Regulation of Growth Cone Actin Filaments by ... - Growth Cones

Regulation of Growth Cone Actin Filaments by Guidance Cues Gianluca Gallo,1 Paul C. Letourneau2 1

Department of Neurobiology and Anatomy, Drexel University College of Medicine, 2900 Queen Lane, Philadelphia, Pennsylvania 19129

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Department of Neuroscience, University of Minnesota, 6-145 Jackson Hall, 321 Church Street, Minneapolis, Minnesota 55455

Received 7 April 2003; accepted 8 April 2003

ABSTRACT:

The motile behaviors of growth cones at the ends of elongating axons determine pathways of axonal connections in developing nervous systems. Growth cones express receptors for molecular guidance cues in the local environment, and receptor-guidance cue binding initiates cytoplasmic signaling that regulates the cytoskeleton to control growth cone advance, turning, and branching behaviors. The dynamic actin filaments of growth cones are frequently targets of this regulatory signaling. Rho GTPases are key mediators of signaling by guidance cues, although much remains to be learned about how growth cone responses are orchestrated by Rho GTPase signaling to change the dynamics of polymerization, transport, and disassembly of actin filaments. Binding of neurotrophins to Trk and p75 receptors on growth cones triggers changes in actin filament dynamics to regulate several aspects of growth cone behaviors. Activation of Trk receptors mediates

INTRODUCTION Growth cones are specialized motile structures at the ends of developing axons. The activity of growth Correspondence to: P. C. Letourneau ([email protected]. edu). Contract grant sponsor: NIH (G.G. and P.C.L.). Contract grant sponsor: NSF (P.C.L.). Contract grant sponsor: Minnesota Medical Foundation (P.C.L.). Contract grant sponsor: Spinal Cord Research Foundation (G.G.). © 2003 Wiley Periodicals, Inc. DOI 10.1002/neu.10282

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local accumulation of actin filaments, while neurotrophin binding to p75 triggers local decrease in RhoA signaling that promotes lengthening of filopodia. Semaphorin IIIA and ephrin-A2 are guidance cues that trigger avoidance or repulsion of certain growth cones, and in vitro responses to these proteins include growth cone collapse. Dynamic changes in the activities of Rho GTPases appear to mediate responses to these cues, although it remains unclear what the changes are in actin filament distribution and dynamic reorganization that result in growth cone collapse. Growth cones in vivo simultaneously encounter positive and negative guidance cues, and thus, growth cone behaviors during axonal pathfinding reflect the complex integration of multiple signaling activities. © 2003 Wiley Periodicals, Inc. J Neurobiol 58: 92–102, 2004

Keywords: growth cone; actin filaments; Rho GTPases; pathfinding; cytoskeleton

cones is the main determinant of axon guidance and elongation. As an axon extends through the complex extracellular environment in vivo, its growth cone samples the local environment and responds to a variety of molecular guidance cues. Growth cones sample their environment by extending slender fingerlike projections called filopodia and veil-like structures termed lamellipodia. Both lamellipodia and filopodia are strictly dependent on the polymerization and organization of actin filaments (F-actin; Fig. 1). Evidence indicates that F-actin is a major intracellular target for the effects of extracellular guidance cues that alter growth cone behavior.

Guidance Cues Regulate Growth Cone Actin

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Figure 1 Growth cones. The left panel shows a phase contrast image of the tip of an embryonic chick dorsal root ganglion axon extending in vitro. The original growth cone has undergone branching, giving rise to two separate growth cones, each with a distinct central (C) and peripheral (P) domain. The right panel shows the same growth cone but reveals actin filaments (red) and microtubules (green) stained with rhodamine phalloidin and an antitubulin antibody, respectively.

Early pharmacological studies identified F-actin as a major determinant of axon elongation rates (Marsh and Letourneau, 1984; Letourneau et al., 1987). More recent work in the field of axon elongation and guidance has focused on the individual roles of actinassociated proteins in regulating axon extension and guidance (Kuhn et al., 2000). In order to appreciate the roles of individual proteins in controlling growth cone motility, it is important to first understand the dynamics of F-actin (see Jay, 2000 for a review; Fig. 2). F-actin is polymerized from monomeric G-actin

subunits. Actin polymerization in growth cones occurs predominantly at the leading edge (e.g., the tips of filopodia and the edge of lamellipodia). Following polymerization F-actin is then retrogradely transported toward the center of the growth cone. While undergoing retrograde transport, F-actin is dynamic, exchanging subunits at filament ends through a process termed filament turnover. As F-actin approaches the center of the growth cone, filaments are depolymerized, and actin subunits are recycled for further F-actin polymerization. Additionally, the cellular

Figure 2 Actin dynamics in growth cones. The panels show a growth cone from an embryonic chick dorsal root ganglion neuron transfected with a plasmid carrying a chimeric green fluorescent protein-␤ actin gene (kind gift of Dr. J. Bamburg, Colorado State University). Images were acquired at 3 s intervals and are shown as multiples of 15 s between panels (numbers in bottom right of each panel denote seconds).

