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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Nat Rev Neurosci. Author manuscript; available in PMC Oct 1, 2011.
Published in final edited form as:
PMCID: PMC3133775
NIHMSID: NIHMS302328

Second messengers and membrane trafficking direct and organize growth cone steering

Abstract

Graded distributions of extracellular cues guide developing axons toward their targets. A network of second messengers, Ca2+ and cyclic nucleotides, shapes cue-derived information into either attractive or repulsive signals that steer growth cones bidirectionally. Emerging evidence suggests that such guidance signals create a localized imbalance between exocytosis and endocytosis, which in turn redirects membrane, adhesion and cytoskeletal components asymmetrically across the growth cone to bias the direction of axon extension. These recent advances allow us to propose a unifying model of how the growth cone translates shallow gradients of environmental information into polarized activity of the steering machinery for axon guidance.

The formation of functional neuronal networks depends crucially on the spatial accuracy of axon growth and navigation. Each growing axon in the developing nervous system is tipped by a growth cone, a specialized amoeboid structure that is able to interpret secreted and membrane-bound molecular “guidance cues” that direct its migration along the correct path. A series of these events deliver the axon to an approximate target region, which is followed typically by axonal arborization and contacts with appropriate postsynaptic partners at particular subcellular locations1. Such specificity of synaptic connections within the target region relies on multiple distinct mechanisms including further cue-mediated axon guidance2,3. In this way, guidance cues in the microenvironment play crucial roles in neuronal network formation.

It is widely accepted that graded distribution of guidance cues controls the direction of axon growth (FIG. 1a). Such gradients can be generated by diffusion of a secreted cue away from its source of synthesis4 or by differential expression of non-diffusible cues5. When a growth cone migrates in a guidance cue gradient, the side of the growth cone facing higher concentrations of the cue will experience higher receptor occupancy. This asymmetric receptor occupancy polarizes the growth cone for turning either toward increasing concentrations of the cue (attraction) or away from the cue (repulsion), via intracellular generation of second messengers such as Ca2+ and cyclic nucleotides611. An extracellular shallow gradient can be transformed into steeply graded12 or, in extreme cases, compartmentalized signals11,13 inside the growth cone. The asymmetrically produced second messengers orchestrate multiple cellular machineries including membrane trafficking, adhesion dynamics and cytoskeletal reorganization to execute bidirectional turning of the growth cone (FIG. 1b).

Figure 1
Overview of signaling and mechanical events during bidirectional growth cone guidance

These basic mechanisms could be sufficient to explain short-distance guidance of axons such as those of local circuit neurons. However, additional complex mechanisms are required for long projecting axons that are guided by intermittently positioned sources of attractants en route, referred to as “intermediate targets”14. To leave an intermediate target after passing through this once attractive region, the growth cone must change its responsiveness and even reverse the polarity of guidance. Such switching can be accomplished through multiple mechanisms including an induction of different second messenger profiles that direct opposing steering machinery15,16.

Extracellular diffusible molecules showing axon guidance activities in vitro have been regarded as “guidance cues” and have been well documented (TABLE 1), even if their functional significance in vivo is less clear. This review will seek to synthesize these findings and provide an integrated picture of how axon guidance works in situ. We first suggest, based largely on in vitro data, the second messenger network models explaining how the growth cone translates shallow gradients of guidance information into either attractive or repulsive turning. Second, we examine recently identified target molecules and mechanisms that link the second messenger system with the steering apparatus. In addition to established mechanisms that act in parallel to remodel the cytoskeleton and substrate adhesions1721, we here propose the hierarchical organization model of cellular machineries in which asymmetric membrane trafficking redirects cytoskeletal and adhesion components in the growth cone to drive its bidirectional turning. Finally, we provide our viewpoint on how the growth cone makes guidance decisions in vivo where it encounters and integrates multiple cues simultaneously to navigate through complex environmental terrain with high fidelity.

Table 1
Ca2+ and cyclic nucleotide signaling elicited by guidance cues

Involvement of second messengers in signal transduction

Second messenger networks involving Ca2+ influx and secondary Ca2+ release controlled by cyclic AMP (cAMP) and cyclic GMP (cGMP) are the primary means by which growth cones interpret and amplify signals encountered in the local environment. Here we explain how second messenger networks are shaped into growth cone guidance signals.

Generation of second messengers

In vitro studies indicate that Ca2+ signals are almost universally required for growth cone guidance with a few notable exceptions (TABLE 1). For all of the guidance cues that have been shown to elicit asymmetric Ca2+ elevations across the growth cone when applied directionally to induce axon turning, higher Ca2+ concentrations are observed on the side of the growth cone facing the source of the cues, regardless of whether the cues are attractive or repulsive (FIG. 1). Thus, localized Ca2+ elevations on one side of the growth cone can act as both attractive and repulsive signals22,23.

