Rac1-mediated endocytosis during ephrin-A2- and semaphorin 3A-induced growth cone collapse

Abstract

Negative guidance molecules are important for guiding the growth of axons and ultimately for determining the wiring pattern in the developing nervous system. In tissue culture, growth cones at the tips of growing axons collapse in response to negative guidance molecules, such as ephrin-A2 and semaphorin 3A. The small GTPase Rac1 is involved in growth cone collapse, but the nature of its role is not clear. Rac1 activity assays showed that Rac1 is transiently inactivated after treatment with ephrin-A2. Ephrin-induced growth cone collapse, however, correlated with resumption of Rac1 activity. We demonstrate that Rac1 is required for endocytosis of the growth cone plasma membrane and reorganization of F-actin but not for the depolymerization of F-actin during growth cone collapse in response to ephrin-A2 and semaphorin 3A. Rac1, however, does not regulate constitutive endocytosis in growth cones. These findings show that in response to negative guidance molecules, the function of Rac1 changes from promoting actin polymerization associated with axon growth to driving endocytosis of the plasma membrane, resulting in growth cone collapse. Furthermore, Rac1 antisense injected into the embryonic chick eye in vivo caused the retinotectal projection to develop without normal topography in a manner consistent with Rac1 having an obligatory role in mediating ephrin signaling.

Fig. 1.

Ephrin-A2 collapses growth cones of axons from the temporal side of the retina and alters Rac1 activity. A, Time-lapse sequence of a growth cone before (−1 min) and after treatment with 1.0 μg/ml ephrin-A2 (+1 to +12 min). Note that growth cone collapse, as evidenced by loss of lamellipodia and filopodia, was evident at 6 min after addition of ephrin-A2 and that the axon had retracted significantly by 12 min after treatment. B, Immunoblot showing levels of activated Rac1 in retina at various times after ephrin treatment. Dissociated cells from temporal retina were treated with 1.0 μg/ml ephrin-A2 for 1–12 min. GTP-bound Rac1 was affinity purified from lysed cells using the Rac1-binding domain of p21-activated kinase 1.NT, No treatment. C, Quantification of Rac1 activity levels as a function of time after treatment with ephrin-A2. Rac1 activity is expressed as percentage of the activity of untreated cells. At 3 min after ephrin-A2 treatment, Rac1 activity was reduced but returned to baseline levels by 6–12 min. The time course of retinal growth cone collapse is shown relative to changes in Rac1 activity levels. D, Quantification of growth cone F-actin content as a function of time after treatment with ephrin-A2. F-actin levels are normalized to data from untreated growth cones. Growth cone F-actin content was significantly reduced after 12 min of treatment with ephrin. The time course of Rac1 inactivation is reproduced from that in C to allow a direct comparison of the two variables. Significant difference from control: *p < 0.01; **p < 0.001.

Fig. 2.

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.

Fig. 3.

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.

Fig. 4.

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.

Fig. 5.

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.

Fig. 6.

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.

Fig. 7.

Model of the dynamics of Rac1 function in an axonal growth cone. A, Axon extension requires polymerization of actin in the process of forming filopodia and lamellipodia. Actin polymerization is driven in part by Rac1 activity at the leading edge of the growth cone. B, Activation of EphA receptors after binding ephrin-A2 results in an initial loss of activated Rac1 and cessation of axon extension. C, Subsequently, Rac1 activity returns and mediates endocytosis of the plasma membrane and F-actin reorganization during collapse of the growth cone. Rac1 activity is required for growth cone collapse. Growth cone collapse also involves depolymerization of F-actin, a process that is independent of Rac1 activity.