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function of F-actin is dependent on its organization. In filopodia F-actin is organized as bundles of filaments, while in a lamellipodium F-actin forms a meshwork of connected and branched filaments. Thus, as an example, the formation of a filopodium is a complex process, involving an initial F-actin nucleation event, followed by elongation of filaments and their organization into a filament bundle (Svitkina et al., 2003). The rate of filopodial tip extension is determined by both the rate of F-actin polymerization at the filopodial tip and the retrograde displacement of polymerized filaments toward the base of the filopodium (Mallavarapu and Mitchison, 1999). Similarly, the extension of the edge of a lamellipodium is determined by the balance of F-actin polymerization and retrograde filament transport (Lin and Forscher, 1995). Thus, in order to understand the mechanisms by which guidance cues direct axon growth by modulating growth cone activity, it is important to determine how guidance cues affect F-actin dynamics and organization. A variety of proteins have been identified that regulate F-actin in growth cones. For this discussion we will focus largely on the Rho-family GTPases (RhoA, Rac1, and Cdc-42) and on myosin II. Rhofamily GTPases were originally identified as regulators of the actin cytoskeleton in fibroblasts (Ridley and Hall, 1992). Injection of Rac1, Cdc-42, and RhoA resulted in the generation of lamellipodia, filopodia, and stress fibers, respectively. Thus, these GTPases are capable of coordinating the elaboration of these important cellular structures. While growth cones exhibit filopodia and lamellipodia, they do not form stress fibers. In non-neuronal cells RhoA activity promotes stress fiber formation by activation of the mechano-enzyme myosin II (Katoh et al., 2001). In neurons RhoA effectors (Rho-kinase) increase myosin II activity and promote growth cone collapse and axon retraction (Katoh et al., 1998; Wahl et al., 2000; Jurney et al., 2002). In growth cones Rac1 and Cdc-42 have been found to regulate both filopodia and lamellipodia and are involved in axon extension and guidance (Kuhn et al., 1998; Brown et al., 2000). Actin depolymerizing factor (ADF) is involved in the regulation of growth cone F-actin depolymerization and the rate of F-actin turnover. Recent studies have implicated ADF activity in mediating the effects of guidance cues on growth cones and in the regulation of axon extension (Meberg and Bamburg, 2000; Aizawa et al., 2001). While myosin II has been implicated in the responses of growth cones to guidance cues, myosin II is also important for the maintenance and dynamics of growth cone morphology (Bridgman et al., 2001). Additionally, antisense experiments in-

dicate that myosin II is required for axon extension (Wylie et al., 1998; Wylie and Chantler, 2001). The long term objective of our research is to investigate the roles of these proteins in the responses to several guidance cues with the aim of understanding how their coordinated activity alters F-actin dynamics and organization, resulting in changes in growth cone motility that determine axon guidance, growth cone behavior, and axon extension. In order to understand guidance-cue-mediated regulation of axon guidance it is necessary to first appreciate the relationship between growth cone behavior and the mechanism of axon extension. Growth cones are described as having two domains: the peripheral (P) and the central (C) domains (Fig. 1). The Pdomain includes the dynamic motility of lamellipodia and filopodia. The C-domain does not exhibit protrusive activity and contains mostly organelles and the distal ends of axonal microtubules. In the axon microtubules are tightly bundled, but in the C-domain the tips of axonal microtubules splay out. The extension of axons consists of three phases: protrusion, engorgement, and consolidation (Goldberg and Burmeister, 1986). Protrusion involves the formation of lamellipodia and filopodia, events mediated by polymerization and reorganization of F-actin. Engorgement refers to the advance of microtubules and organelles from the C-domain into the P-domain. Engorgement occurs in regions of the growth cone that previously underwent protrusion. Following engorgement the region of the growth cone that was formerly the base of the C-domain collapses around the microtubules and forms a new extent of axon shaft. Thus, guidance cues could direct axon extension by modulating either the protrusion or engorgement phase. In the following sections we review and discuss advances in the understanding of the cytoskeletal mechanisms of guidance-cue-mediated growth cone guidance.