How do Ca2+ elevations of the same polarity elicit bidirectional turning? This most likely relies on the gating of differential sets of Ca2+ channels. The generation of cytoplasmic Ca2+ signals takes advantage of a steep gradient of Ca2+ concentrations across the plasma membrane that drives Ca2+ influx through various cation channels such as transient receptor potential type C (TRPC) channels and L-type voltage-dependent Ca2+ channels (L-VDCCs) (REF. 24). Ca2+ influx can be further amplified and extended in space and duration by secondary Ca2+ release from the endoplasmic reticulum (ER) through ryanodine receptors (RyRs) or inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs). The limited diffusion of cytoplasmic Ca2+ (REF. 25) allows spatial confinement of Ca2+ elevation to the vicinity of single open channels or discrete clusters of open channels, referred to as Ca2+ nanodomains or microdomains, respectively26.

These diverse spatiotemporal patterns of Ca2+ signals contribute to the growth cone's bidirectional responses to various guidance cues (TABLE 1 and FIG. 2). High amplitude Ca2+ produced by Ca2+-induced Ca2+ release (CICR) through RyRs or IP3-induced Ca2+ release (IICR) through IP3Rs are sufficient to initiate growth-cone turning toward the side with Ca2+ signals6,11,13 and can mediate cue-induced attraction (FIG. 2a,c,e). Conversely, repulsion is triggered by lower-amplitude Ca2+ signals that are mostly generated by Ca2+ influx through plasma membrane channels and not accompanied by CICR or IICR (REFS.7,27) (TABLE 1 and FIG. 2b,f). The amplitude and nanodomain or microdomain localization of this Ca2+ signal can vary depending on the gating of differential sets of Ca2+ channels, and mediate bidirectional growth cone turning23. The differences in amplitude can be detected by at least two Ca2+ effectors: Ca2+/calmodulin-dependent protein kinase II (CaMKII) and the Ca2+/calmodulin-dependent protein phosphatase, calcineurin (REF. 28). They are able to do so by virtue of their differing affinity for Ca2+-calmodulin: calcineurin has a higher affinity for Ca2+-calmodulin than CaMKII (REFS. 29,30). In this Ca2+ affinity model, low-amplitude Ca2+ signals activate calcineurin over CaMKII for repulsion whereas high-amplitude Ca2+ signals preferentially activate CaMKII for attraction28. However, the source of Ca2+ signals can be another determinant of the turning direction, because Ca2+ signals of equivalent amplitude with or without CICR elicit growth cone attraction or repulsion, respectively27.

Figure 2
Second messenger systems for growth cone guidance

To reconcile these seemingly contradictory observations, we propose to refine the Ca2+ affinity model28 by assuming that Ca2+ effectors for repulsion and attraction are localized to differential subcellular compartments: high-affinity Ca2+ effectors such as calcineurin exist diffusely in the growth cone cytosol whereas low-affinity Ca2+ effectors such as CaMKII are localized in close proximity to ER Ca2+ channels. Both calmodulin and CaMKII have been shown to associate, although not exclusively31, with RyRs and IP3Rs (REFS. 32,33), and thus are favorably situated for the detection of Ca2+ release from the ER. In this refined model, Ca2+ influx through plasma membrane channels alone would activate repulsive, but not attractive effectors, leading to growth cone repulsion. Conversely, attractive turning would require activation of attractive effectors by means of Ca2+ release from the ER, abundant Ca2+ influx across the plasma membrane that can elevate cytoplasmic Ca2+ concentrations at the ER, or combinations of the two. Consistent with this model, for example, myelin-associated glycoprotein (MAG)-induced repulsion depends on low-amplitude Ca2+ release from the ER (REF. 7) and possibly Ca2+ influx through TRPC1 channels (REF. 34) (FIG. 2d), but MAG-induced attraction requires high-amplitude Ca2+ signals accompanied by CICR (REF. 7; T. Tojima, R. Itofusa and H. Kamiguchi, unpublished data).

Asymmetric cyclic nucleotide signaling can also mediate axon guidance. For example, activation of the cAMP pathway on one side of the growth cone is sufficient to trigger attractive turning35,36. Such instructive cAMP signaling has been implicated in pituitary adenylate cyclase-activating polypeptide-induced attraction35 and netrin-1-induced attraction36, although there is no direct evidence for asymmetric cAMP distribution across the growth cone in gradients of these cues. The extracellular guidance cue Semaphorin 3A (Sema3A) stimulates the production of cGMP preferentially on the side of the growth cone facing the Sema3A source, leading to a Ca2+ influx pattern for repulsive turning10. Among several guidance cues that have been shown to influence cyclic nucleotide levels, attractive and repulsive cues tend to elevate cAMP and cGMP, respectively (TABLE 1 and FIG. 2).

