regulation of growth cone actin filaments by guidance cues

Regulation of Growth Cone Actin Filaments byGuidance Cues

Gianluca Gallo,1 Paul C. Letourneau2

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

2 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 conesat the ends of elongating axons determine pathways ofaxonal connections in developing nervous systems.Growth cones express receptors for molecular guidancecues in the local environment, and receptor-guidancecue binding initiates cytoplasmic signaling that regulatesthe cytoskeleton to control growth cone advance, turn-ing, and branching behaviors. The dynamic actin fila-ments of growth cones are frequently targets of thisregulatory signaling. Rho GTPases are key mediators ofsignaling by guidance cues, although much remains tobe learned about how growth cone responses are orches-trated by Rho GTPase signaling to change the dynamicsof polymerization, transport, and disassembly of actinfilaments. Binding of neurotrophins to Trk and p75receptors on growth cones triggers changes in actinfilament dynamics to regulate several aspects of growthcone behaviors. Activation of Trk receptors mediates

local accumulation of actin filaments, while neurotro-phin binding to p75 triggers local decrease in RhoAsignaling that promotes lengthening of filopodia. Sema-phorin IIIA and ephrin-A2 are guidance cues that trig-ger avoidance or repulsion of certain growth cones, andin vitro responses to these proteins include growth conecollapse. Dynamic changes in the activities of RhoGTPases appear to mediate responses to these cues,although it remains unclear what the changes are inactin filament distribution and dynamic reorganizationthat result in growth cone collapse. Growth cones in vivosimultaneously encounter positive and negative guid-ance cues, and thus, growth cone behaviors during ax-onal pathfinding reflect the complex integration of mul-tiple signaling activities. © 2003 Wiley Periodicals, Inc. J

Neurobiol 58: 92–102, 2004

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

INTRODUCTION

Growth cones are specialized motile structures at theends of developing axons. The activity of growth

cones is the main determinant of axon guidance andelongation. As an axon extends through the complexextracellular environment in vivo, its growth conesamples the local environment and responds to avariety of molecular guidance cues. Growth conessample their environment by extending slender fin-gerlike projections called filopodia and veil-like struc-tures termed lamellipodia. Both lamellipodia andfilopodia are strictly dependent on the polymerizationand organization of actin filaments (F-actin; Fig. 1).Evidence indicates that F-actin is a major intracellulartarget for the effects of extracellular guidance cuesthat alter growth cone behavior.

Correspondence to: P. C. Letourneau ([email protected]).

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

92

Early pharmacological studies identified F-actin asa major determinant of axon elongation rates (Marshand Letourneau, 1984; Letourneau et al., 1987). Morerecent work in the field of axon elongation and guid-ance has focused on the individual roles of actin-associated proteins in regulating axon extension andguidance (Kuhn et al., 2000). In order to appreciatethe roles of individual proteins in controlling growthcone motility, it is important to first understand thedynamics 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 oc-curs predominantly at the leading edge (e.g., the tipsof filopodia and the edge of lamellipodia). Followingpolymerization F-actin is then retrogradely trans-ported toward the center of the growth cone. Whileundergoing retrograde transport, F-actin is dynamic,exchanging subunits at filament ends through a pro-cess termed filament turnover. As F-actin approachesthe center of the growth cone, filaments are depoly-merized, and actin subunits are recycled for furtherF-actin polymerization. Additionally, the cellular

Figure 1 Growth cones. The left panel shows a phase contrast image of the tip of an embryonicchick dorsal root ganglion axon extending in vitro. The original growth cone has undergonebranching, 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) andmicrotubules (green) stained with rhodamine phalloidin and an antitubulin antibody, respectively.

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

Guidance Cues Regulate Growth Cone Actin 93

function of F-actin is dependent on its organization. Infilopodia F-actin is organized as bundles of filaments,while in a lamellipodium F-actin forms a meshworkof connected and branched filaments. Thus, as anexample, the formation of a filopodium is a complexprocess, involving an initial F-actin nucleation event,followed by elongation of filaments and their organi-zation into a filament bundle (Svitkina et al., 2003).The rate of filopodial tip extension is determined byboth the rate of F-actin polymerization at the filopo-dial tip and the retrograde displacement of polymer-ized filaments toward the base of the filopodium (Mal-lavarapu and Mitchison, 1999). Similarly, theextension of the edge of a lamellipodium is deter-mined by the balance of F-actin polymerization andretrograde filament transport (Lin and Forscher,1995). Thus, in order to understand the mechanismsby which guidance cues direct axon growth by mod-ulating growth cone activity, it is important to deter-mine how guidance cues affect F-actin dynamics andorganization.