REGULATIONS OF F-ACTIN BY GUIDANCE CUES Among their important developmental roles, neurotrophins regulate the development of neuronal shape. During development of the retinotectal projection, brain-derived neurotrophic factor (BDNF) promotes the formation of retinal ganglion cell axon filopodia and branches (Cohen-Corey and Fraser, 1995). BDNF also regulates aspects of synaptogenesis and synaptic activity (Lu and Figurov, 1997). Similarly, nerve growth factor (NGF) is involved in sensory axon terminal arborization in the skin (Diamond et al.,


1992) and is also required for sensory axon elongation during development (Tucker et al., 2001). Neurotrophins can also determine the direction of axon extension in vitro (Letourneau, 1978; Gundersen and Barrett, 1979; Song et al., 1997). The roles of neurotrophins in axon guidance in vivo have not yet been fully established. However, the introduction of a source of exogenous neurotrophins can redirect regenerating axon growth in vivo (Politis et al., 1982). Thus, it is of interest to identify the signaling pathways and cytoskeletal mechanism responsible for the morphogenetic effects of neurotrophins. Neurotrophins bind to two classes of receptors, the Trk high-affinity receptors and the p75 low-affinity receptor (Kaplan and Miller, 2000). Neurotrophins bind Trk receptors specifically (e.g., NGF binds the TrkA receptor, while BDNF binds the TrkB receptor). All neurotrophins bind the p75 receptor. However, the responses elicited by neurotrophin binding to the p75 receptor differ for individual neurotrophins. Using an in vitro approach, we have shown that both growth cone turning towards a localized source of NGF and NGF-mediated sprouting of axonal filopodia require NGF binding to the TrkA receptor (Gallo et al., 1997; Gallo and Letourneau, 1998). The p75 receptor is partially involved in NGF-mediated growth cone turning, but not in NGF-mediated axonal filopodia sprouting. Neurotrophins have rapid effects on growth cone and axonal morphology. One of the most prominent responses to neurotrophins is the formation of filopodia at the growth cone and along the axon. Neurotrophins can also promote lamellipodial formation. In this respect, BDNF causes the formation of both filopodia and lamellipodia in Xenopus spinal neurons and in chick sensory neurons (Gallo and Letourneau, 1998; Gibney and Zheng, 2003), while NGF promotes mostly filopodial formation in chick sensory neurons (Gallo and Letourneau, 1998). Thus, individual neurotrophins can have different morphogenetic effects on neurons. Filopodia act as “long distance” sensors for the growth cone. The regulation of filopodial length is important in determining the extent of the environment that a growth cone can directly sample. For example, assume that a growth cone has an average filopodial length of R microns. Then, the area around the growth cone that the filopodial tips can sample is approximated by ␲R2/2 where R is the average length of the filopodia (x). Thus, an increase in average filopodial length translates into an increase in sampling area that is given by the square of the increase in filopodial length (R). In other words, an increase in filopodial length of 30% would result in an increase of

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the sampling area by 69%. We investigated the effects of neurotrophins on growth cone filopodial length. Treatment of chicken sensory growth cones with neurotrophins resulted in an average 20 –30% increase in filopodial length within 30 min. The effects of neurotrophins on filopodial length occurred at high concentrations, consistent with a role for the p75 lowaffinity receptor. We tested the hypothesis that the p75 receptor mediates neurotrophin-induced increases in filopodial length. The p75 receptor is found over the entire surface of growth cones, including filopodia. Treatment of growth cones from sensory ganglia of the p75 ⫺/⫺ KO mouse did not elicit increases in filopodial length. Conversely, neurotrophin treatment of ciliary growth cones, which express only the p75 receptor and not Trk receptors, resulted in increases in filopodial length. Thus, the p75 receptor is required and sufficient to mediate the effects of neurotrophins on filopodial length. Barde and colleagues (Yamashita et al., 1999) have proposed that the p75 receptor in the unbound state activates the RhoA GTPase. Following neurotrophin binding, the p75 receptor no longer activates RhoA. Thus, neurotrophin binding to the p75 receptor decreases the levels of active RhoA in growth cones. Thus, we tested the hypothesis that the p75-mediated effects of neurotrophins on filopodial length are mediated through a regulation of RhoA. Inactivation of RhoA using C3 exozyme mimics the effects of neurotrophins on filopodial length, and the introduction of constitutively active RhoA into growth cones blocks the neurotrophin-induced increases in filopodial length. Thus, these data indicate that the p75 receptor is a regulator of growth cone filopodial length. The formation of axonal filopodia is important because it is the first step in the formation of axon collateral branches. Axonal filopodia formation in response to neurotrophins is dependent on actin filament polymerization, as evidenced by the block of filopodia formation by pharmacological agents that inhibit actin polymerization (Gallo and Letourneau, 1998). Interestingly, when neurotrophin-coated beads contact axons in the presence of cytochalasin D, a drug that blocks barbed-end F-actin polymerization, filopodial formation is blocked but F-actin accumulates at the site of axon-bead contact. This may reflect the activation of F-actin nucleation by neurotrophins. If neurotrophins induced the de novo nucleation of filaments, cytochalasin D would block growth of the filaments, thus resulting in a patch of F-actin instead of the long filaments required for filopodia extension. The ARP2/3 complex is an important nucleator of F-actin that also caps the pointed ends of filaments (Welch, 1999). Thus, NGF may induce ARP2/3-me-