Second messenger crosstalk for directional switching and signal amplification

Axon pathfinding involves not only attraction or repulsion, but often a switch from one to the other. Cyclic nucleotides can act as such a switch by modulating Ca2+ channel activities37. The Ca2+ release from the ER that determines growth cone turning polarity is regulated counteractively by cAMP and cGMP pathways11,27,38: cAMPv facilitates ligand-dependent activation of RyRs and IP3Rs for an attractive response. Conversely, cGMP inactivates RyRs and blocks CICR although the involvement of cGMP in the regulation of IICR during axon guidance remains to be determined. Biochemical and molecular structural studies have shown that the gating of ER Ca2+ channels can be modulated by phosphorylation by the cAMP- and cGMP-dependent kinases protein kinase A (PKA) and protein kinase G (PKG) (REFS. 32,33). The roles of PKA and PKG described in this review, which are based on experimental results using non-specific pharmacological agents, should be confirmed with recently developed cyclic nucleotide analogs and RNA interference technology that can specifically target these kinases36,39,40.

Evidence from other biological systems helps in the development of a hypothetical model of how growth cones can translate and amplify shallow concentration gradients of guidance cues into steeper gradients of intracellular signals. In addition to the pathway by which cAMP facilitates Ca2+ mobilization, Ca2+ in turn stimulates the production of cAMP via Ca2+-dependent adenylate cyclases (ACs) such as AC1 and AC8 (REF. 41). Indeed, AC8 has been shown to increase cAMP levels in retinal ganglion cells downstream of Ca2+-calmodulin activation42. Furthermore, recent work has demonstrated that Ca2+ depletion from the ER, an experimental equivalent of the consequence of CICR or IICR, stimulates AC-mediated cAMP production by a mechanism that is independent of the cytoplasmic Ca2+ concentration43. If this store-operated cAMP production pathway operates in growth cones, it could mediate CICR- and IICR-elicited cAMP elevations. Such positive-feedback mechanisms would potentially participate in amplifying attractive guidance signals. Both mathematical modeling of cAMP and IICR regulation and imaging of spontaneous cAMP and Ca2+ in neuronal cell bodies have shown the existence of positive-feedback regulation, further opening the possibility that this might occur in growth cones44.

This positive-feedback augmentation of guidance signals can be terminated by multiple mechanisms. The cytoplasmic Ca2+ concentration can regulate the open probability of RyRs and IP3Rs biphasically: open probability increases as Ca2+ concentrations elevate, but further Ca2+ increase above threshold levels of 0.2 to hundreds of micromolars begins to inhibit opening of these Ca2+ channels32,33,45. Also, cyclic nucleotide levels can be self-suppressed via cyclic nucleotide-dependent phosphodiesterases (PDEs) (REF. 46). It is likely that growth cones employ these mechanisms to limit the amplitude of guidance signals.

Importantly, intracellular signals may also be refined via reciprocal inhibition pathways between cAMP and cGMP. Shelly et al47 recently identified such pathways in neuronal growth cones: cAMP elevation causes cGMP reduction via cGMP-specific PDE5 whereas cGMP elevation causes cAMP reduction via cAMP-specific PDE4. These reciprocal inhibition pathways allow attractive and repulsive signals to reduce cGMP and cAMP, respectively, and possibly contribute to signal amplification by inhibiting these counteractive regulators of ER Ca2+ channels. This may force the guidance machinery toward a clear-cut decision between attractive or repulsive response. It is generally thought that gradient amplification mechanisms in chemotactic cells involve both “local augmentation” and “global inhibition” of the guidance signal, and indeed, such mechanisms appear to be employed by growth cones48. This has led to the development of a molecular model that utilizes the positive feedback loops described in this and subsequent sections.

Bidirectional growth cone turning: a molecular model

Although guidance cues signal through structurally diverse receptors, many participating second messengers are shared. This has allowed us to propose a general model for second messenger networks that facilitate attractive and repulsive turning. Signaling networks including positive-feedback loops and reciprocal inhibition help to explain how a growth cone shapes Ca2+ signals for precise navigational responses to shallow gradients of attractive cues (FIG. 3). In this hypothetical model, netrin-1, for example, induces primary Ca2+ influx through TRPC1 channels (REFS. 9,49) and elevates the cAMP concentration15,36,50,51, preferentially on the side of the growth cone facing the netrin-1 source. The cAMP elevation would cause cGMP reduction via reciprocal inhibition47. The resultant increase in the ratio of cAMP to cGMP pushes L-VDCCs and RyRs to the active state and facilitates the generation of secondary Ca2+ entry through these channels27,37,38, which in turn stimulates further production of cAMP (REFS. 4143). Similarly, second messenger crosstalk shapes attractive guidance signals downstream of nerve growth factor (NGF) (REFS. 11,5254) and brain-derived neurotrophic factor (BDNF) (REFS. 8,52,55) (FIG. 3a). It is tempting to speculate that these positive-feedback loops act as the machinery for “local augmentation” and produce high-amplitude CICR or IICR on one side of the growth cone for attractive guidance.