A variety of proteins have been identified thatregulate F-actin in growth cones. For this discussionwe will focus largely on the Rho-family GTPases(RhoA, Rac1, and Cdc-42) and on myosin II. Rho-family GTPases were originally identified as regula-tors of the actin cytoskeleton in fibroblasts (Ridleyand Hall, 1992). Injection of Rac1, Cdc-42, and RhoAresulted in the generation of lamellipodia, filopodia,and stress fibers, respectively. Thus, these GTPasesare capable of coordinating the elaboration of theseimportant cellular structures. While growth cones ex-hibit filopodia and lamellipodia, they do not formstress fibers. In non-neuronal cells RhoA activity pro-motes stress fiber formation by activation of themechano-enzyme myosin II (Katoh et al., 2001). Inneurons RhoA effectors (Rho-kinase) increase myosinII activity and promote growth cone collapse and axonretraction (Katoh et al., 1998; Wahl et al., 2000;Jurney et al., 2002). In growth cones Rac1 and Cdc-42have been found to regulate both filopodia and lamel-lipodia and are involved in axon extension and guid-ance (Kuhn et al., 1998; Brown et al., 2000). Actindepolymerizing factor (ADF) is involved in the reg-ulation of growth cone F-actin depolymerization andthe rate of F-actin turnover. Recent studies have im-plicated ADF activity in mediating the effects ofguidance cues on growth cones and in the regulationof axon extension (Meberg and Bamburg, 2000;Aizawa et al., 2001). While myosin II has been im-plicated in the responses of growth cones to guidancecues, myosin II is also important for the maintenanceand dynamics of growth cone morphology (Bridgmanet al., 2001). Additionally, antisense experiments in-

dicate that myosin II is required for axon extension(Wylie et al., 1998; Wylie and Chantler, 2001). Thelong term objective of our research is to investigatethe roles of these proteins in the responses to severalguidance cues with the aim of understanding howtheir coordinated activity alters F-actin dynamics andorganization, resulting in changes in growth conemotility that determine axon guidance, growth conebehavior, and axon extension.

In order to understand guidance-cue-mediated reg-ulation of axon guidance it is necessary to first appre-ciate the relationship between growth cone behaviorand the mechanism of axon extension. Growth conesare described as having two domains: the peripheral(P) and the central (C) domains (Fig. 1). The P-domain includes the dynamic motility of lamellipodiaand filopodia. The C-domain does not exhibit protru-sive activity and contains mostly organelles and thedistal ends of axonal microtubules. In the axon mi-crotubules are tightly bundled, but in the C-domainthe tips of axonal microtubules splay out. The exten-sion of axons consists of three phases: protrusion,engorgement, and consolidation (Goldberg and Bur-meister, 1986). Protrusion involves the formation oflamellipodia and filopodia, events mediated by poly-merization and reorganization of F-actin. Engorge-ment refers to the advance of microtubules and or-ganelles from the C-domain into the P-domain.Engorgement occurs in regions of the growth conethat previously underwent protrusion. Following en-gorgement the region of the growth cone that wasformerly the base of the C-domain collapses aroundthe microtubules and forms a new extent of axonshaft. Thus, guidance cues could direct axon exten-sion by modulating either the protrusion or engorge-ment phase. In the following sections we review anddiscuss advances in the understanding of the cytoskel-etal mechanisms of guidance-cue-mediated growthcone guidance.

REGULATIONS OF F-ACTIN BYGUIDANCE CUES

Among their important developmental roles, neuro-trophins regulate the development of neuronal shape.During development of the retinotectal projection,brain-derived neurotrophic factor (BDNF) promotesthe formation of retinal ganglion cell axon filopodiaand branches (Cohen-Corey and Fraser, 1995). BDNFalso regulates aspects of synaptogenesis and synapticactivity (Lu and Figurov, 1997). Similarly, nervegrowth factor (NGF) is involved in sensory axonterminal arborization in the skin (Diamond et al.,

94 Gallo and Letourneau

1992) and is also required for sensory axon elongationduring development (Tucker et al., 2001). Neurotro-phins can also determine the direction of axon exten-sion in vitro (Letourneau, 1978; Gundersen and Bar-rett, 1979; Song et al., 1997). The roles ofneurotrophins in axon guidance in vivo have not yetbeen fully established. However, the introduction of asource of exogenous neurotrophins can redirect regen-erating axon growth in vivo (Politis et al., 1982).Thus, it is of interest to identify the signaling path-ways and cytoskeletal mechanism responsible for themorphogenetic effects of neurotrophins.