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diated filament nucleation, which in the presence of CD is expected to result in the accumulation of short filaments. It will therefore be of interest to characterize at the ultrastructural level the filaments that form in response to NGF in the presence of CD and to further determine if neurotrophins activate F-actin nucleation. An important aspect of the biology of F-actin is its rapid filament turnover rates. As demonstrated by the effects of jasplakinolide, a peptide that binds F-actin and prevents its depolymerization, filament turnover is required for growth cone motility (Gallo et al., 2002). This observation is consistent with the idea that cellular protrusions are driven by the polymerization and turnover of F-actin instead of the reorganization of pre-existing filaments into filopodia and lamellipodia. Filopodial F-actin bundles have significantly slower turnover rates than lamellipodial Factin. Filopodial F-actin is estimated to have a halflife of 25 min relative to 0.5–3 min for lamellipodial F-actin (Mallavarapu and Mitchison, 1999). However, filopodia are transient structures that form, extend, and retract fully on a scale of minutes. Thus, although the F-actin in filaments has significantly lower turnover rates than in lamellipodia, additional mechanisms must exist that disassemble filopodial F-actin bundles during filopodial retraction. The stabilization of filopodia by guidance cues has been suggested to be an important aspect of growth cone guidance. For example, when growth cones of embryonic grasshopper pioneer neurons make filopodial contact with a guidepost cell, the contacting filopodium is stabilized and does not retract (Bentley and O’Connor, 1994). Similarly, when chick sensory growth cone filopodia contact a bead coated with NGF, the growth cone turns toward the direction of contact (Gallo et al., 1997). Importantly, filopodia that contact the NGF bead are stabilized against retraction. A similar observation was made for the contact of spontaneously formed axonal filopodia with NGF beads at a distance from the axon. Thus, guidance cues encountered in a spatially heterogeneous contact (e.g., a guidepost cell or a guidance-cue-coated bead) activate mechanisms that selectively stabilize the contacting filopodium. These observations suggest that spatially restricted guidance cues may elicit growth cone turning by inhibiting the cellular mechanisms that are responsible for filopodial retraction. This may involve the inhibition of F-actin retrograde flow in the filopodium or the blockade of filament severing and depolymerization. Interestingly, the axonal filopodia of gelsolin knockout mice have significantly longer life spans than wild-type (Lu et al., 1997). Gelsolin is an actin filament severing protein, consistent with the sugges-

tion of a role for F-actin filament severing in filopodial retraction. Thus, guidance cues that result in selective stabilization of filopodia may do so by inhibiting filopodial F-actin disassembly by severing proteins like gelsolin. A role for the attenuation of F-actin retrograde flow in axon guidance has also been demonstrated. Following contact with a positive guidance cue, like the surface of another neuron, Aplysia growth cones exhibit decreased rates of Factin retrograde flow in the direction of contact (Lin et al., 1994). Thus, a combination of the inhibition of retrograde flow and additional mechanisms involved in filopodial retraction, such as F-actin severing, may be responsible for the selective stabilization of filopodia by guidance cues. When growth cones encounter a soluble gradient of an attractant guidance cue they turn towards the direction of greater concentration. During this type of guidance, filopodial numbers have been shown to be greater in the direction of the gradient (Zheng et al., 1996). Similarly, filopodial formation is more frequent in the region of the growth cone that contacts a guidepost cell (Bentley and O’Connor, 1994). During turning towards a contact with an NGF-bead, selective extension of filopodia has also been observed (Gallo et al., 1997). The selective extension of filopodia in regions of growth cones exposed to guidance cues occurs in concert with the inhibition of filopodial extension away from the site of guidance cue contact. Thus, guidance cues polarize filopodial dynamics resulting in increased rates of filopodial formation towards the cue and decreased filopodial formation away from the guidance cue. The interaction between filopodial F-actin bundles and microtubule tips is of great relevance to the mechanism of growth cone guidance. The tips of axonal microtubules extend into the body of the growth cone and undergo dynamic instability. Microtubule dynamic instability is required for growth cone turning in response to guidance cues (Williamson et al., 1996; Challacombe et al., 1997; Gallo and Letourneau, 2000). Blocking microtubule dynamic instability with pharmacological reagents prevents growth cone turning at substratum borders and in response to localized sources of neurotrophins. Conversely, asymmetric microtubule elongation/stability results in growth cone turning in the direction of greatest microtubule elongation/stability (Buck and Zheng, 2002). As first indicated by electron microscopic observations of fixed growth cones (Letourneau, 1983), simultaneous live imaging of actin and microtubules revealed that microtubules could associate with filopodial F-actin bundles (Schaefer et al., 2002). The interaction of microtubules and F-actin bundles is promoted by blocking