Figure 3
Second messenger network shapes attractive and repulsive Ca2+ signals

Conversely, molecular mechanisms underlying “global inhibition” remain more elusive. Recent work47 has provided insight into the global inhibition mechanisms: when the cAMP pathway is activated within a single growth cone of a hippocampal neuron, all other neurites of that neuron exhibit cAMP reduction and cGMP elevation presumably involving active transport of unidentified regulators of cyclic nucleotides. If this self-down-regulation of cAMP can operate within an even shorter range, one would imagine that attractant-induced cAMP elevation on one side of the growth cone results in cAMP reduction and cGMP elevation on the other side. This would block the generation of secondary Ca2+ signals on the side facing lower attractant concentrations, thereby contributing to the confinement of attractive Ca2+ signals to the leading side of the growth cone.

During repulsive guidance, cGMP levels are elevated by Sema3A (REF. 10) and probably by netrin-1 (REF. 37). The cGMP elevation likely causes cAMP reduction via reciprocal inhibition47. In this situation, cue-induced primary Ca2+ influx would fail to trigger further Ca2+ entry through L-VDCCs, RyRs or IP3Rs (REFS. 11,27,37,38) (FIG. 3a). Such Ca2+ influx without Ca2+ release from the ER acts as a repulsive signal. The signaling network for repulsive guidance may lack a positive-feedback loop between Ca2+ and cyclic nucleotides (FIG. 3b), which is consistent with the concept that the amplitude of repulsive Ca2+ signals is low. Thus, an intriguing model that remains to be thoroughly tested is that amplification of repellent gradients occurs downstream of Ca2+ signals.

There are exceptions, however, such as Sema3A, which attracts growth cones if cGMP levels increase to levels that are high enough to activate PKG, which induces membrane depolarization and high-amplitude Ca2+ influx through voltage-dependent Ca2+ channels (REF. 56). The proposed model (FIG. 3b) can be scrutinized by simultaneous imaging of multiple second messengers and analyses of their spatiotemporal dynamics in growth cones during attraction and repulsion.

Steering machinery for growth cone guidance

In order to accomplish growth cone turning, the external signals that have been interpreted, amplified and transduced need to result in changes in the growth cone morphology. The classic machinery that drives such morphological changes includes the cytoskeleton and adhesion complexes, where the former facilitates plasma membrane protrusion and the latter mediates membrane anchoring to extracellular matrix. Adhesion receptors like integrins link to the underlying actin cytoskeleton at adhesion complexes by means of adaptor proteins including talin (REF. 57). Together, these machineries cooperate to drive migration. Accumulating evidence highlights the importance of their asymmetric reorganization during bidirectional guidance (FIG. 1b): the growth cone turns preferentially toward the side with cytoskeletal and adhesion complex assembly or away from the side with disassembly. Thus, effector processes that control cytoskeletal and adhesion dynamics must be regulated asymmetrically across the growth cone to initiate chemotactic turning. A growing number of effector proteins have been implicated in growth cone guidance, including Rho-family GTPases (REFS. 13,5862), actin-depolymerizing factor/cofilin (REFS. 63,64), Enabled/vasodilator-stimulated phosphoprotein (REF. 65), Mical (REFS.66,67), adenomatous polyposis coli (REF. 68), calpain (REF. 69), focal adhesion kinase (FAK) and Src-family kinases (REFS. 7078). Local translation or degradation of cytoskeletal components and their regulators also participates in growth cone guidance7985.

Remarkably, several recent studies8689 have demonstrated that regulated membrane trafficking is a critical effector process for chemotactic guidance by gradients of diffusible cues. In these circumstances, membrane trafficking becomes asymmetric across the growth cone immediately after the onset of guidance signals, preceding any detectable changes in cytoskeletal dynamics. These findings are consistent with the hypothesis that regulated membrane trafficking facilitates the subsequent cytoskeletal remodeling that is necessary for axon guidance, although future studies are warranted to determine the causal relationship between these cellular processes. In the following sections, we will examine how asymmetric membrane trafficking can be an instructive step in the initiation of growth cone steering.

Exocytosis and endocytosis in the growth cone

Developing neurons undergo dramatic morphological changes that require alterations in the surface area by means of exocytosis and endocytosis9095. Axon extension depends on the expansion of growth cone plasma membrane92, which can be mediated by vesicle-associated membrane protein 7 (VAMP7)-dependent exocytosis96. Regulated exocytosis can also occur locally in the growth cone, whereby synaptic protein-containing vesicles migrate along filopodia and fuse with the filopodial surface97. Conversely, growth cone collapse is accompanied by a reduction in the surface area via endocytosis87,93,94. These findings have led to the hypothesis that axon guidance involves asymmetric membrane trafficking across the growth cone98,99.

