Anatomical Science International (2004) 79, 173–183

Blackwell Publishing, Ltd.Special Review Based on a Presentation made at the 16th International Congress of the IFAA

Regulating filopodial dynamics through actin-depolymerizing factor/cofilinJoseph Fass,1 Scott Gehler,2 Patrick Sarmiere,1 Paul Letourneau2 and James R. Bamburg1

1 Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado and 2 Department of Neuroscience, University of Minnesota, Minneapolis, Minnesota, USA

AbstractThe regulation of filopodial dynamics by neurotrophins and other guidance cues plays an integral role ingrowth cone pathfinding. Filopodia are F-actin-based structures that explore the local environment, generateforces and play a role in growth cone translocation. Here, we review recent research showing that theactin-depolymerizing factor (ADF)/cofilin family of proteins mediates changes in the length and number ofgrowth cone filopodia in response to brain-derived neurotrophic factor (BDNF). Although inhibition of myosincontractility also causes filopodial elongation, the elongation in response to BDNF does not occur through amyosin-dependent pathway. Active ADF/cofilin increases the rate of cycling between the monomer andpolymer pools and is critical for the BDNF-induced changes. Thus, we discuss potential mechanisms bywhich ADF/cofilin may affect filopodial initiation and length change via its effects on F-actin dynamics in lightof past research on actin and myosin function in growth cones.

Key words: actin-depolymerizing factor, cofilin, filopodia, growth cone.

Introduction

Neuronal growth cones rely on dynamic filopodiato explore their surroundings, as well as for forcegeneration related to motility (Heidemann et al.,1990). Filopodia are generally short (0.5 to severalµm) membranous protrusions containing a core ofcolinear, bundled F-actin filaments, but also containmyosin and components of focal contacts (Bridgman& Dailey, 1989; Steketee & Tosney, 2002). The F-actinin filopodial bundles is cross-linked with fascin (Cohanet al., 2001) and is very stable, having a half-lifemore than 10-fold longer (approximately 25 min)than lamellipodial F-actin (Mallavarapu & Mitchison,1999).

Regulation of filopodia in neurons is importantfor both growth cone guidance and neurite branchformation, both of which determine the ultimatearborization and connectivity in the nervous system(Goodman & Shatz, 1993; Gallo & Letourneau, 1999).Contact with guidance cues directs both of these

processes; growth cone morphology and the behaviorof its actin and microtubule cytoskeletal structuresis modified downstream of guidance molecules(Gallo et al., 1997; Gallo & Letourneau, 2004; Zhou& Cohan, 2004) and axonal collateral branching(following extensive filopodial sprouting) can resultfrom contact with beads coated with nerve growthfactor (NGF; Gallo & Letourneau, 1998). In addition, den-dritic filopodia are precursors to dendritic spines,post-synaptic structures whose diverse and dynamicmorphology is thought to underlie learning and mem-ory (Sala, 2002). Neurotrophins have been implicatedin modulating the formation and morphology of bothdendritic filopodia and spines (Shimada et al., 1998;Matsutani & Yamamoto, 2004). Furthermore, a pointmutation that impairs secretion of the neurotrophinbrain-derived neurotrophic factor (BDNF) is corre-lated with hippocampal functional impairment inhumans and animals and plasticity impairment inhippocampal cell culture models (Egan et al., 2003;Hariri et al., 2003), providing a potential link betweenfilopodial regulation and adult cognitive function.

Growth cone filopodia explore their local environ-ment via extension, waving and retraction. Whenadhered at their tips, they can exert a pulling force(Heidemann et al., 1990; Smith, 1994; Suter et al.,1998; Suter & Forscher, 2000) that can either moveattached objects (such as beads or other neuriteshafts) rearwards towards the growth cone or pull

Correspondence: James R. Bamburg, Department of Biochemistry & Molecular Biology, 1870 Campus Delivery, Colorado State University, Fort Collins, CO 80523-1870, USA. Email: jbamburg@lamar.colostate.eduPresented at the 16th International Congress of the IFAA, held at the International Conference Hall, Kyoto, 22–27 August 2004.Received 3 September 2004; accepted 13 September 2004.

