Rac1-Mediated Endocytosis during Ephrin-A2- and Semaphorin 3A-Induced Growth Cone Collapse

Rac1 is required for ephrin-A2-induced growth cone collapse. A, Time-lapse sequence of a retinal growth cone treated first with 2.0 μg/ml Rac1 inhibitory peptide (0 min–60 min; rac inhib pep) and then with 1.0 μg/ml ephrin-A2 for 15 min. B, Pseudocolor images of retinal growth cones stained with rhodamine-phalloidin to reveal F-actin. All images were acquired using identical acquisition parameters. Warmer colors indicate higher levels of actin. Treatment with the Rac1 inhibitory peptide (1 hr, 2.0 μg/ml) did not alter F-actin levels or organization in growth cones. Treatment with ephrin-A2 (12 min, 1.0 μg/ml) caused F-actin to depolymerize and aggregate in the distal axon, a structure that we refer to as the collapse bulb. Treatment with Rac1 inhibitory peptide first, followed by addition of ephrin-A2, resulted in levels of F-actin in the growth cone that were similar to those seen in the growth cones treated with ephrin-A2 alone. However, F-actin did not reorganize into a collapse bulb. C, Quantification of Rac1 inhibitory peptide treatments and growth cone response to ephrin-A2. The Rac1 inhibitory peptide blocked ephrin-induced growth cone collapse in a dose-dependent manner. Growth cones were treated with the peptide for 1 hr before ephrin-A2 treatment. Significant difference from culture without inhibitory peptide treated with ephrin: *p < 0.001.

Reduction of Rac1 protein level using an antisense oligonucleotide inhibits ephrin-A2-induced growth cone collapse.A, Immunoblot showing levels of Rac1 after oligonucleotide treatment. Rac1 antisense treatment reduced the expression of Rac1 protein. Dissociated cells from the temporal side of the retina were cultured in the presence of 40 μmmissense control or 10–40 μm Rac1 antisense oligonucleotides for 24 hr. B, Quantification of growth cone collapse in oligonucleotide-treated cultures. Reduction of Rac1 expression inhibits the ability of ephrin-A2 to induce growth cone collapse. Temporal retinal explants were cultured for 24 hr in the presence of missense or Rac1 antisense oligonucleotides and then treated with 1.0 μg/ml ephrin-A2 for 15 min.

Inhibition of Rac1 signaling but not expression of constitutively active Rac1 blocks DRG growth cone collapse in response to ephrin-A2. A, Quantification of DRG growth cone response to ephrin-A2. Ephrin-A2 induced growth cone collapse in all culture conditions. E9 lumbrosacral DRG were cultured overnight in NGF, neurotrophin-3 (NT3), or BDNF and then treated with 2.0 μg/ml ephrin-A2 for 15 min. B, Role of Rac1 signaling in ephrin-A2 induced collapse. Inhibition of Rac1 signaling blocked ephrin-induced growth cone collapse. DRG explants were raised in BDNF and then treated with Rac1 inhibitory peptide (rac inhib pep; 1 hr, 2.0 μg/ml) before exposure to ephrin-A2 (15 min, 2.0 μg/ml). C, Role of constitutively active Rac1 in growth cone collapse. Viral infection did not change the percentage of spontaneously collapsed growth cones (p > 0.05). After ephrin-A2 treatment (15 min, 2.0 μg/ml), growth cones collapsed to a similar extent regardless of viral infection. DRG neurons were dissociated and infected with adenovirus engineered to express either constitutively active Rac1 (V12) or lacZ as a control and then cultured for 3 d. Additional control cultures were not infected with viruses (NT). More growth cones were spontaneously collapsed in all groups after 3 d in vitro versus 1 d in vitro. Significant difference form control: *p < 0.05; **p < 0.01.

Ephrin-A2 and semaphorin 3A induce endocytosis of the growth cone plasma membrane in a Rac1-dependent manner. A, Endocytosis of labeled dextran after ephrin-A2 treatment. Endocytotic activity was elevated at 12 min after treatment with ephrin-A2. The time course of changes in Rac1 activity is reproduced from Figure 1C to allow a direct comparison. Retinal growth cones were treated with ephrin-A2 plus 2.5 mg/ml rhodamine-labeled dextran for 3–12 min. The number of dextran-containing vesicles in the distal 20 μm of axons was then counted (3 experiments with >80 growth cones sampled at each time point). The increase in dextran labeling is expressed relative to the number of dextran-labeled vesicles in time-matched controls treated with dextran alone. B, Role of Rac1 in ephrin-A2 induced endocytosis. A 12 min treatment with 1.0 μg/ml ephrin-A2 resulted in a large increase in the number of endocytotic vesicles in the distal tip of axons compared with those in cultures treated with vehicle alone. Inhibition of Rac1 signaling prevented the increase in endocytotic vesicles in response to ephrin-A2. Staining the membrane with DiO showed retinal growth cone morphology, and endocytosis was revealed by accumulation of rhodamine-labeled dextran. All images were obtained using identical acquisition parameters. The DiO images were embossed to better reveal growth cone morphology. C, Quantification of endocytosis in retinal growth cones. Ephrin-A2 significantly increased endocytosis in the distal 20 μm of retinal axons, and the Rac1 inhibitory peptide (rac inhib pep) blocked the effects of ephrin-A2 on endocytosis. Treatment with the Rac1 inhibitory peptide alone did not alter the number of dextran-labeled vesicles (p > 0.5).D, Quantification of endocytosis in DRG growth cones. As with retinal ganglion cells, ephrin-A2 increased the number of dextran-labeled vesicles, and the Rac1 inhibitory peptide blocked the effect of ephrin-A2. Similarly, a 30 min exposure to semaphorin 3A (sema 3A) increased the number of dextran-labeled vesicles in a Rac1-dependent manner. Treatment with the Rac1 peptide did not affect basal endocytosis (p > 0.5). Differences in absolute number of dextran-labeled vesicles between retinal and DRG growth cones likely reflect a difference in growth cone size, DRG growth cones being significantly smaller. Significant difference from control: *p < 0.001; **p < 0.0001.

Rac1 activity is required for normal development of retinotectal topography in vivo. Rac1 antisense or missense oligonucleotide was injected into one eye of chick embryos on E6 and E8. On E10, a retrogradely transported axon tracer, DiI, was injected into the posterior portion of the optic tectum contralateral to the oligonucleotide-treated eye. Embryos were fixed on E11. A, Coronal section from the brain of a Rac1 antisense-treated embryo stained with an antibody that labels retinal axons. The asterisk denotes the optic tectum contralateral to the Rac1 antisense-injected eye. Arrowspoint to stained retinal axons in the optic fiber layer. Rac1 antisense treatment did not inhibit retinal axon growth across the tectum.B, C, Tracings of the outlines of flat mounted-retinas injected with missense (B) or Rac1 antisense (C) oligonucleotide. Dotsrepresent retrogradely labeled cells. In the retina injected with the Rac1 antisense oligonucleotide, no organized topography could be distinguished.