It is now evident that regulated exocytosis and endocytosis occur asymmetrically across the growth cone in response to guidance cue gradients8689 (FIG. 4). A gradient of chemoattractants promotes vesicle transport and subsequent VAMP2-mediated exocytosis on the gradient side of the growth cone. Conversely, a chemorepellent gradient causes asymmetric clathrin-mediated endocytosis on the side facing the gradient. It is likely that differential regulation of these opposing forces remodels the composition of the surface membrane and associated molecules locally to initiate turning. Regulated membrane trafficking can participate in multiple steps in the axon guidance process such as receptor-mediated signal transduction and downstream mechanical processes.

Figure 4
Asymmetric membrane trafficking drives bidirectional growth cone turning

The participation of exocytic and endocytic trafficking in the control of signaling events in the growth cone has been well documented. For example, the growth cone's responses to guidance cues can be modulated by membrane trafficking that alters the surface availability of relevant receptors such as deleted in colorectal cancer, uncoordinated 5 and neuropilin 1 (REFS. 100102). The growth cone can adjust its sensitivity to guidance cues by the process of adaptation103, which depends on endocytosis-mediated desensitization102 and protein synthesis-dependent resensitization102,103: endocytosis of the relevant receptors transiently desensitizes the growth cone to collapse-inducing activities of Sema3A and netrin-1 (REF. 102). Receptor endocytosis may be necessary for the full generation of intracellular guidance signals from early endosomes104106. Also, Ca2+-permeable cation channels packaged into VAMP-positive vesicles are carried to the growth cone, which leads to changes in morphology107.

In addition to a role in signaling, emerging evidence suggests that membrane trafficking can be a primary and instructive driving machinery for growth cone turning86,89. First, asymmetric exocytosis and endocytosis are necessary for growth cone attraction and repulsion, respectively, induced by direct Ca2+ manipulation that can bypass receptor activation and any upstream signaling events. Second, when the turning polarity is switched between attraction and repulsion by modulating second messenger profiles, the balance of exocytosis and endocytosis is also reversed. Importantly, the reversed turning polarity also depends on the newly acquired dominant trafficking mechanism, with attractive Ca2+ signals favoring exocytosis and repulsive Ca2+ signals triggering endocytosis. Finally, asymmetric perturbation of the balance of exocytosis and endocytosis is sufficient to initiate turning toward the side with more exocytosis or less endocytosis. Taken together, these findings indicate that the balance of exocytosis and endocytosis can dictate bidirectional growth cone turning downstream of second messenger signaling.

How do second messengers control membrane trafficking?

Asymmetric Ca2+ signals attract and repel growth cones via CaMKII and calcineurin, respectively28 (FIG. 5a). Both the magnitude and subcellular localization of Ca2+ signals might dictate which effector pathways are activated, and consequently favor either exocytosis or endocytosis. During attractive turning, Ca2+ signals promote microtubule-based centrifugal transport of membrane vesicles and their subsequent VAMP2-mediated exocytosis on the side with elevated Ca2+ (REFS. 86,108). The attractive Ca2+ signals might locally activate CaMKII, which could phosphorylate motor proteins like myosin-V and KIF17 and trigger release of these motors from cargo vesicles109,110. After releasing these motors, the vesicles could be incorporated into a readily-releasable pool of vesicles docked beneath the plasma membrane, followed by Ca2+-dependent activation of synaptotagmins and other effector proteins on the vesicle membrane that regulate exocytosis111. Although the exact mechanisms remain to be elucidated, it is likely that attractive Ca2+ signals facilitate at least two distinct but successive processes: vesicle translocation into the growth cone periphery and regulated exocytosis. Repulsive Ca2+ signals elicit asymmetric clathrin-mediated endocytosis in a calcineurin-dependent manner89. The role of calcineurin in clathrin-mediated endocytosis has been extensively studied in presynaptic terminals112. Calcineurin dephosphorylates, and thereby activates, dephosphins, a group of at least eight endocytic-adaptor proteins113,114. In synaptic transmission, activated dephosphins facilitate synaptic vesicle endocytosis after exocytic neurotransmitter release. In growth cones, the requirement for calcineurin activity in repulsive turning28 and clathrin-mediated endocytosis89 suggests that dephosphins play a similar role in axon guidance. Taken collectively, repulsive Ca2+ signals facilitate clathrin-mediated endocytosis via calcineurin, whereas attractive Ca2+ signals promote exocytic trafficking possibly via CaMKII (FIG. 5a).

Figure 5
Working model of the growth cone steering machinery: membrane trafficking as a master organizer

In addition to regulating the Ca2+ signal within growth cones, cyclic nucleotides can regulate membrane trafficking. For example, cAMP activation of PKA elicits phosphorylation of synapsin I (REF. 115), which then dissociates from the cytoplasmic surface of VAMP2-positive synaptic vesicles116. This potentiates regulated exocytosis in presynaptic terminals116 and facilitates vesicle translocation from the growth cone center toward the periphery117. Furthermore, the importance of synapsin phosphorylation in growth cone motility has been supported by the observation that expression of a synapsin mutant mimicking constitutive phosphorylation by PKA is sufficient to stimulate axon extension even in the presence of a PKA inhibitor118. These results suggest that cAMP might collaborate with Ca2+ to facilitate centrifugal vesicle transport and exocytosis during growth cone attraction. cGMP elevation can accelerate synaptic vesicle endocytosis via elevating presynaptic levels of phosphatidylinositol 4,5-biphosphate (REF. 119), but the role of cGMP in membrane trafficking in growth cones remains undefined.