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material from the growth cone central region antero-gradely, resulting in engorgement of the filopodiumand advance of the growth cone along the path ofthe filopodium (Goldberg & Burmeister, 1986; Smith,1994; Letourneau, 1996). Engorgement of a filopo-dium into a neurite is a proposed mechanism forinteractions with guidepost cells during grasshopperpioneer axon extension (Sabry et al., 1991) and hasbeen observed during interactions between Aplysiagrowth cones and restrained (immobilized) Aplysiacell adhesion molecule (apCAM)-coated beads(Suter et al., 1998), as well as between chick sym-pathetic neuron growth cones and other cells orbeads coated with polyornithine or laminin (Smith,1994). This filopodial behavior seems analogous tothe entire growth cone, in which there is an inverserelationship between retrograde flow and growthcone advance (Lin & Forscher, 1995). This relation-ship has led to a model for forward motility wherebya cellular process will either be retracted at sometime after protrusion or adhere to some stablesubstrate, allowing the same forces that would havecaused retraction to result in forward movement ofmore proximal cellular components. Generation ofpulling force is dependent upon motor proteins (fora review, see Brown & Bridgman, 2004), presumablyisoforms of myosin attached to actin filaments withopposite orientations (Wylie et al., 1998; Bridgmanet al., 2001; Wylie & Chantler, 2001; Bridgman,2002; Brown & Bridgman, 2003b). However, it is asyet unclear which specific isoforms of myosin areinvolved directly in filopodial retraction or, for thatmatter, engorgement.

Nevertheless, filopodial behavior is not simplythe result of motor-based rearrangement of staticactin structures. F-actin throughout the growth coneis constantly turning over, via polymerization at theleading edge, retrograde flow of F-actin and depo-lymerization within the transitional zone (Forscher &Smith, 1988; Lin & Forscher, 1995; Welnhofer et al.,1997; Schaefer et al., 2002). One of the major agentsresponsible for this turnover is the actin-depolymerizingfactor (ADF)/cofilin (AC) family of proteins (reviewedby Sarmiere & Bamburg, 2004). The AC proteinsbind preferentially to F-actin on which the associatedATP has been hydrolyzed to ADP (Carlier et al., 1997)and promote both severing of filaments (Bamburget al., 1999; Ichetovkin et al., 2000, 2002) and depo-lymerization from the pointed end. Once in the mon-omer state, profilin induces a rapid exchange ofATP for ADP on the G-actin and, because AC proteinspreferentially bind ADP–actin monomers, they arefree to recycle to F-actin to repeat their dynamizingeffects (Pollard et al., 2000). Profilin-bound ATP–G-actinreadily participates in barbed-end polymerization;thus, rather than simply promoting depolymerization

of F-actin, active AC proteins can participate inenhancing the rate of cycling, or turnover, betweenG- and F-actin in many systems (Carlier et al., 1997;Lappalainen & Drubin, 1997; Rosenblatt et al., 1997;Theriot, 1997), including within neuronal growth cones(Meberg & Bamburg, 2000).

How might AC proteins be involved specifically infilopodial regulation? Recently, Gehler et al. (2004a)demonstrated that BDNF signals in retinal gangliongrowth cones by inhibiting Rho GTPase. Workingdownstream of Rho, Gehler et al. have found thatBDNF effects on filopodia are mediated throughmodulation of AC protein activity and, although myosinII-dependent alterations in filopodia length can occurin these growth cones, BDNF does not work throughmodulating myosin II activity (Gehler et al., 2004b).These experiments will be reviewed first and we willthen address their potential implications for how ACproteins may function in filopodial initiation, extensionand retraction.

AC regulation of filopodia

Gehler et al. previously demonstrated that BDNFbinding to the p75 neurotrophin receptor (p75NTR)enhances filopodial length by decreasing the activityof RhoA (Gehler et al., 2004a). Their results showedthat several neurotrophins (BDNF, nerve growthfactor (NGF), and neurotrophin (NT)-3) cause increasesin filopodial length in chick retinal, dorsal rootganglion (DRG) and ciliary growth cones (Gehleret al., 2004a). Furthermore, these length changes weremimicked and pre-empted by treatment with eitherof two p75-specific antibodies: CHEX and AB1554.Neurotrophin-induced filopodial length changes werenot observed in growth cones of p75–/– mouse retinalor DRG neurons, yet these growth cones had longerfilopodia, similar to neurotrophin-treated p75+/+ growthcones, implicating unoccupied p75NTR in downregu-lation of filopodial length. Finally, both p75–/– andBDNF-treated neurons displayed reduced levels ofRhoA activity. More recent experiments have dis-sected the signaling downstream of Rho, beginningwith the rho-associated coiled-coil kinase (ROCK),also called Rho kinase (ROK) or Rho-associatedkinase (Gehler et al., 2004b).