Neurotrophins bind to two classes of receptors, theTrk high-affinity receptors and the p75 low-affinityreceptor (Kaplan and Miller, 2000). Neurotrophinsbind Trk receptors specifically (e.g., NGF binds theTrkA receptor, while BDNF binds the TrkB receptor).All neurotrophins bind the p75 receptor. However, theresponses elicited by neurotrophin binding to the p75receptor differ for individual neurotrophins. Using anin vitro approach, we have shown that both growthcone turning towards a localized source of NGF andNGF-mediated sprouting of axonal filopodia requireNGF binding to the TrkA receptor (Gallo et al., 1997;Gallo and Letourneau, 1998). The p75 receptor ispartially involved in NGF-mediated growth cone turn-ing, but not in NGF-mediated axonal filopodia sprout-ing.

Neurotrophins have rapid effects on growth coneand axonal morphology. One of the most prominentresponses to neurotrophins is the formation of filop-odia at the growth cone and along the axon. Neuro-trophins can also promote lamellipodial formation. Inthis respect, BDNF causes the formation of both filop-odia and lamellipodia in Xenopus spinal neurons andin chick sensory neurons (Gallo and Letourneau,1998; Gibney and Zheng, 2003), while NGF promotesmostly filopodial formation in chick sensory neurons(Gallo and Letourneau, 1998). Thus, individual neu-rotrophins can have different morphogenetic effectson neurons.

Filopodia act as “long distance” sensors for thegrowth cone. The regulation of filopodial length isimportant in determining the extent of the environ-ment that a growth cone can directly sample. Forexample, assume that a growth cone has an averagefilopodial length of R microns. Then, the area aroundthe growth cone that the filopodial tips can sample isapproximated by �R2/2 where R is the average lengthof the filopodia (x). Thus, an increase in averagefilopodial length translates into an increase in sam-pling area that is given by the square of the increasein filopodial length (R). In other words, an increase infilopodial length of 30% would result in an increase of

the sampling area by 69%. We investigated the effectsof neurotrophins on growth cone filopodial length.Treatment of chicken sensory growth cones with neu-rotrophins resulted in an average 20–30% increase infilopodial length within 30 min. The effects of neu-rotrophins on filopodial length occurred at high con-centrations, consistent with a role for the p75 low-affinity receptor. We tested the hypothesis that thep75 receptor mediates neurotrophin-induced increasesin filopodial length. The p75 receptor is found overthe entire surface of growth cones, including filopo-dia. Treatment of growth cones from sensory gangliaof the p75 �/� KO mouse did not elicit increases infilopodial length. Conversely, neurotrophin treatmentof ciliary growth cones, which express only the p75receptor and not Trk receptors, resulted in increases infilopodial length. Thus, the p75 receptor is requiredand sufficient to mediate the effects of neurotrophinson filopodial length. Barde and colleagues (Yamashitaet al., 1999) have proposed that the p75 receptor in theunbound state activates the RhoA GTPase. Followingneurotrophin binding, the p75 receptor no longer ac-tivates RhoA. Thus, neurotrophin binding to the p75receptor decreases the levels of active RhoA ingrowth cones. Thus, we tested the hypothesis that thep75-mediated effects of neurotrophins on filopodiallength are mediated through a regulation of RhoA.Inactivation of RhoA using C3 exozyme mimics theeffects of neurotrophins on filopodial length, and theintroduction of constitutively active RhoA intogrowth cones blocks the neurotrophin-induced in-creases in filopodial length. Thus, these data indicatethat the p75 receptor is a regulator of growth conefilopodial length.

The formation of axonal filopodia is importantbecause it is the first step in the formation of axoncollateral branches. Axonal filopodia formation in re-sponse to neurotrophins is dependent on actin filamentpolymerization, as evidenced by the block of filopodiaformation by pharmacological agents that inhibit actinpolymerization (Gallo and Letourneau, 1998). Inter-estingly, when neurotrophin-coated beads contact ax-ons in the presence of cytochalasin D, a drug thatblocks barbed-end F-actin polymerization, filopodialformation is blocked but F-actin accumulates at thesite of axon-bead contact. This may reflect the acti-vation of F-actin nucleation by neurotrophins. If neu-rotrophins induced the de novo nucleation of fila-ments, cytochalasin D would block growth of thefilaments, thus resulting in a patch of F-actin insteadof the long filaments required for filopodia extension.The ARP2/3 complex is an important nucleator ofF-actin that also caps the pointed ends of filaments(Welch, 1999). Thus, NGF may induce ARP2/3-me-


diated filament nucleation, which in the presence ofCD is expected to result in the accumulation of shortfilaments. It will therefore be of interest to character-ize at the ultrastructural level the filaments that formin response to NGF in the presence of CD and tofurther determine if neurotrophins activate F-actinnucleation.