actin retrograde flow, which allows microtubules to extend along F-actin bundles and invade filopodia. The invasion of filopodia by microtubules is likely the first step in directing the movement of intracellular organelles and cytoplasm during growth cone turning. Significantly, causing asymmetric loss of F-actin filopodial bundles in growth cones results in microtubule redistribution towards the region of the growth cone containing the largest amount of F-actin bundles and thus turning away from the direction lacking F-actin bundles (Zhou et al., 2002). Thus, the interaction between F-actin bundles and microtubules is a basic mechanism in growth cone guidance. The reorganization of microtubules by asymmetric F-actin bundle formation/stabilization may also have consequences for asymmetric growth cone motility observed during turning (see previous paragraphs). Microtubule dynamics have been shown to regulate cell surface protrusion. In growth cones, attenuation of microtubule dynamics results in impaired lamellipodial protrusion (Gallo, 1998). Similarly, in nonneuronal cells microtubule tips promote lamellipodial motility through the activity of the GTPase Rac1 (Waterman-Storer et al., 1999), which has a wellestablished role in lamellipodial formation. Growth cones turn toward a source of the microtubule-stabilizing drug taxol, which results in asymmetric redistribution of microtubules toward the source of taxol (Buck and Zheng, 2002). Additionally, growth cone turning towards a source of taxol requires the activity of GTPases. Thus, an asymmetric distribution of Factin bundles across the growth cone is expected to result in an asymmetry in microtubule-tip-mediated promotion of surface motility. These observations may explain the asymmetric loss of protrusive activity in the direction opposite to that of growth cone turning described in previous paragraphs. The interactions between microtubules and F-actin bundles are a frontier in understanding the mechanism of growth cone guidance.

MODIFICATION OF THE F-ACTIN CYTOSKELETON IN RESPONSE TO NEGATIVE GUIDANCE CUES The term “growth cone collapse” describes the loss of lamellipodia and filopodia in response to a negative guidance cue. For example, following treatment with Semaphorin IIIA (SemaIIIA) sensory growth cones collapse and no longer exhibit protrusive P-domain activity. When growth cones make filopodial contact with a localized source of SemaIIIA (e.g., a bead coated with SemaIIIA) the whole growth cone does

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not collapse, just the portion of the growth cone near the contacting filopodium (Fan and Raper, 1995). Growth cones have been observed to collapse in vivo (Halloran and Kalil, 1994). However, negative guidance cues likely steer growth cones by inducing partial or local collapse. Fan et al. (1993) investigated the effects of SemaIIIA on the cytoskeleton of growth cones and noted a loss of F-actin that coincided with growth cone collapse. Similarly, F-actin depolymerization induced by pharmacological agents also causes growth cone collapse. Thus, the effects of growth cone collapsing cues may be mediated largely by the depolymerization of F-actin. While the simplicity of this model for the mechanism of growth cone collapse is attractive, recent observations indicate that growth cone collapse involves a more complex series of events. In the standard protocol for investigating growth cone collapse cultures are treated with a collapsing reagent (e.g., SemaIIIA), and the percentage of collapsed growth cones is determined. While useful, this approach does not allow for a careful analysis of the events underlying growth cone collapse, and additional important information can be obtained from live imaging of growth cone responses. For example, the static growth cone collapse assay cannot determine whether growth cones that do not appear collapsed maintained protrusive activity after treatment with a collapsing agent. Growth cone collapse is mediated by several signaling pathways, including the Rho-GTPases (Liu and Strittmatter, 2001), ADF (Aizawa et al., 2001), and kinases (GSK-3␤, FYN, cdk5; Eickholt et al., 2002; Sasaki et al., 2002). Jurney et al. (2002) found that inhibition of Rac1 activity blocks growth cone collapse in response to ephrin-A2. However, while growth cones with decreased Rac1 activity did not collapse, time-lapse imaging revealed that no further extension or retraction of lamellipodia and filopodia occurred following treatment with ephrin-A2. Thus, although not collapsed by ephrin-A2, growth cones became “frozen” in place. This demonstrates that growth cone collapse is independent of the maintenance of growth cone protrusive activity (the extension/retraction of filopodia and lamellipodia). Additionally, ephrin-A2 did not cause growth cone collapse when Rac1 signaling activity was blocked, although a similar amount of F-actin depolymerization occurred as when growth cones were treated with ephrin-A2 alone. These results indicate that growth cone collapse and F-actin depolymerization are independent processes. Pfenninger and colleagues (de La Houssaye et al.,