Rho-associated kinase is well characterized asan upregulator of myosin II-dependent contractilityin non-muscle cells (e.g. Hirose et al., 1998) that actsthrough either direct activation of myosin light chainkinase or inhibition of the phosphatase that removesthe phosphate from the light chain (Amano et al.,1996; Kimura et al., 1996). In addition, ROCK regu-lates AC activity via the activation of specific kinases,namely LIMK1 and 2 (named for the proteins lin-11,islet-1 and Mec-3, which contain similar LIM domains)

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and testicular kinase (TESK) 1 and 2, which phos-phorylate and inactivate AC proteins at serine 3(Arber et al., 1998; Yang et al., 1998; Toshima et al.,2001a, 2001b). The dephosphorylation (activation)of AC is regulated both by general phosphatases(Meberg et al., 1998; Samstag & Nebl, 2003) andmore specific phosphatases, such as those of theslingshot family, of which six isoforms have beenidentified in mammalian cells (Niwa et al., 2002;Endo et al., 2003; Ohta et al., 2003; Nagata-Ohashiet al., 2004). Although the mechanisms regulatingthe slingshot phosphatases are not all known, one ofthese isoforms, namely slingshot-1L (SSH-1L), requiresbinding to F-actin for activity (Ohta et al., 2003;Nagata-Ohashi et al., 2004) and is also phosphor-ylated and inactivated by p21-activated kinase 4(PAK4; J. Soosairajah et al., unpubl. obs., 2004) andother as yet unidentified kinases. In addition, thedephosphorylation of AC is regulated by its bindingto members of the 14-3-3 family of phosphoprotein-binding partners (Gohla & Bokoch, 2002). BothLIMK1 and slingshot also bind 14-3-3 family mem-bers and because the consequences of theseinteractions are not well understood, the effects ofthe Rho signaling pathway on AC activity may be quitecomplex. However, a summary of the known regula-tory pathways is given in Fig. 1.

Gehler et al. (2004b) treated cultured retinalganglion neurons with a number of inhibitors or loadedthe neurons with purified proteins using the Chariot™reagent (ActiveMotif, Carlsbad, CA, USA). Inhibitorsincluded the ROCK inhibitors Y-27632, HA-1077 andH-1152 and blebbistatin, a small molecule inhibitorof myosin II (Straight et al., 2003). Proteins loaded

included the AC-binding scaffolding protein 14-3-3!and several mutants of Xenopus AC (XAC): a non-severing mutant XAC KK95,96QQ (KKQQ), a non-phosphorylatable and, therefore, constitutively activeXAC(S3A) (XACA3) and a pseudophosphorylatedand, therefore, less active XAC(S3E) (XACE3), whichacts in a dominant negative manner in polarizationof cultured fibroblasts (Dawe et al., 2003).

The BDNF-induced increase in growth conefilopodial length (Gehler et al., 2004a, 2004b) couldbe mimicked by ROCK inhibition or introduction ofXACA3 and blocked by introduction of XACE3 orthe non-severing XAC KKQQ mutants or 14-3-3!. Nofurther increases in filopodial length were observedwhen BDNF was used to treat cells pretreated witha ROCK inhibitor or preloaded with XACA3. Immu-nostaining specific for inactive (phosphorylated) ACrevealed similar reductions throughout the growthcone after BDNF or ROCK inhibitor treatment; thesereductions were blocked in cells loaded with 14-3-3!, suggesting 14-3-3! prevented the filopodiallength increase through its ability to inhibit activationof endogenous AC. Finally, slingshot immunostainingwas punctate throughout the growth cone and, insome cases, was observed along and at the tips offilopodia, although its involvement in BDNF signalingwas not assessed directly.