Polarized remodeling of adhesion and cytoskeletal machinery

Growth cone turning depends on membrane trafficking, adhesion dynamics and cytoskeletal reorganization. While these mechanical processes can function independently, membrane trafficking might also polarize the activity of adhesion and cytoskeletal machinery by redistributing their components and regulators. For example, clathrin-mediated endocytosis retrieves integrins from the cell surface and sorts them into early endosomes for subsequent degradation or recycling back to the cell surface120. In non-neuronal cells, clathrin mediates β1-integrin endocytosis, a process that is required for focal adhesion disassembly and efficient cell migration121. Endocytosed β1-integrin is also present in VAMP2-positive vesicles in these cells, and depletion of VAMP2 reduces the amount of β1-integrin on the cell surface, which inhibits cell adhesion and chemotactic migration on a fibronectin substrate122. These findings indicate that cell adhesion to extracellular matrix can be controlled by endocytic and exocytic trafficking of integrins. An analogous mechanism might operate in axon guidance whereby growth cone repulsion would be driven by clathrin-mediated endocytosis of β1-integrin and asymmetric adhesion-complex disassembly88, whereas growth cone attraction would require exocytic trafficking of VAMP2-positive vesicles86 that could carry β1-integrin (REF. 122) (FIG. 5a). In this way, integrins may be transported to the growth cone periphery in a targeted manner and undergo exocytosis in regions where integrin-containing adhesions assemble, such as the lamellipodia and filopodia97,123,124. Importantly, asymmetric alterations in growth cone-substrate adhesion by blocking β1-integrin are sufficient to initiate growth cone turning away from the side with reduced β1-integrin function88. Thus, asymmetric trafficking of β1-integrin and potentially other adhesion molecules would be expected to cause polarized adhesion and turning of growth cones.

Downstream of repulsive guidance signals, endocytic removal of integrins must be coupled spatiotemporally with adhesion complex disassembly that often accompanies cell retraction and detachment from extracellular matrix57. As expected, a gradient of the chemorepellent Sema3A causes polarized endocytosis89 and asymmetric growth cone detachment from a laminin substrate as assessed by internal reflection microscopy125. Moreover, activation of the Ca2+-calcineurin pathway leads to disappearance of phosphorylated FAK, a component of integrin-based adhesion machinery, and growth cone detachment from a laminin substrate73. These findings imply that repulsive guidance signals can orchestrate multiple effector processes including dispersal of adhesion structures and selective retrieval of integrins. One possible mechanism underlying this orchestration is that Ca2+-dependent effectors modify components of integrin-containing adhesions for subsequent integrin internalization. For example, spontaneous Ca2+ transients in growth cone filopodia promote repulsive turning when generated asymmetrically, through local activation of the Ca2+-dependent protease calpain (REF. 69) that cleaves several components of the adhesion machinery such as FAK (REF. 126), Src (REF. 127) and talin (REF. 128). Thus, calpain may mediate the linkage between Ca2+-induced integrin endocytosis and integrin disengagement from the underlying cytoskeleton.

Cytoskeleton-associated proteins can also be a cargo of intracellular vesicles. Activation of the GTPase Rac can occur on early endosomes in non-neuronal cells129. Intracellular vesicles containing the activated Rac then move toward the cell periphery where actin-based membrane protrusions form129. Similarly, the GTPase Cdc42 can localize to intracellular vesicles and be delivered to the leading edge for directed cell migration130. Proteomic analyses have identified a large number of proteins including actin, tubulin, Rac and actin-related protein 2/3 complex that associate with VAMP2-positive synaptic vesicle and clathrin-coated vesicle preparations isolated from the brain131,132, supporting the notion of membrane trafficking as a means for the spatial restriction of cytoskeletal remodeling. Furthermore, phosphatidylinositol 3,4,5-trisphosphate (PIP3) is delivered to neuronal growth cones through microtubule-based anterograde vesicle transport133, where PIP3 facilitates axon formation via multiple signaling cascades including Cdc42 and Rac1 activation134, opening the possibility that asymmetric delivery of lipid mediators across the growth cone might participate in axon guidance. Therefore, it is reasonable to speculate that polarized trafficking of cytoskeletal regulators in the growth cone causes asymmetric cytoskeletal remodeling and growth cone turning (FIG. 5a). Another possibility is that local exocytosis and endocytosis influence the rate of actin polymerization by decreasing and increasing tension in the growth cone plasma membrane, respectively. This possibility is consistent with the Brownian-ratchet model of actin polymerization135 and has been supported by experimental results that the plasma membrane tension is inversely correlated with the rate of lamellipodial protrusion136.