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1999) investigated the role of eicosanoids in growth cone collapse in response to thrombin and SemaIIIA. Both growth cone collapsing cues increase eicosanoid production through the activity of 12/15-lipoxygenase, and generation of eicosanoids is necessary for growth cone collapse. Addition of eicosanoids to the culture medium mimics the effects of the collapsing agents. Similar to the findings of Jurney et al. (2002), Mikule et al. (2002) report that growth cones treated with SemaIIIA under conditions of inhibited eicosanoid synthesis remain spread and do not collapse, but F-actin depolymerization is not blocked. These results confirm that growth cone collapse involves more than simply F-actin depolymerization. These results indicate that hypotheses for the mechanism of growth cone collapse should be revisited. We suggest the hypothesis that growth cone collapse is the result of the activation of several distinct signaling pathways that modify particular growth cone components and that actin filament depolymerization is not the primary cause of growth cone collapse. Hereafter, we will refer to these cellular effects as “modules”. Additionally, we propose that the modules activated by negative guidance cues need to be orchestrated in order to bring about collapse, and blockade of one or more of these modules can prevent growth cone collapse. The question thus becomes: what does it take to collapse a growth cone? Based on published observations it is possible to make a “grocery list” of the recognized modules involved in growth cone collapse in response to guidance cues: F-actin depolymerization; the cessation of protrusive activity; F-actin reorganization; loss of attachment to the substratum; and endocytosis. Which modules are necessary for growth cone collapse? Based on previous considerations, F-actin depolymerization is not sufficient. The loss of substratum adhesion is unlikely to be necessary for Factin depolymerization as growth cones that remain adherent to the substratum and do not collapse can still undergo F-actin depolymerization. Similarly, endocytosis is not required for F-actin depolymerization, because blocking ephrin-A2-induced endocytosis by inactivating Rac1 does not stop F-actin depolymerization in response to ephrin-A2. F-actin reorganization may be an important component of growth cone collapse. Unlike untreated growth cones in which F-actin is concentrated in filopodia and lamellipodia of the P-domain, the remaining F-actin in collapsed growth cones is aggregated in the C-domain of the growth cone. Blocking of Rac1 activity prevents this reorganization in response to ephrin-A2, and the remaining F-actin is in the form of actin bundles. Zhou and Cohan (2001) showed that F-actin

bundle loss occurs during growth cone collapse, but that F-actin meshworks are less affected during collapse. Thus, reorganization of F-actin may be a required module for growth cone collapse. Negative guidance cues induce an increase in endocytosis at the growth cone during collapse (Fournier et al., 2000; Jurney et al., 2002). The increase in endocytosis is not due to depolymerization of F-actin, loss of growth cone morphology, or the inhibition of protrusive activity, because growth cone collapse induced by pharmacological depolymerization of F-actin does not result in increased endocytotic activity. It is presently unknown whether endocytosis is of mechanistic relevance to the process of growth cone collapse. A consideration against a direct role for endocytosis in growth cone collapse is that neurotrophins promote growth cone filopodial and lamellipodial extension but also cause increased endocytosis. We suggest that the increase in endocytotic activity during growth cone collapse may reflect the internalization of receptors bound to the collapse-inducing ligands (e.g., SemaIIIA or ephrin-A2). Receptor internalization following ligand binding has been well characterized for a number of growth factors including neurotrophins (Barker et al., 2002), and the internalization may be a required component of guidance cue signaling. Thus, it will be of interest to further investigate the role of endocytosis in the signaling of negative guidance cues and to determine whether it is required for the induction of growth cone collapse. Growth cone collapsing guidance cues decrease substratum attachment (de La Houssaye et al., 1999; Mikule et al., 2002). It is unlikely that substratum attachment is required for continued protrusive activity of individual filopodia or lamellipodia, because both structures can extent away from the substratum, indicating independence from substratum attachment. However, substratum attachments must be broken for the retraction of filopodia and lamellipodia. Thus, loss of substratum attachment may be an important aspect of collapse. Positive guidance cues promote and polarize protrusive activity in the direction of growth cone migration. Conversely, the cessation of protrusive activity is likely to be of fundamental importance to the actions of negative guidance cues. Thus, asymmetric inhibition of protrusion can direct growth cone migration away from a negative guidance cue due to continued protrusion on the side of the growth cone unaffected by the guidance cue. Inhibition of protrusion could be due to several effects on the cytoskeleton, such as the blockade of F-actin nucleation, changes in polymerization rates, or the severing of filaments. To our knowledge no information is presently available re-