Gehler et al. also assessed ROCK and XACeffects on filopodial number in the same way, but theresults were somewhat more ambiguous (Gehleret al., 2004b). Inhibition of ROCK and loading withXACA3 mostly recapitulated BDNF-induced increasesin filopodial number, but further increases wereobserved when these treatments were combined with

Figure 1. Schematic representation of theregulatory interactions between several actinregulatory proteins and the actin polymerization/depolymerization cycle. p21-activated kinase4 (PAK4) activates (phosphorylates) LIMkinase 1 (LIMK1) and inactivates (phos-phorylates) slingshot-1L (SSH-1L) on a siteother than the C-terminal tail domain (J.Soosairajah et al., unpubl. obs., 2004).Slingshot inactivates (dephosphorylates)LIMK1 and activates (dephosphorylates)actin-depolymerizing factor (ADF)/cofilin (AC),resulting in a net increase in AC activity andactin filament turnover. The other players inthis pathway are F-actin and 14-3-3!. F-Actinis necessary for slingshot activity; theinteraction of SSH-1L, which has beenphosphorylated in its C-terminal tail domain by an unidentified kinase, and F-actin is inhibited by 14-3-3" and 14-3-3! (Nagata-Ohashiet al., 2004; J. Soosairajah et al., unpubl. obs., 2004). Stimulatory interactions (arrows) and inhibitory interactions (blocked lines) areshown. Active AC increases F-actin turnover rate by binding preferentially to ADP–actin subunits (white) as opposed to ADP·Pi-actin(gray) and ATP-actin subunits (black). Bound AC increases F-actin severing and pointed end depolymerization. Free ADP–G-actinmonomers can exchange their bound nucleotide (enhanced by profilin) and engage in polymerization (predominantly at the barbedend).

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BDNF. Similarly, XACE3 loading did not completelyblock the effects of BDNF treatment on filopodialnumber, whereas loading of XAC KKQQ or 14-3-3!did. These inconsistencies may be the result ofunequal protein loading, partial activity of XACE3 orthe presence of endogenous AC proteins. However,taken together, these results suggest that whereasBDNF controls filopodial length solely through ACregulation, the effects of BDNF on filopodial numberare only partially AC dependent and may involve anAC-independent pathway.

Given the downregulation of Rho and ROCK byBDNF, another pathway through which filopodialelongation and numbers could be affected is throughmodulation of myosin II activity. To investigate therole of myosin II, Gehler et al. (2004b) used bleb-bistatin, an inhibitor of non-muscle myosin II, bothalone and in concert with the various treatmentsalready described. Treatment of growth cones withblebbistatin resulted in concentration-dependentincreases in filopodial length and number but, over-all, the morphological responses differed from thoseseen following BDNF treatment. Time-lapse imagingrevealed that 20 µmol/L blebbistatin increasedfilopodial extension rates, while gradually reducingretraction rates. Paradoxically, blebbistatin alsoreduced the fraction of time filopodia spent extend-ing or retracting. In addition, blebbistatin treatmentcaused lamellipodial retraction, as well as filopodialdetachment, bending and buckling. In contrast,BDNF treatment did not result in lamellipodial retrac-tion and filopodia remained substrate bound. Treat-ment with BDNF resulted in enhanced filopodialextension rates, and no change in either retractionrates or in the fraction of time filopodia spent extend-ing or retracting.

Further evidence that the effects of BDNF onfilopodial length are not mediated via myosin II camefrom experiments combining blebbistatin treatmentwith BDNF, ROCK inhibitors and XAC mutants(Gehler et al., 2004b). Blebbistatin (20 µmol/L) causedadditive increases in filopodial length when usedin concert with BDNF, ROCK inhibitor or XACA3loading, whereas XACE3 loading did not blockblebbistatin-induced increases in filopodial length.Blebbistatin did not cause changes in filopodial numberat concentrations lower than 50 µmol/L, at whichadditivity with other treatments was assessed. Thus,results from Gehler et al. (2004b) suggest that BDNFacts to regulate growth cone filopodia independentlyof myosin II contractility.

