trkA receptor mediates growth cone turning toward a localized source of nerve growth factor

Abstract

We have developed an in vitro system for studying the interaction of chick dorsal root ganglion neuronal growth cones with a localized source of nerve growth factor (NGF) covalently conjugated to polystyrene beads. Growth cones rapidly turned and migrated under NGF-coated beads in a process that involved the initial formation of persistent contact with a bead, followed by directed flow of cytoplasm toward the point of contact. A role for the local activation of the high-affinity NGF receptor trkA was suggested by a strong inhibition of the turning response by (1) the addition of an antibody against the extracellular portion of trkA, (2) the elevation of the background concentration of NGF to saturate trkA, or (3) the presence of a concentration of the drug K252a that inhibits trkA activation. NGF binding to the pan-neurotrophin receptor p75 is also involved but is not required for turning. These data show a new role for both the trkA and the p75 receptors: the mediation of local events in the guidance of nerve growth cones.

Fig. 1.

Growth cone behavior during interactions with beads. A, Example of growth cone contact with an NGF-coated bead in the presence of 0.05 ng/ml background NGF. Initial filopodial contact was established (0 min). The filopodial contact underwent darkening/thickening (1.5 min). Engorgement of the filopodial contact by the growth cone cytoplasm occurred (5.5 min), and a new growth cone was subsequently formed underneath the bead (9 min,arrowheads point to filopodia extending from underneath the bead). B, Example of growth cone contact with a cyto-C-coated bead. Although filopodial contacts occurred, the growth cones did not turn toward the bead. C, Example of NGF-coated bead being picked up by the growth cone, followed by turning toward the initial position of the bead. The growth cone established an initial filopodial contact with the NGF-coated bead (0 min), and subsequently the bead was translocated from its original position onto the nerve fiber (5–7.5 min, arrow indicates the direction in which the bead was displaced). The growth cone then proceeded to migrate in the direction of contact with the bead (12.5 min), even though the bead was no longer present at its original location. D, Example of aborted turn in the presence of 500 nm KT5926. The growth cone initially exhibited behavior characteristic of a turning response (0–11 min, compare withA). However, the turn was subsequently aborted (20 min), and the growth cone continued migrating past the bead. Diameter of a bead, 10 μm.

Fig. 2.

Immunocytochemical visualization of trkA and p75 receptors on the growth cones of DRG neurons cultured in 0.05 ng/ml NGF. A, Actin filaments in a growth cone and the edge of a non-neuronal cell (top left) labeled with fluorescein-phalloidin. B, The same cells as inA labeled with 7 μg/ml of the anti-p75 CHEX. The growth cone is strongly labeled, but not the non-neuronal cell.Arrows in A and B indicate filopodia that are labeled by anti-p75. C, Actin filaments in a growth cone and the edge of a non-neuronal cell (top right) labeled with fluorescein-phalloidin.D, The same cells as in C labeled with 25 μg/ml of the anti-trkA CTA. The neurite and growth cone are labeled, but not the non-neuronal cell. Arrows inC and D indicate filopodia that are labeled by anti-trkA. Scale bar, 10 μm.

Fig. 3.

Inhibition of the turning response toward NGF-coated beads by the application of trkA and p75 receptor antibodies. In the presence of the trkA antibodies (CTA, 25 μg/ml), growth cones did not turn toward NGF-coated beads more than toward cyto-C beads. Both 14 (C1) and 70 (C2) μg/ml antibodies against p75 (Chex) diminished, but did not abolish, the turning response of growth cones toward NGF-coated beads. Antibodies against trkB (20 μg/ml) or L1 (25 μg/ml) did not affect the percentage of growth cones that turned toward NGF-coated beads. The percentage of growth cones that turned toward NGF-coated beads in the absence of antibodies (no Ab) is provided for comparison purposes. All experiments were performed with a 0.05 ng/ml background NGF concentration.

Fig. 4.

Soluble NGF and BDNF affect growth cone turning toward NGF-coated beads. A, Increasing the background NGF concentration decreased the percentage of growth cones that turned toward NGF-coated beads. The percentage of growth cones that turned toward cyto-C-coated beads in 0.05 ng/ml NGF is presented for comparison purposes and establishes the basal level of turning toward protein-coated beads. B, Adding 100 ng/ml BDNF to the culture medium (0.05 ng/ml NGF background) resulted in a decrease in the percentage of growth cones that turned toward NGF-coated beads, whereas 10 ng/ml BDNF did not have a similar effect. The percentage of growth cones that turned toward NGF-coated beads in 0.05 ng/ml NGF is presented for comparison.

Fig. 5.

K252a, but not its analog KT5926, inhibited the turning response toward NGF-coated beads. In the presence of 100 nm K252a, the percentage of growth cones that turned was not greater than that observed with cyto-C beads (see previous figures). Whereas 100 nm KT5926 had no effect on growth cone turning toward NGF-coated beads, 500 nm caused a decrease in the percentage of growth cones that turned. The vehicle for K252a and KT5926, DMSO at a concentration of 2.5 μg/ml, did not affect growth cone turning. All experiments were performed with a background NGF concentration of 0.05 ng/ml.

Fig. 6.

Working model for the mechanism of growth cone turning toward NGF-coated beads. See the Discussion for details.