An important aspect of the biology of F-actin is itsrapid filament turnover rates. As demonstrated by theeffects of jasplakinolide, a peptide that binds F-actinand prevents its depolymerization, filament turnoveris required for growth cone motility (Gallo et al.,2002). This observation is consistent with the ideathat cellular protrusions are driven by the polymeriza-tion and turnover of F-actin instead of the reorgani-zation of pre-existing filaments into filopodia andlamellipodia. Filopodial F-actin bundles have signif-icantly slower turnover rates than lamellipodial F-actin. Filopodial F-actin is estimated to have a half-life of 25 min relative to 0.5–3 min for lamellipodialF-actin (Mallavarapu and Mitchison, 1999). However,filopodia are transient structures that form, extend,and retract fully on a scale of minutes. Thus, althoughthe F-actin in filaments has significantly lower turn-over rates than in lamellipodia, additional mecha-nisms must exist that disassemble filopodial F-actinbundles during filopodial retraction. The stabilizationof filopodia by guidance cues has been suggested tobe an important aspect of growth cone guidance. Forexample, when growth cones of embryonic grasshop-per pioneer neurons make filopodial contact with aguidepost cell, the contacting filopodium is stabilizedand does not retract (Bentley and O’Connor, 1994).Similarly, when chick sensory growth cone filopodiacontact a bead coated with NGF, the growth coneturns toward the direction of contact (Gallo et al.,1997). Importantly, filopodia that contact the NGFbead are stabilized against retraction. A similar ob-servation was made for the contact of spontaneouslyformed axonal filopodia with NGF beads at a distancefrom the axon. Thus, guidance cues encountered in aspatially heterogeneous contact (e.g., a guidepost cellor a guidance-cue-coated bead) activate mechanismsthat selectively stabilize the contacting filopodium.These observations suggest that spatially restrictedguidance cues may elicit growth cone turning byinhibiting the cellular mechanisms that are responsi-ble for filopodial retraction. This may involve theinhibition of F-actin retrograde flow in the filopodiumor the blockade of filament severing and depolymer-ization. Interestingly, the axonal filopodia of gelsolinknockout mice have significantly longer life spansthan wild-type (Lu et al., 1997). Gelsolin is an actinfilament severing protein, consistent with the sugges-

tion of a role for F-actin filament severing in filopo-dial retraction. Thus, guidance cues that result inselective stabilization of filopodia may do so by in-hibiting filopodial F-actin disassembly by severingproteins like gelsolin. A role for the attenuation ofF-actin retrograde flow in axon guidance has alsobeen demonstrated. Following contact with a positiveguidance cue, like the surface of another neuron,Aplysia growth cones exhibit decreased rates of F-actin retrograde flow in the direction of contact (Lin etal., 1994). Thus, a combination of the inhibition ofretrograde flow and additional mechanisms involvedin filopodial retraction, such as F-actin severing, maybe responsible for the selective stabilization of filop-odia by guidance cues.

When growth cones encounter a soluble gradientof an attractant guidance cue they turn towards thedirection of greater concentration. During this type ofguidance, filopodial numbers have been shown to begreater in the direction of the gradient (Zheng et al.,1996). Similarly, filopodial formation is more fre-quent in the region of the growth cone that contacts aguidepost cell (Bentley and O’Connor, 1994). Duringturning towards a contact with an NGF-bead, selec-tive extension of filopodia has also been observed(Gallo et al., 1997). The selective extension of filop-odia in regions of growth cones exposed to guidancecues occurs in concert with the inhibition of filopodialextension away from the site of guidance cue contact.Thus, guidance cues polarize filopodial dynamics re-sulting in increased rates of filopodial formation to-wards the cue and decreased filopodial formationaway from the guidance cue.

The interaction between filopodial F-actin bundlesand microtubule tips is of great relevance to the mech-anism of growth cone guidance. The tips of axonalmicrotubules extend into the body of the growth coneand undergo dynamic instability. Microtubule dy-namic instability is required for growth cone turningin response to guidance cues (Williamson et al., 1996;Challacombe et al., 1997; Gallo and Letourneau,2000). Blocking microtubule dynamic instability withpharmacological reagents prevents growth cone turn-ing at substratum borders and in response to localizedsources of neurotrophins. Conversely, asymmetric mi-crotubule elongation/stability results in growth coneturning in the direction of greatest microtubule elon-gation/stability (Buck and Zheng, 2002). As first in-dicated by electron microscopic observations of fixedgrowth cones (Letourneau, 1983), simultaneous liveimaging of actin and microtubules revealed that mi-crotubules could associate with filopodial F-actin bun-dles (Schaefer et al., 2002). The interaction of micro-tubules and F-actin bundles is promoted by blocking


actin retrograde flow, which allows microtubules toextend along F-actin bundles and invade filopodia.The invasion of filopodia by microtubules is likely thefirst step in directing the movement of intracellularorganelles and cytoplasm during growth cone turning.Significantly, causing asymmetric loss of F-actinfilopodial bundles in growth cones results in micro-tubule redistribution towards the region of the growthcone containing the largest amount of F-actin bundlesand thus turning away from the direction lackingF-actin bundles (Zhou et al., 2002). Thus, the inter-action between F-actin bundles and microtubules is abasic mechanism in growth cone guidance.