One major question arising from the resultsdescribed above is: what direct effect is increasedAC activity having on growth cone F-actin? Recentwork by Condeelis and colleagues has shown thatactivated cofilin and Arp2/3 act in concert to

increase the number of free barbed ends, thus allow-ing a burst of actin polymerization that drives lamel-lipodial protrusion and can thereby determine thedirection of lamellipodia-driven migration (Ichetovkinet al., 2002; DesMarais et al., 2004; Ghosh et al.,2004). The finding of Gehler et al. (2004b) that thenon-severing KKQQ mutant blocked BDNF-inducedchanges as well as the XACE3 mutant suggests thatsevering is also important in filopodial length andnumber regulation and, on a qualitative basis, BDNFtreatment was seen to induce an increase in lamel-lipodial area (S. Gehler & P. Letourneau, unpubl.obs., 2004). However, it is not known whether BDNFinduced bulk polymerization or depolymerization inthe experiments of Gehler et al. (2004b).

Whereas Gehler et al. (2004b) applied BDNF insolution, it seems likely that growth cones wouldencounter more localized sources of neurotrophinsduring development. However, even in the case ofhomogeneous activation, Gehler et al. did not exam-ine whether AC proteins are differentially activatedor targeted to different compartments of the growthcone or F-actin populations. Phosphorylated ACstaining appears fairly homogeneous throughout thegrowth cone (once thickness is accounted for; seeFig. 2A) and does not disappear preferentially fromany particular region after exposure to BDNF. How-ever, simultaneous tracking of both total AC proteinsand their activation state is required in order toascertain whether localized activation of AC proteinsis occurring in response to even homogeneousexposure to BDNF. In addition, there may be othermechanisms localizing the effects of AC in thegrowth cone, such as protection of some fraction ofthe F-actin population by tropomyosin (DesMarais etal., 2002).

Nevertheless, the work of Gehler et al. (2004b)strongly suggests that BDNF regulates filopodial lengthsolely through regulation of AC protein activation,whereas filopodial number may involve additionalsignaling pathways. However, it seems unlikely(although not implausible) that length and number offilopodia are regulated directly (i.e. that there is adirect feedback pathway from some sensor of lengthor number to a mechanism for controlling length ornumber). Rather, we hypothesize that the numberand length of filopodia present at any time dependson several mechanisms: initiation, extension andretraction. Thus, we ask which of the processescausing filopodia to arise or change in length maybe affected by AC regulation.

Initiation

Data from melanoma cells suggest membrane-boundenabled/vasodilator-stimulated phosphoprotein (Ena/

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VASP) family proteins accumulate in a specific areaof the leading edge membrane, where they competewith capping proteins for barbed ends of actinfilaments in the lamellar meshwork and, thus, allowlocalized elongation and consolidation (throughbundling of the elongating filaments; e.g. by fascin)of the affected filaments, a process called conver-gent elongation (Svitkina et al., 2003). Svitkina et al.(2003) termed the initial structures resulting fromthis process ‘#-precursors’ due to the arrowhead-shaped concentration of local actin filaments(Fig. 3). The #-precursors have not yet beenobserved in growth cones and growth cones differfrom migrating cells in important aspects, such asthe distribution of Arp2/3, which is enriched in theperipheral ruffling lamella of fibroblasts but, in growthcones, is enriched in the central domain (Strasser etal., 2004). However, there is evidence to suggest thatthe convergent elongation model may extend toneurite growth cones, where Ena/VASP is locatedat the tips of filopodia and is essential for filopodialformation in response to Netrin-1 (Lebrand et al., 2004).

Another proposed mechanism for growth conefilopodial initiation involves actin filament nucleationfrom growth cone structures called focal rings(Steketee et al., 2001). These structures are foundat the bases of filopodia and may be associated withdefining the site of the initial nub, which developsinto a filopodium. Convergent elongation and focalring-dependent filopodial initiation are not mutuallyexclusive mechanisms, because focal rings could

precede the formation of localized Ena/VASP patchesor form as a consequence.

Regardless, there are no data concerning thedependence of either of these hypothesizedmechanisms on the rate of actin turnover, whichis increased in growth cones expressing XAC(wt)(Meberg & Bamburg, 2000) and is presumablyincreased as a result of the AC activation followingBDNF treatment and ROCK inhibition and decreasedfollowing loading with XACE3 or KKQQ mutants. Inthe case of the convergent elongation hypothesis,one may imagine that more rapid turnover may allowmore frequent convergence of peripheral actinfilaments to form #-precursor structures that eithermeet membrane-associated concentrations of initia-tion factors, such as Ena/VASP, or bring togetherbarbed-end associated proteins that then further theinitiation process.