We would like to propose a mechanistic model for bidirectional axon guidance, in which asymmetric membrane trafficking acts, downstream of second messengers, as a “master organizer” to spatially localize the growth cone driving machinery (FIG. 5b). In response to attractive Ca2+ signals, exocytic trafficking of VAMP2-containing vesicles would deliver positive regulators of adhesion complexes, cytoskeletal machinery and bulk membrane preferentially to the side of the growth cone with elevated Ca2+. Conversely, repulsive Ca2+ signals enhance endocytic removal of adhesion molecules, cytoskeletal components and membrane to reshape the growth cone. In this way, attractive and repulsive Ca2+ signals are transformed into exocytosis and endocytosis, respectively, which in turn potentiate asymmetric traction and protrusion forces for growth cone turning through polarized targeting of the driving machinery.

Future prospects: how does a growth cone integrate multiple guidance signals?

A growth cone must integrate signals of multiple guidance cues when navigating through complex environmental terrain in vivo. For example, commissural axon pathfinding along the dorsal-ventral axis of the spinal cord requires the release of multiple cues from either the ventral midline floor plate or the dorsal roof plate. The floor-plate-derived attractants netrin-1 (REF. 137) and Sonic hedgehog (REF. 138), and the roof-plate-derived repellents bone morphogenetic proteins (REF. 139) and possibly draxin (REF. 140) cooperate to guide commissural axons toward the ventral midline. After midline crossing, commissural axons turn rostrally, guided by counter gradients of the repellent Sonic hedgehog (REF. 141) and an attractive Wnt activity142,143 along the longitudinal axis. Another example comes from studies of the thalamocortical tract where axons are guided by counter gradients of the repellent ephrin-A5 (REF. 144) and the attractant netrin-1 (REF. 145). The growth cone's detection of multiple attractants and repellents with complementary gradient polarities likely serves to ensure correct pathway choices.

It is well known that guidance cues can regulate axonal responses to other guidance cues at the level of receptors146 or second messengers147. In addition to this hierarchical and regulatory interaction, recent work has demonstrated a “push and pull” mechanism in which both a repellent and an attractant play instructive and mutually supportive roles by confronting the same growth cone from opposite sides148. This study has also provided in vitro evidence that simultaneous presentation of two opposing cues as counter gradients improves the fidelity of growth cone turning.

Because many guidance cues signal through cytoplasmic Ca2+, counter gradients of two opposing cues could cause Ca2+ elevations on both sides of the growth cone (FIG. 6). For the growth cone to turn in this situation, spatial asymmetry in the type of Ca2+ and its downstream signaling should form across its axis. One plausible mechanism for this asymmetry is that attractive and repulsive cues differentially regulate cyclic nucleotide signaling such that cAMP and cGMP levels have counter gradients. These counter gradients could be generated as a result of attractant/repellent-induced cAMP/cGMP increases, the reciprocal inhibition between cAMP and cGMP, and the self down-regulation of cAMP as discussed in the previous section. The high cAMP/cGMP ratio on the attractant side would facilitate CICR and IICR while the low cAMP/cGMP ratio on the repellent side would prevent Ca2+ release from the ER, which causes the reciprocal distribution of attractive and repulsive Ca2+ signals in the growth cone. These reciprocal Ca2+ signals would elicit the counter gradients of exocytic and endocytic activities that cause asymmetric alterations in actin assembly and growth cone adhesiveness. In this way, the growth cone could translate multiple guidance signals into turning behavior with high fidelity. Although future studies are necessary to elucidate the growth cone's responses to various combinations of guidance cues, one could speculate that a growth cone would bifurcate in response to attractants on both sides or would stall in response to repellents on both sides. Coexistence of an attractant and a repellent on the same side of the growth cone can be counterbalancing149 but may induce attraction or repulsion depending on whether the positive-feedback loop between Ca2+ and cAMP has been turned on.

Figure 6
Hypothetical model for growth cone guidance by multiple cues

Conclusions

Pioneering work in the 1990s led to the discovery of many important axon guidance cues and their receptors. Subsequent studies in the 2000s elucidated signaling mechanisms by which guidance cues polarize the growth cone for turning, e.g., specific ion channels responsible for asymmetric Ca2+ signals, a second messenger network that controls switching between attraction and repulsion, and Ca2+-dependent enzymes involved in axon guidance. More recent work has identified target molecules and mechanisms that link the second messenger system with cellular machinery for growth cone motility. Whereas Ca2+ and cyclic nucleotides can influence the machinery via multiple pathways, we propose that asymmetric membrane trafficking plays a fundamental role in transforming guidance signals into polarized activity of adhesion and cytoskeletal dynamics for bidirectional turning. These revelations, along with future progress in growth cone research will contribute not only to developmental neurobiology but also to understanding mechanisms of human disorders with aberrant axon connectivity150,151 and to technological innovation for guiding regenerating axons to their appropriate targets after injury to the adult central nervous system152.