garding aspects of F-actin in collapsed growth cones, such as filament length and orientation, numbers of filament barbed ends available for polymerization, or the details of filament organization. However, it is likely that filament turnover is altered during growth cone collapse. Aizawa et al. (2001) found that the activity of ADF, which regulates filament turnover, is required for growth cone collapse in response to SemaIIIA. Similarly, Gallo et al. (2002a) report that the F-actin present in growth cones collapsed by ephrin-A2 exhibits slower turnover than F-actin in untreated growth cones. Thus, although collapsing signals decrease the F-actin content of growth cones, the remaining actin filaments are not as dynamic as in untreated growth cones. Collapse is a complex process that affects growth cones at several levels. In the future, it will be important to identify further modules and to determine the functional relationships between modules. Additionally, the detailed reorganization of the components of collapsed growth cones needs to be further clarified to understand the mechanisms responsible for collapse.

MECHANISM OF AXON RETRACTION Growth cone collapse in response to inhibitory guidance cues halts axon extension. However, growth cone collapsing cues can also cause axon retraction. The mechanism of axon retraction is not well understood, although it is important during development and in the response of axons to injury. For example, during development of the retinal projection, axons extend to inappropriate targets and many branches are subsequently pruned to remove incorrect projections (O’Leary, 1992). Following injury axons retract from the site of injury (Selzer, 2003). Blocking injuryinduced axon retraction may be an important target for therapeutic strategies aimed at minimizing damage to the nervous system. Therefore, understanding the mechanism of axon retraction has relevance to both development and the response of the nervous system to injury. Axon retraction could occur through two separate, but not mutually exclusive, mechanisms: depolymerization of the axonal cytoskeleton; and the reconfiguration and retraction of the cytoskeleton resulting from changes in intracellular forces. Recent evidence indicates that the latter mechanism may be prevalent. Ahmad et al. (2000) found that inhibition of the microtubule motor dynein caused axon retraction. Retracting axons often assume a sinusoidal morphology and contain a dense, curved bundle of microtubules. Axon retraction in response to the inhibition of dyenin

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was dependent on actomyosin contractility, indicating that axon extension is regulated by a balance of forces generated by microtubule- and actin-based motor proteins. Gallo et al. (2002a) investigated the effects on axon elongation of blocking F-actin turnover with a cell permeable compound called jasplakinolide, which binds to F-actin and prevents filament depolymerization. Treatment with jasplakinolide resulted in rapid axon retraction, characterized by sinusoidal bending of axons, similar to what was observed by Ahmad et al. (2000) following the block of dyenin activity. Jasplakinolide-induced axon retraction was found to depend on the endogenous actomyosin contractility in axons. These observations indicate that F-actin turnover in axons and growth cones prevents endogenous myosin II activity from causing axon retraction. Collectively, these studies indicate that axon retraction is regulated by the activities of motor proteins that generate forces on the cytoskeleton. Guidance cues regulate the force generation mechanisms of axons to induce retraction. Gallo et al. (2002a) found that retinal axon retraction in response to ephrin-A2 is dependent on RhoA signaling. RhoA regulates myosin II activity in neurons, and introduction of RhoA into neuronlike cells causes axon retraction. He et al. (2002) report that during nitric oxide (NO)-induced axon retraction microtubules are not depolymerized but undergo reconfiguration, resulting in the formation of sinusoidal bends in the microtubule array. As discussed above, the formation of curvatures in the axonal array is likely to be a general feature of force-mediated axon retraction. These studies demonstrate that guidance cues can regulate the activity of cytoskeletal motors resulting in the reorganization of the cytoskeleton and axon retraction. The small GTPase Rac1 is also required for axon retraction in response to ephrin-A2 (Jurney et al., 2002). The best characterized function of Rac1 is to promote lamellipodial formation. Paradoxically, Rac1 activity is also required for growth cone collapse and axon retraction in response to some guidance cues. It will thus be of interest to further define the pathway through which Rac1 acts during growth cone responses to negative guidance cues.