Extension

Once nascent filopodia have grown beyond the#-precursor or bud stage, what determines theirultimate length or, rather, the length distribution of allthe filopodia on a growth cone at a certain timepoint? G-Actin adds to the tips of filopodia (Okabe& Hirokawa, 1991), where bundled F-actin filamentsare thought to cause membrane protrusion by anelastic Brownian ratchet mechanism (Mogilner &Oster, 2003; Upadhyaya & van Oudenaarden, 2004).By observing photobleached or photoactivated marks

Figure 2. Brain-derived neurotrophic factor (BDNF)-induced reduction of phosphorlyated actin-depolymerizing factor (ADF)/cofilin(pAC) levels in retinal growth cones. Treatment with BDNF for up to 30 min resulted in a progressive reduction in the intensity of pACstaining (A,C,E,G). Growth cones were counterstained with Alexa Fluor 568-phalloidin (B,D,F,H).

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on filopodial actin bundles, Mallavarapu and Mitchison(1999) revealed two distinct, seemingly independentmechanisms of control over filopodial length versustime. They determined that both retraction rate (indi-cated by retrograde motion of the fiduciary marks)and elongation rate (indicated by an increase in therelative distance between a fiduciary mark and theend of the filopodium) governed changes in the posi-tion of the filopodium tip (i.e. total filopodial length,assuming lack of lamellipodial advance or retraction).Their data also suggested that there was a strongercorrelation between length change and elongation(polymerization) rate than between length changesand retraction rates. However, these studies wereperformed in untreated growth cones; drug or growthfactors could, potentially, shift dominance betweenthese two processes.

The BDNF-induced filopodial length increasesreported by Gehler et al. (2004b) could have beendue to either a reduction in retraction rate or anincrease in polymerization. One possibility is thatincreased AC activity may lead to an increased G-actin concentration, but this is not necessarily thecase. However, DesMarais et al. (2004) found thatby blocking either Arp2/3 or cofilin function theycould prevent an increase in free barbed ends andconcomitant lamellipodial protrusion in rat adenocar-cinoma cells. Conceivably, activation of cofilin (and/or ADF) leads to a greater prevalence of severingand then minus-end depolymerization, whereas acti-vation of both cofilin and Arp2/3 leads, more often,to severing and then protection of the free pointedends, limiting minus-end depolymerization andpreserving free barbed ends. The former would cause

Figure 3. Platinum replica electron microscopy (top) of the leading edge of a mouse melanoma B16F1 cell showing a filopodium(arrow) and a #-precursor (arrowhead). Some filaments converging to the vertex of the #-precursor are highlighted in cyan.Fluorescence images of the same cell expressing a green fluorescent protein (GFP)-fascin chimera (green) costained withrhodamine-phalloidin (red) are shown at the bottom. The GFP-fascin is accumulated in the mature filopodium, but not in the #-precursor. A boxed region from a lower magnification view (lower left) is enlarged in the lower right panel; the electron micrograph(top) depicts a region corresponding to the boxed area in the light microscopic image at the lower right. Bar, 250 nm. (Imagescourtesy of T. M. Svitkina, University of Pennsylvania.)

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an overall shift from F-actin to G-actin, whereas thelatter would cause the reverse. Thus, the BDNF-mediated increase in AC activity alone may have ledto increased turnover as well as somewhat elevatedG-actin levels, which then increased the rate ofpolymerization at filopodial tips.

Alternatively, AC activity could ‘chop up’ the actinmeshwork surrounding the base of a filopodium.Presumably it is myosin-generated tension producedbetween this actin meshwork and the filaments ofa filopodial actin bundle that results in retrogrademotion of that filopodium (Jay, 2000) and a highlysevered actin meshwork with less structural integritymay serve as a poorer base for tension generation.Less tension leads to less retrograde flow and,thus, with even a constant rate of polymerization atfilopodial tips, filopodial length would increase. If thisis the mechanism underlying increased filopodiallength, it is evidently not entirely effective; as shownin Fig. 2, there is no marked decrease in F-actindensity evident after BDNF treatment, during the sametime that filopodial length is increasing. Thus, it isreasonable that inhibition of myosin II via blebbistatinwould further decrease the rate of retrograde ‘reelingin’ of filopodia, further increasing length.

