This study addressed the question of whether asymmetrical protein synthesis has a role in growth cone turning. Our results indicate that new β-actin protein is synthesized in response to netrin-1 and that an external gradient of netrin-1 causes a polarized increase of β-actin on the side of the growth cone nearest to the source. The increase occurs just 5 minutes after addition of netrin-1 and is abolished by inhibitors of translation. Significantly, β-actin AMOs block attractive but not repulsive turning suggesting that β-actin synthesis is particularly important for directional guidance towards a positive cue.
The finding that the β-actin AMOs abolished the netrin-1-induced increases in β-actin in growth cones indicates that the AMOs effectively inhibited β-actin translation over the time period examined (5-60 minutes). Overall, the AMOs caused a 20-25% drop in β-actin QIF signal. This relatively small knockdown of β-actin probably reflects the large maternal pool of actin that is recycled for the first 24-36 hours of embryonic development and likely predominates in the pioneering population of retinal axons, which develop just 28 hours post-fertilization. Indeed, the knockdown in β-actin QIF signal was higher in older growth cones than in younger ones (approximately 40% in stage 35/36 versus 20% in stage 24, data not shown). Importantly, AMO-containing axons grew at a normal rate and exhibited appropriate chemotropic responses to Sema3A and netrin-1 under repulsive conditions. It could be argued that attractive turning is more sensitive to β-actin levels (and hence actin polymerization rates) than axon extension or repulsive turning, and the β-actin AMO knocks down baseline β-actin translation levels just enough to block attractive turning but not extension or repulsion. Such an argument, however, would predict that the AMO would at least reduce axon extension and repulsive turning, while in fact we observed that extension and repulsion remained normal. Together, these lines of evidence indicate that β-actin translation is specifically required in attractive turning, not undirected growth, repulsive turning, or general responsiveness to netrin-1.
What role does β-actin translation play in attractive turning? At first glance it would seem that β-actin synthesis is not necessary for actin polymerization given that the pool of unpolymerized actin in growth cones is thought to be large34
. Indeed, it has been estimated that β-actin synthesis provides only 7% of the total actin needed for polymerization in migrating fibroblasts19
, making it unlikely that asymmetrical β-actin synthesis makes the growth cone turn by sheer mass alone. It has been proposed that newly synthesized β-actin can polymerize or nucleate polymerization more efficiently than “older” actin due to chaperone binding to the nascent β-actin chain11,35
and protecting it from glutathionylation, which restricts the rate of polymerization36
. Thus, given that the rate-limiting step of actin polymerization is nucleation, an appealing model is that local β-actin synthesis provides spatially localized nucleation sites for actin polymerization11,19,37
. The restricted size of the growth cone compartment would concentrate newly synthesized β-actin monomers in a small volume, thereby contributing to rapid formation of nucleation sites. We therefore suggest that a gradient of netrin-1 elicits spatially biased actin polymerization by inducing the asymmetric actin nucleation sites on the side of the growth cone closest to the pipette (see Supplementary Fig. 3
). The netrin-1-stimulated increase in the β-actin signal is particularly evident in filopodia, suggesting that the new actin contributes to actin-filament bundles in these structures that radiate from the less polarized actin mesh network in the body of the growth cone38
. As β-actin synthesis occurs at least 10 minutes before overt turning, we hypothesize that asymmetrical β-actin translation prefigures the turn itself.
Asymmetrical synthesis requires spatial regulation of β-actin translation. One possible mechanism is transport of β-actin mRNA to the side closest to the source of the gradient. Previous studies have shown that the neurotrophin NT-3 elicits the transport of the β-actin mRNA-ZBP1 complex into growth cones14
and serum stimulation induces the transport of β-actin mRNA to the leading edge of fibroblasts39
. An independent study shows that both ZBP1 and β-actin mRNA become asymmetrically distributed in the growth cones of Xenopus
spinal neurons upon BDNF stimulation (J. Zheng, personal communication). In agreement, our experiments show that Vg1RBP binds β-actin mRNA and that netrin-1 stimulation induces transport of Vg1RBP granules into filopodia, asymmetrically if netrin-1 is presented in a gradient. We also find that sites of filopodial contact can induce the transport of Vg1RBP-eGFP granules into filopodia, suggesting that external cues recruit RNA-binding proteins and their mRNA cargo to local sites of stimulation. Although our dynamic imaging studies could not demonstrate that the Vg1RBP-eGFP granules moving into filopodia were specifically transporting β-actin mRNA, fixed samples showed that Vg1RBP colocalizes with β-actin mRNA and the netrin-1-induced increase in filopodial Vg1RBP is accompanied by an increase in filopodial β-actin mRNA.