MODULATION OF GROWTH CONE RESPONSES BY THE COMBINED SIGNALING OF POSITIVE AND NEGATIVE GUIDANCE CUES In vivo, growth cones are simultaneously exposed to a number of different guidance cues. Thus, it is necessary to understand how growth cones integrate mul-

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tiple, often opposing, signals to reach their targets. Target-derived BDNF promotes the formation of retinal ganglion cell axon arbors. Conversely, NO produced by the target cells of retinal axons promotes the retraction of inappropriately targeted axons. BDNF and NO are encountered by retinal axons in vivo during the same period of development. Therefore, we investigated the responses of retinal growth cones to the combined signaling of BDNF and NO (Ernst et al., 2000). Treatment with NO alone caused growth cone collapse and axon retraction. However, pretreatment with BDNF blocked the effects of NO on growth cones and axons. In order to investigate the mechanism of the protective effects of BDNF against NOinduced growth cone collapse we tested the role of cAMP and protein kinase A in mediating the effects of BDNF (Gallo et al., 2002b). Neurotrophin-induced cAMP signaling was previously found to be required for the ability of neurotrophins to allow axons to overcome inhibitory myelin-derived signals (Qiu et al., 2002), and cyclic nucleotides have been shown to convert the actions of guidance cues from attraction to repulsion, and vice-versa (Song et al., 1997, 1998). BDNF induced a rapid, but transient, activation of PKA signaling in retinal ganglion cells that is required for the establishment of the protective effects of BDNF against NO-induced growth cone collapse. While PKA signaling is required for the initiation of the protective effects of BDNF, it is not required for the maintenance of the protective effects, as blocking PKA activity after the initial transient increase in activity did not diminish the protective effects of BDNF. However, removal of BDNF resulted in the time-dependent loss of the protective effects of BDNF. Finally, PKA activation alone did not mimic the effects of BDNF. Thus, PKA activity in BDNF signaling acts as a required “switch” to turn on a protective mechanism that is subsequently maintained by additional BDNF-activated signaling pathways. Neurotrophins modulate SemaIIIA-induced growth cone collapse. Dontchev and Letourneau (2002) found that the response of sensory growth cones to SemaIIIA was dependent on the concentration of NGF the neurons were exposed to prior to treatment with SemaIIIA. The protective effects of NGF against SemaIIIA-induced growth cone collapse were partial and depended on the concentrations of NGF and SemaIIIA. Thus, NGF modulates the responsiveness of growth cones to SemaIIIA. The modulation is not due to decreased cell surface expression of SemaIIIA receptors. Experiments aimed at testing the role of PKA in the effects of NGF and SemaIIIA revealed that PKA activation mimics the protective effects of NGF. Inhibition of PKA signaling reversed the protective effects of NGF. These results

further demonstrate that neurotrophins can modulate the responsiveness of growth cones to negative guidance cues through PKA-based mechanisms. It will be of interest to define the pathways downstream of PKA activation that are responsible for the protective effects of neurotrophins against negative guidance cues.

FUTURE DIRECTIONS Although advances have been made in our understanding of the regulation of actin filaments in growth cones, much remains to be learned about how all the activities that determine actin filament organization and function are differentially regulated by guidance cues. Actin filaments are dynamic structures that are nucleated, polymerized or depolymerized, stabilized, and organized on demand to accommodate the directions obtained from extracellular guidance cues. A number of fundamental issues require additional investigation in order to further appreciate the complexities of growth cone behavior. For example: what are the mechanisms by which filopodia and lamellipodia are initiated? How are they selectively stabilized during guidance? What are the functional relationships between microtubules and actin filaments? At what level does the regulation of growth cone behavior and the cytoskeleton by multiple guidance cues occur? What cellular events are required for growth cone collapse in response to guidance cues? What mechano-enzymes are involved in growth cone behaviors and what are their roles? These questions will be best answered by the use of methods including live visualization of actin filaments and associated regulatory proteins in growth cones, biochemical and molecular biological approaches, and selective activation/inhibition of molecular species in living cells (e.g., microCALI). As the growth cone is a dynamic system with many inter-related elements, it will be of great importance to simultaneously track multiple events in growth cones in order to begin to determine the relationships between cytoskeletal and signaling systems. A major task for the future will be to integrate the emergent knowledge into a comprehensive model of growth cone biology. The authors acknowledge valuable contributions from members of the Letourneau laboratory, Scott Gehler, Vassil Dontchev, Florence Roche, and Eric Veien.

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