Retraction

As mentioned above, we must interpret the resultsof Gehler et al. (2004b) on filopodial retraction andextension with caution; these may be the result of acombination of changes in polymerization rate at thetip and retrograde ‘reeling in’ of the filopodial actinbundle with respect to the substrate (and growthcone). However, although BDNF does signal throughROCK (Gehler et al., 2004b), which is known to affectmyosin activity, there seems to be compensatorysignaling to limit the effects of BDNF to AC proteins.We are led to this conclusion by the finding of Gehleret al., (2004b) that enhancing AC activity (whichshould not affect myosin activation and, thus,retrograde F-actin flow) was sufficient by itself toreproduce the filopodial changes caused by BDNFsignaling.

The balance of forces and relative motions ofgrowth cone cytoskeletal structures during differentbehaviors is poorly understood. Certainly, tensiongeneration within growth cones is important, becausetreatment with the broader-acting and less-specificmyosin inhibitor 2,3-butanedione monoxime (BDM)retards retrograde flow and leads to increasedoutgrowth (Lin et al., 1996). Retrograde flow occursin the actin meshwork of lamellipodia (Forscher &Smith, 1988; Welnhofer et al., 1997) and filopodia(Mallavarapu & Mitchison, 1999) and must, ultimately,result from motor-based tension generation between

surface adhesions and F-actin within the growthcone or neurite shaft. Filopodia can also form surfaceadhesions and it appears that these adhesionsserve different functions (including regulation oflamellipodial veil advance between adjacent filopo-dia) according to their locations along a filopodium(Steketee & Tosney, 2002). The idea that filopodiaspecifically, and the growth cone in general, caneither retrogradely pull in protrusions (filopodia orlamellipodial) or grab hold of substrate adhesionsand generate tension leading to growth cone advanceis known as the clutch hypothesis (Mitchison &Kirschner, 1988; Jay, 2000), but the locations anddynamics of ‘clutching’ (i.e. the force distribution overtime within growth cones) are not well characterized.

Many classes of non-muscle myosins are foundin neuronal growth cones and their roles in neuriteoutgrowth and growth cone motility have beenreviewed recently (Brown & Bridgman, 2003a; Brown& Bridgman, 2004). Briefly, although myosin II hasbeen the best studied of they myosin classes,there is still controversy over the roles of its specificisoforms (Brown & Bridgman, 2003a). Myosin IIbappears be responsible for retrograde flow of F-actinin growth cone lamellipodia and myosin IIa may func-tion redundantly. In addition, myosin IIa may havea role in stabilizing nascent focal contacts (Wylie& Chantler, 2001). Myosin 1B (MYO1B) and D arepresent in neurons, but have not been well studied,whereas there is some controversial evidence thatMYO1C (and not myosin II) is responsible for retro-grade flow (Diefenbach et al., 2002). Although someevidence initially suggested that myosin V resistsfilopodial retraction (Wang et al., 1996), other studieshave refuted this and, instead, implicated myosin Vain organelle transport (Evans et al., 1997); myosinVb and c are not well studied in the nervous system.Myosin VI is also found in growth cones, but its physiol-ogical function has not been well studied.

Blebbistatin, a small molecule inhibitor of myosinII (Cheung et al., 2002) that works by indirectly inter-fering with actin binding (Kovacs et al., 2004), wasrecently assessed against non-muscle myosins andfound to inhibit human non-muscle myosin II but nothuman myosins Ib, Va and X (Straight et al., 2003).Thus, myosin VI cannot be ruled out as a possiblemediator of blebbistatin-induced filopodial changesobserved by Gehler et al. (2004b). The observationthat blebbistatin increased filopodial length impliesa reduction of retrograde flow of filopodial F-actinbundles, by inhibition of myosin IIa or b. Meanwhile,Gehler et al. (2004b) reported that filopodia detach-ment and ‘bending and buckling’ increased inresponse to blebbistatin treatment. Detachment mayhave been a result of loss of focal contact stabilizationvia myosin IIa inhibition, whereas the bending and

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buckling behavior suggests continued activity ofsome myosin class within the filopodia (perhapsmyosin VI). Alternatively, this behavior may have sim-ply been an effect of random variation of myosin IIinhibition.