The reorganization of microtubules by asymmetricF-actin bundle formation/stabilization may also haveconsequences for asymmetric growth cone motilityobserved during turning (see previous paragraphs).Microtubule dynamics have been shown to regulatecell surface protrusion. In growth cones, attenuationof microtubule dynamics results in impaired lamelli-podial protrusion (Gallo, 1998). Similarly, in non-neuronal cells microtubule tips promote lamellipodialmotility through the activity of the GTPase Rac1(Waterman-Storer et al., 1999), which has a well-established role in lamellipodial formation. Growthcones turn toward a source of the microtubule-stabi-lizing drug taxol, which results in asymmetric redis-tribution of microtubules toward the source of taxol(Buck and Zheng, 2002). Additionally, growth coneturning towards a source of taxol requires the activityof GTPases. Thus, an asymmetric distribution of F-actin bundles across the growth cone is expected toresult in an asymmetry in microtubule-tip-mediatedpromotion of surface motility. These observationsmay explain the asymmetric loss of protrusive activityin the direction opposite to that of growth cone turn-ing described in previous paragraphs. The interactionsbetween microtubules and F-actin bundles are a fron-tier in understanding the mechanism of growth coneguidance.

MODIFICATION OF THE F-ACTINCYTOSKELETON IN RESPONSE TONEGATIVE GUIDANCE CUES

The term “growth cone collapse” describes the loss oflamellipodia and filopodia in response to a negativeguidance cue. For example, following treatment withSemaphorin IIIA (SemaIIIA) sensory growth conescollapse and no longer exhibit protrusive P-domainactivity. When growth cones make filopodial contactwith a localized source of SemaIIIA (e.g., a beadcoated with SemaIIIA) the whole growth cone does

not collapse, just the portion of the growth cone nearthe contacting filopodium (Fan and Raper, 1995).Growth cones have been observed to collapse in vivo(Halloran and Kalil, 1994). However, negative guid-ance cues likely steer growth cones by inducing par-tial or local collapse.

Fan et al. (1993) investigated the effects ofSemaIIIA on the cytoskeleton of growth cones andnoted a loss of F-actin that coincided with growthcone collapse. Similarly, F-actin depolymerization in-duced by pharmacological agents also causes growthcone collapse. Thus, the effects of growth cone col-lapsing cues may be mediated largely by the depoly-merization of F-actin. While the simplicity of thismodel for the mechanism of growth cone collapse isattractive, recent observations indicate that growthcone collapse involves a more complex series ofevents.

In the standard protocol for investigating growthcone collapse cultures are treated with a collapsingreagent (e.g., SemaIIIA), and the percentage of col-lapsed growth cones is determined. While useful, thisapproach does not allow for a careful analysis of theevents underlying growth cone collapse, and addi-tional important information can be obtained fromlive imaging of growth cone responses. For example,the static growth cone collapse assay cannot deter-mine whether growth cones that do not appear col-lapsed maintained protrusive activity after treatmentwith a collapsing agent.

Growth cone collapse is mediated by several sig-naling pathways, including the Rho-GTPases (Liu andStrittmatter, 2001), ADF (Aizawa et al., 2001), andkinases (GSK-3�, FYN, cdk5; Eickholt et al., 2002;Sasaki et al., 2002). Jurney et al. (2002) found thatinhibition of Rac1 activity blocks growth cone col-lapse in response to ephrin-A2. However, whilegrowth cones with decreased Rac1 activity did notcollapse, time-lapse imaging revealed that no furtherextension or retraction of lamellipodia and filopodiaoccurred following treatment with ephrin-A2. Thus,although not collapsed by ephrin-A2, growth conesbecame “frozen” in place. This demonstrates thatgrowth cone collapse is independent of the mainte-nance of growth cone protrusive activity (the exten-sion/retraction of filopodia and lamellipodia). Addi-tionally, ephrin-A2 did not cause growth conecollapse when Rac1 signaling activity was blocked,although a similar amount of F-actin depolymeriza-tion occurred as when growth cones were treated withephrin-A2 alone. These results indicate that growthcone collapse and F-actin depolymerization are inde-pendent processes.