Online Summary

  1. During neural development, graded distribution of extracellular cues in the microenvironment causes asymmetric generation of second messengers across the growth cone in order to guide the axon along its correct path. Asymmetrically generated Ca2+ signals are sufficient to initiate growth cone turning toward the side with higher Ca2+ concentrations (attraction) or with lower Ca2+ concentrations (repulsion).
  2. Gating of differential sets of Ca2+ channels, which are regulated counteractively by cyclic AMP and cyclic GMP, can be responsible for switching between growth cone attraction and repulsion. We propose that high-amplitude Ca2+ elevation involving Ca2+ release from the endoplasmic reticulum (ER) mediates attractive guidance, whereas low-amplitude Ca2+ influx that does not trigger substantial Ca2+ release from the ER mediates repulsive guidance.
  3. Shallow concentration gradients of guidance cues can shape growth cone Ca2+ signals for precise navigational responses. This process may depend on second messenger networks including positive-feedback augmentation between Ca2+ and cyclic AMP.
  4. Repulsive Ca2+ signals cause asymmetric clathrin-mediated endocytosis across the growth cone with more endocytosis on the side with elevated Ca2+. Attractive Ca2+ signals promote centrifugal transport of membrane vesicles and their subsequent exocytosis mediated by vesicle-associated membrane protein 2 on the side with elevated Ca2+.
  5. We propose that asymmetric membrane trafficking is an early and instructive step in the initiation of growth cone turning. Localized imbalance between endocytosis and exocytosis may trigger redistribution of adhesion molecules, cytoskeletal components and bulk membrane, which would potentiate asymmetric traction and protrusion forces essential for turning.
  6. The growth cone in complex environmental terrain in vivo must integrate multiple guidance signals simultaneously to navigate with high fidelity. Spatiotemporally regulated interactions among Ca2+ and cyclic nucleotides potentially play crucial roles in this integration process, which will need to be scrutinized by quantitative imaging of multiple second messengers in navigating growth cones.

Acknowledgements

We apologize to investigators whose work could not be cited owing to space limitations. The authors' work is supported by RIKEN Brain Science Institute (H.K.), Grants-in-Aid from MEXT (H.K. and T.T.), JST PRESTO program (T.T.), funding by the National Institutes of Health (J.R.H.), and a John M. Nasseff, Sr., Career Development Award in Neurologic Surgery Research from the Mayo Clinic (J.R.H).

glossary

Local augmentation
A theoretical reaction of chemotactic cells for gradient sensing, in which signals are augmented locally in the cellular area containing higher receptor occupancy.
Global inhibition
Another theoretical reaction for gradient sensing. The locally augmented signals can be further isolated by spreading antagonistic signals over the whole cell.
Adaptation
The ability of a growth cone to readjust its sensitivity over a wide range of guidance cue concentrations during long-distance chemotaxis.
Early endosome
An intracellular membrane compartment where endocytosed molecules are sorted and directed to the plasma membrane for recycling or to lysosomes for degradation. Early endosomes can also serve as a platform where internalized receptors generate signals.
Rac and Cdc42
Rho-family GTPases that link extracellular signals to cytoskeletal rearrangements. Rac and Cdc42 can, for example, promote actin assembly through their major effector actin-related protein 2/3 complex.

Biographies

• 

Takuro Tojima obtained his Ph.D. (2002) from Graduate School of Science, Hokkaido University, Japan. He joined the Laboratory for Neuronal Growth Mechanisms directed by Hiroyuki Kamiguchi at RIKEN Brain Science Institute. He is also supported by JST PRESTO program. His research interests are in the molecular mechanisms of axon guidance and regeneration.

• 

Jacob Hines earned a Ph.D. in Biomedical Sciences (2010) from the Mayo Clinic, working in the Developmental and Regenerative Neurobiology laboratory led by John Henley. His research interests are in the mechanisms of cell migration in the developing nervous system.

• 

John Henley earned a Ph.D. in Biomedical Sciences (1997) from the Mayo Clinic and did postdoctoral training in molecular neurobiology with Mu-ming Poo at UC San Diego and UC Berkeley. He joined the faculty of the College of Medicine, Mayo Clinic (2006) and directs the Developmental and Regenerative Neurobiology laboratory (http://mayoresearch.mayo.edu/mayo/research/henley_lab/).

• 

Hiroyuki Kamiguchi obtained his M.D. (1989) from Keio University, Japan, and was board certified in neurosurgery (1995). He completed his Ph.D. (1996) at Keio University and postdoctoral training at Case Western Reserve University, USA. He joined the faculty of RIKEN Brain Science Institute in 1999 and studies developmental mechanisms of neuronal circuits.

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