Differential regulation of AC proteins in turning behavior

Could differential AC regulation across the growthcone be a mechanism of achieving directional guid-ance? It has been observed previously that filopodialextension and retraction rates vary across growthcones (Bray & Chapman, 1985; Mallavarapu &Mitchison, 1999) and that calcium signaling canresult from contacts experienced by individualfilopodia (Gomez et al., 2001). The AC proteins canbe regulated downstream of intracellular calcium(Meberg, 2000). In addition, individual filopodia canrespond to contact with guidance cues (Smith, 1994;Gallo & Letourneau, 2004) and filopodial sproutinghas been shown to be necessary for turning inresponse to a guidance cue (Zheng et al., 1996).This suggests a potential link between AC activity,filopodia and turning and, indeed, preliminary datasuggest that AC activity is increased concomitantlywith filopodial sprouting on the far side of a growthcone turning away from a negative guidance cue(Fig. 4).

Alternatively, a recent study found that growthcone turning on a uniform substrate was significantlycorrelated with lamellipodial size and not with filopo-dial length or number (Wang et al., 2003). Further-more, preferential laser inactivation on one side ofthe growth cone of myosin Ic, but not myosin V,interfered with turning behavior. Although this studysuggests a dominant role for both motor activationand lamellipodial area in growth cone turning, theturning was not a result of guidance cue signaling.Thus, it remains to be seen whether turning depends onasymmetric motor protein activation or lamellipodialprotrusion during physiologically relevant guidancebehavior.

Conclusions

Filopodia are dynamic structures and similar distri-butions of lengths and numbers of filopodia couldresult from different regulatory processes. In order tomore fully understand the regulation of filopodialbehavior, lifetime analysis of individual filopodia (i.e.‘birth’ and ‘death’ times, length histories, locations)should be used to characterize the responses todifferent guidance cues.

The results of Gehler et al. (2004b) document therole of AC in the regulation of the length and numberof growth cone filopodia; however, the mechanismsunderlying this control remain unclear. Activation ofAC may increase filopodial length either by increas-ing the G-actin pool, thus enhancing polymerizationat filopodial tips, or by modifying the actin meshworkwithin the transitional zone, making motor-basedretraction of filopodial F-actin bundles less efficient.Filopodial number may be regulated by AC proteinsvia enhancement of convergent elongation of lamel-lipodial F-actin filaments underlying the plasma mem-brane, thus making filopodial initiation more frequent.However, this mechanism has not yet been demon-strated fully in neurons. Further studies that use pho-tobleaching, photoactivation or speckle microscopyto track F-actin within filopodia and lamellipodia willbe helpful in pinning down the mechanism by whichAC regulates filopodial length and number.

Acknowledgments

We gratefully acknowledge grant support from theNational Institutes of Health (NIH; NS43115, J.N.F.;GM35126, NS40371, J.R.B.; HD19950, P.C.L.), theAlzheimer’s Association (IIRG-01–2730, J.R.B.), theChristopher Reeve Paralysis Foundation (SB-0110–2,P.D.S.), the National Science Foundation (NSF;IBN0080932, P.C.L.) and the Minnesota MedicalFoundation (P.C.L.). S.G. is a trainee on an NIH EyeInstitute Training Grant. The authors thank Tatyana

Figure 4. Ratio imaging of total cofilin to phosphorylated actin-depolymerizing factor (ADF)/cofilin (AC) in neuronal growthcones. The upper panels show a typical growth cone in theabsence of any externally applied guidance cue. The white/redcolors show the region of highest AC activity (lowest levels ofphosphorylated AC) normalized for every growth cone to aregion in the distal neurite shaft where each immunofluorescentimage is adjusted to the same intensity (to correct fordifferences between antibody affinities). Bottom panels showa growth cone turning away from an aggrecan stripe, whichcontains rhodamine dextran for visualization; there is anaccumulation of the active AC away from the repulsiveguidance cue. (P.D. Sarmiere, unpubl. data., 2004).

ADF/cofilin and filopodial dynamics 181

M. Svitkina, University of Pennsylvania, for kindlyproviding unpublished data, and Chi Pak, ColoradoState University, for helpful discussions.

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