Pfenninger and colleagues (de La Houssaye et al.,


1999) investigated the role of eicosanoids in growthcone collapse in response to thrombin and SemaIIIA.Both growth cone collapsing cues increase eicosanoidproduction through the activity of 12/15-lipoxygen-ase, and generation of eicosanoids is necessary forgrowth cone collapse. Addition of eicosanoids to theculture medium mimics the effects of the collapsingagents. Similar to the findings of Jurney et al. (2002),Mikule et al. (2002) report that growth cones treatedwith SemaIIIA under conditions of inhibited eico-sanoid synthesis remain spread and do not collapse,but F-actin depolymerization is not blocked. Theseresults confirm that growth cone collapse involvesmore than simply F-actin depolymerization.

These results indicate that hypotheses for themechanism of growth cone collapse should be revis-ited. We suggest the hypothesis that growth conecollapse is the result of the activation of several dis-tinct signaling pathways that modify particular growthcone components and that actin filament depolymer-ization is not the primary cause of growth cone col-lapse. Hereafter, we will refer to these cellular effectsas “modules”. Additionally, we propose that the mod-ules activated by negative guidance cues need to beorchestrated in order to bring about collapse, andblockade of one or more of these modules can preventgrowth cone collapse. The question thus becomes:what does it take to collapse a growth cone? Based onpublished observations it is possible to make a “gro-cery list” of the recognized modules involved ingrowth cone collapse in response to guidance cues:F-actin depolymerization; the cessation of protrusiveactivity; F-actin reorganization; loss of attachment tothe substratum; and endocytosis.

Which modules are necessary for growth conecollapse? Based on previous considerations, F-actindepolymerization is not sufficient. The loss of sub-stratum adhesion is unlikely to be necessary for F-actin depolymerization as growth cones that remainadherent to the substratum and do not collapse canstill undergo F-actin depolymerization. Similarly, en-docytosis is not required for F-actin depolymeriza-tion, because blocking ephrin-A2-induced endocyto-sis by inactivating Rac1 does not stop F-actindepolymerization in response to ephrin-A2. F-actinreorganization may be an important component ofgrowth cone collapse. Unlike untreated growth conesin which F-actin is concentrated in filopodia and la-mellipodia of the P-domain, the remaining F-actin incollapsed growth cones is aggregated in the C-domainof the growth cone. Blocking of Rac1 activity pre-vents this reorganization in response to ephrin-A2,and the remaining F-actin is in the form of actinbundles. Zhou and Cohan (2001) showed that F-actin

bundle loss occurs during growth cone collapse, butthat F-actin meshworks are less affected during col-lapse. Thus, reorganization of F-actin may be a re-quired module for growth cone collapse.

Negative guidance cues induce an increase in en-docytosis at the growth cone during collapse(Fournier et al., 2000; Jurney et al., 2002). The in-crease in endocytosis is not due to depolymerizationof F-actin, loss of growth cone morphology, or theinhibition of protrusive activity, because growth conecollapse induced by pharmacological depolymeriza-tion of F-actin does not result in increased endocytoticactivity. It is presently unknown whether endocytosisis of mechanistic relevance to the process of growthcone collapse. A consideration against a direct role forendocytosis in growth cone collapse is that neurotro-phins promote growth cone filopodial and lamellipo-dial extension but also cause increased endocytosis.We suggest that the increase in endocytotic activityduring growth cone collapse may reflect the internal-ization of receptors bound to the collapse-inducingligands (e.g., SemaIIIA or ephrin-A2). Receptor in-ternalization following ligand binding has been wellcharacterized for a number of growth factors includ-ing neurotrophins (Barker et al., 2002), and the inter-nalization may be a required component of guidancecue signaling. Thus, it will be of interest to furtherinvestigate the role of endocytosis in the signaling ofnegative guidance cues and to determine whether it isrequired for the induction of growth cone collapse.

Growth cone collapsing guidance cues decreasesubstratum attachment (de La Houssaye et al., 1999;Mikule et al., 2002). It is unlikely that substratumattachment is required for continued protrusive activ-ity of individual filopodia or lamellipodia, becauseboth structures can extent away from the substratum,indicating independence from substratum attachment.However, substratum attachments must be broken forthe retraction of filopodia and lamellipodia. Thus, lossof substratum attachment may be an important aspectof collapse.