Another mechanism for spatial regulation of β-actin synthesis is the asymmetrical activation of translation. We found that a gradient of netrin-1 induced a significant asymmetry in the phosphorylation of the translation initiation factor 4EBP, suggesting a corresponding asymmetry in the global rate of translation. The translation of β-actin mRNA, like that of most eukaryotic mRNAs, is cap-dependent40
, making it subject to asymmetrical activation of translation initiation factors. Both attractants and repellents stimulate activation of translation initiation factors10,41
, though they presumably induce synthesis of different proteins. Indeed, in contrast to our result that netrin-1 stimulates synthesis of β-actin, the repellents Sema3A and Slit2 stimulate synthesis of RhoA and cofilin respectively, proteins that promote actin depolymerization8,9
. An external gradient can activate signalling cascades asymmetrically to generate an internal gradient of translation activation oriented toward both attractants and repellents, while the identity of the asymmetrically synthesized proteins and, hence, the polarity of the turning response, is determined by mRNA-specific regulation. For ‘Class 1’ (Ca2+
-dependent) guidance cues like netrin-1, Ca2+
is a candidate switch mechanism3
, especially since laminin seems to switch netrin-induced attraction to repulsion by lowering cAMP and thereby reducing Ca2+
. Global translation initiation regulation combined with mRNA-specific regulation is consistent with our finding that enolase protein levels do not change with netrin-1 stimulation, despite both the presence of enolase mRNA and upregulation of global translation initiation10
. Further studies will be needed to test this idea, especially the identity of proteins synthesized in attractive versus repulsive conditions.
mRNA-specific regulation may not entail separate RNA binding proteins. Proteomic and colocalization studies have revealed that RNP complexes consist of multiple RNA binding proteins and can transport several different mRNAs42
. Consistent with this, we have shown that Vg1RBP also interacts with cofilin mRNA, which is translated in response to the chemorepellent Slit-28
. It may be that in attractive conditions, Vg1RBP activates β-actin translation after being phosphorylated by Src, as occurs for the chick homology of Vg1RBP, ZBP120
, while an alternative signaling pathway causes Vg1RBP to activate cofilin-1 translation but suppress β-actin translation in repulsive conditions. The exact sequence of signalling between DCC and Src remains largely unknown, but Src might in turn be activated by DCC through direct binding or via focal adhesion kinase (FAK), which interacts with DCC43
The differential sensitivity to β-actin translation inhibition of attraction versus repulsion and collapse suggests that repulsion and attraction are not simply mirror-symmetrical processes but operate through distinct pathways. This may not be surprising because attraction involves near-side filopodial extension, while repulsion involves near-side filopodial withdrawal16
. Indeed, it is thought that repulsive turning is essentially localized collapse, because guidance cues or drugs that cause collapse when added globally generally elicit repulsive turning when presented asymmetrically44
. Consistent with this, the repellent Slit2 does not induce β-actin synthesis, and indeed causes a significant decrease in β-actin levels8
, possibly caused by depolymerization due to Slit2-induced cofilin synthesis, combined with suppression of β-actin translation. Similarly, we observed that a gradient of netrin-1 in repulsive conditions did not induce near side β-actin synthesis, but rather caused a slight, though non-significant, decrease of β-actin on the near side. This model predicts that although inhibition of β-actin synthesis does not block repulsive turning, de-regulation of β-actin synthesis might. In agreement with this model, an independent study detects a decrease of β-actin on the near side in response to a repulsive gradient and shows that an antisense oligonucleotide directed against the ‘zipcode’ in β-actin mRNA’s 3’ UTR blocks both the asymmetrical decrease of β-actin and repulsive turning (J. Zheng, personal communication).
In summary, we provide evidence that netrin-1 induces a rapid local asymmetric increase in β-actin mRNA translation in retinal growth cones, which is necessary for the attractive turning response and might be achieved via directed transport of Vg1RBP and/or asymmetrical translation initiation. We speculate that newly synthesized β-actin could provide spatially targeted de novo
nucleation sites for actin polymerization and hence direct the migration of the growth cone towards the cue (see Supplementary Fig. 3
). Our data agree with those previously obtained in fibroblasts19
and suggest that similar mechanisms occur to direct migration in the two cell types. Together with the fact that numerous cytoskeletal mRNAs and cytoskeletal-regulatory protein mRNAs have been identified in axons, this suggests that the ability of growth cones to transduce external gradients into matching internal asymmetries of cytoskeletal protein synthesis might be a conserved mechanism.