Positive guidance cues promote and polarize pro-trusive activity in the direction of growth cone migra-tion. Conversely, the cessation of protrusive activity islikely to be of fundamental importance to the actionsof negative guidance cues. Thus, asymmetric inhibi-tion of protrusion can direct growth cone migrationaway from a negative guidance cue due to continuedprotrusion on the side of the growth cone unaffectedby the guidance cue. Inhibition of protrusion could bedue to several effects on the cytoskeleton, such as theblockade of F-actin nucleation, changes in polymer-ization rates, or the severing of filaments. To ourknowledge no information is presently available re-


garding aspects of F-actin in collapsed growth cones,such as filament length and orientation, numbers offilament barbed ends available for polymerization, orthe details of filament organization. However, it islikely that filament turnover is altered during growthcone collapse. Aizawa et al. (2001) found that theactivity of ADF, which regulates filament turnover, isrequired for growth cone collapse in response toSemaIIIA. Similarly, Gallo et al. (2002a) report thatthe F-actin present in growth cones collapsed by eph-rin-A2 exhibits slower turnover than F-actin in un-treated growth cones. Thus, although collapsing sig-nals decrease the F-actin content of growth cones, theremaining actin filaments are not as dynamic as inuntreated growth cones.

Collapse is a complex process that affects growthcones at several levels. In the future, it will be impor-tant to identify further modules and to determine thefunctional relationships between modules. Addition-ally, the detailed reorganization of the components ofcollapsed growth cones needs to be further clarified tounderstand the mechanisms responsible for collapse.

MECHANISM OF AXON RETRACTION

Growth cone collapse in response to inhibitory guid-ance cues halts axon extension. However, growthcone collapsing cues can also cause axon retraction.The mechanism of axon retraction is not well under-stood, although it is important during developmentand in the response of axons to injury. For example,during development of the retinal projection, axonsextend to inappropriate targets and many branches aresubsequently pruned to remove incorrect projections(O’Leary, 1992). Following injury axons retract fromthe site of injury (Selzer, 2003). Blocking injury-induced axon retraction may be an important targetfor therapeutic strategies aimed at minimizing damageto the nervous system. Therefore, understanding themechanism of axon retraction has relevance to bothdevelopment and the response of the nervous systemto injury.

Axon retraction could occur through two separate,but not mutually exclusive, mechanisms: depolymer-ization of the axonal cytoskeleton; and the reconfigu-ration and retraction of the cytoskeleton resultingfrom changes in intracellular forces. Recent evidenceindicates that the latter mechanism may be prevalent.Ahmad et al. (2000) found that inhibition of themicrotubule motor dynein caused axon retraction. Re-tracting axons often assume a sinusoidal morphologyand contain a dense, curved bundle of microtubules.Axon retraction in response to the inhibition of dyenin

was dependent on actomyosin contractility, indicatingthat axon extension is regulated by a balance of forcesgenerated by microtubule- and actin-based motor pro-teins. Gallo et al. (2002a) investigated the effects onaxon elongation of blocking F-actin turnover with acell permeable compound called jasplakinolide,which binds to F-actin and prevents filament depoly-merization. Treatment with jasplakinolide resulted inrapid axon retraction, characterized by sinusoidalbending of axons, similar to what was observed byAhmad et al. (2000) following the block of dyeninactivity. Jasplakinolide-induced axon retraction wasfound to depend on the endogenous actomyosin con-tractility in axons. These observations indicate thatF-actin turnover in axons and growth cones preventsendogenous myosin II activity from causing axonretraction. Collectively, these studies indicate thataxon retraction is regulated by the activities of motorproteins that generate forces on the cytoskeleton.

Guidance cues regulate the force generation mech-anisms of axons to induce retraction. Gallo et al.(2002a) found that retinal axon retraction in responseto ephrin-A2 is dependent on RhoA signaling. RhoAregulates myosin II activity in neurons, and introduc-tion of RhoA into neuronlike cells causes axon retrac-tion. He et al. (2002) report that during nitric oxide(NO)-induced axon retraction microtubules are notdepolymerized but undergo reconfiguration, resultingin the formation of sinusoidal bends in the microtu-bule array. As discussed above, the formation of cur-vatures in the axonal array is likely to be a generalfeature of force-mediated axon retraction. These stud-ies demonstrate that guidance cues can regulate theactivity of cytoskeletal motors resulting in the reor-ganization of the cytoskeleton and axon retraction.

The small GTPase Rac1 is also required for axonretraction in response to ephrin-A2 (Jurney et al.,2002). The best characterized function of Rac1 is topromote lamellipodial formation. Paradoxically, Rac1activity is also required for growth cone collapse andaxon retraction in response to some guidance cues. Itwill thus be of interest to further define the pathwaythrough which Rac1 acts during growth cone re-sponses to negative guidance cues.

MODULATION OF GROWTH CONERESPONSES BY THE COMBINEDSIGNALING OF POSITIVE ANDNEGATIVE GUIDANCE CUES

In vivo, growth cones are simultaneously exposed to anumber of different guidance cues. Thus, it is neces-sary to understand how growth cones integrate mul-


tiple, often opposing, signals to reach their targets.Target-derived BDNF promotes the formation of ret-inal ganglion cell axon arbors. Conversely, NO pro-duced by the target cells of retinal axons promotes theretraction of inappropriately targeted axons. BDNFand NO are encountered by retinal axons in vivoduring the same period of development. Therefore, weinvestigated the responses of retinal growth cones tothe combined signaling of BDNF and NO (Ernst et al.,2000). Treatment with NO alone caused growth conecollapse and axon retraction. However, pretreatmentwith BDNF blocked the effects of NO on growthcones and axons. In order to investigate the mecha-nism of the protective effects of BDNF against NO-induced growth cone collapse we tested the role ofcAMP and protein kinase A in mediating the effectsof BDNF (Gallo et al., 2002b). Neurotrophin-inducedcAMP signaling was previously found to be requiredfor the ability of neurotrophins to allow axons toovercome inhibitory myelin-derived signals (Qiu etal., 2002), and cyclic nucleotides have been shown toconvert the actions of guidance cues from attraction torepulsion, and vice-versa (Song et al., 1997, 1998).BDNF induced a rapid, but transient, activation ofPKA signaling in retinal ganglion cells that is requiredfor the establishment of the protective effects ofBDNF against NO-induced growth cone collapse.While PKA signaling is required for the initiation ofthe protective effects of BDNF, it is not required forthe maintenance of the protective effects, as blockingPKA activity after the initial transient increase inactivity did not diminish the protective effects ofBDNF. However, removal of BDNF resulted in thetime-dependent loss of the protective effects ofBDNF. Finally, PKA activation alone did not mimicthe effects of BDNF. Thus, PKA activity in BDNFsignaling acts as a required “switch” to turn on aprotective mechanism that is subsequently maintainedby additional BDNF-activated signaling pathways.

Neurotrophins modulate SemaIIIA-induced growthcone collapse. Dontchev and Letourneau (2002) foundthat the response of sensory growth cones to SemaIIIAwas dependent on the concentration of NGF the neuronswere exposed to prior to treatment with SemaIIIA. Theprotective effects of NGF against SemaIIIA-inducedgrowth cone collapse were partial and depended on theconcentrations of NGF and SemaIIIA. Thus, NGF mod-ulates the responsiveness of growth cones to SemaIIIA.The modulation is not due to decreased cell surfaceexpression of SemaIIIA receptors. Experiments aimed attesting the role of PKA in the effects of NGF andSemaIIIA revealed that PKA activation mimics the pro-tective effects of NGF. Inhibition of PKA signalingreversed the protective effects of NGF. These results

further demonstrate that neurotrophins can modulate theresponsiveness of growth cones to negative guidancecues through PKA-based mechanisms. It will be of in-terest to define the pathways downstream of PKA acti-vation that are responsible for the protective effects ofneurotrophins against negative guidance cues.

FUTURE DIRECTIONS

Although advances have been made in our under-standing of the regulation of actin filaments in growthcones, much remains to be learned about how all theactivities that determine actin filament organizationand function are differentially regulated by guidancecues. Actin filaments are dynamic structures that arenucleated, polymerized or depolymerized, stabilized,and organized on demand to accommodate the direc-tions obtained from extracellular guidance cues. Anumber of fundamental issues require additional in-vestigation in order to further appreciate the complex-ities of growth cone behavior. For example: what arethe mechanisms by which filopodia and lamellipodiaare initiated? How are they selectively stabilized dur-ing guidance? What are the functional relationshipsbetween microtubules and actin filaments? At whatlevel does the regulation of growth cone behavior andthe cytoskeleton by multiple guidance cues occur?What cellular events are required for growth conecollapse in response to guidance cues? Whatmechano-enzymes are involved in growth cone be-haviors and what are their roles? These questions willbe best answered by the use of methods including livevisualization of actin filaments and associated regula-tory proteins in growth cones, biochemical and mo-lecular biological approaches, and selective activa-tion/inhibition of molecular species in living cells(e.g., microCALI). As the growth cone is a dynamicsystem with many inter-related elements, it will be ofgreat importance to simultaneously track multipleevents in growth cones in order to begin to determinethe relationships between cytoskeletal and signalingsystems. A major task for the future will be to inte-grate the emergent knowledge into a comprehensivemodel of growth cone biology.

The authors acknowledge valuable contributions frommembers of the Letourneau laboratory, Scott Gehler, VassilDontchev, Florence Roche, and Eric Veien.

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