new players in actin polymerization – wh2-domain-containing actin nucleators

New players in actin polymerization –WH2-domain-containing actinnucleatorsBritta Qualmann and Michael M. Kessels

Institute for Biochemistry I, Friedrich-Schiller-University Jena, Nonnenplan 2, 07743 Jena, Germany

Review

Actin nucleators promote the polymerization of thedifferent types of actin arrays formed in a variety ofcellular processes, such as cell migration, cellularmorphogenesis and membrane trafficking processes.Several novel nucleators have been discovered recently.They all contain Wiskott-Aldrich syndrome protein(WASP) homology 2 (WH2 or W) domains for actinnucleation but seem to employ different molecularmechanisms and serve distinct cellular functions. Here,we summarize what is currently known about the differ-ent molecular mechanisms that Spire, Cordon-Bleu andLeiomodin seem to use and, also, the bacterial counter-parts that mimic them (VopF, VopL and TARP). Recentstudies on these WH2 proteins offer unique insight intothe biological problem of actin-filament formation andhow cells use specialized molecular machines to bringabout so many different cytoskeletal structures.

Actin nucleationThe wealth of cellular processes that the actin cytoskeletonis involved in includes cell polarity establishment andmaintenance, cell migration, cell division, cellularadhesion, and the formation of specialized cellmorphologies and tissue architectures. It has long beenrecognized that actin filaments and their turnover (Box 1)are crucial for life in eukaryotes. How cells can generatefilaments in the first place, however, has been amystery fordecades. Examinations of actin assembly in vitro showedthat spontaneous formation of actin nuclei from pure actinmonomers is by orders of magnitude too slow comparedwith rates observed during cellular responses [1].

Considering the structure of actin filaments, nuclei canbe formed in different ways (Figure 1). For the first step,there are already two possibilities, formation of cross-fila-ment or longitudinal dimers. Calculations of associationand dissociation rate constants and binding energiesstrongly suggest that longitudinal dimers are much morelikely [2] (Figure 1). Because even theKd of this muchmorefavorable reaction still is 4.6 M however, this is clearly anobstacle that is hard to overcome spontaneously. Trimerformation (Kd 0.6 mM) is still problematic. Only tetramerformation and further polymerization reactions finallyexhibit Kd values, permitting spontaneous and fast pro-cedures (0.14 mM and 0.12 mM, respectively) [2].

Corresponding author: Kessels, M.M. ([email protected]).

276 0962-8924/$ – see front matter � 2009 El

Today we know that cells express nucleation-promotingfactors to overcome the kinetic barrier of actin nucleation.The first nucleator discovered was the Arp2/3 complex [1](Figure 2). Two actin-related proteins (Arp) assembled inthis complex provide a surface for the addition of actinmonomers (Box 2). Subsequently, formins were recognizedas actin nucleators, too [1] (Figure 2; Box 2). Now, a set ofrecent papers reveal a third group of nucleators comprisingCordon-bleu (Cobl) [3], Leiomodin (Lmod-2) [4] and Spireproteins (for a review, see Ref. [5]) (Figure 3). Here, wesummarize the current work on these new players toprovide an overview of this exciting emerging field.

Whereas a recent review covering Spire and Coblfocused on the structural details of WH2-domain-mediatedactin binding (Box 3) and modulation of actin dynamics,and extensively discussed biophysical and structural con-siderations that could lead to designs of artificial nuclea-tors [6], this review aims to provide an overview of all therecently discovered WH2-domain-based nucleators, themolecular mechanisms they seem to use and the cellbiological processes they are involved in.

The cell biological importance of WH2-domain-mediated actin nucleation is further strengthened byrecent studies revealing that certain pathogenic bacteriaabuse this powerful mechanism and make their ownWH2-domain-based nucleators to highjack actin cytoskeletalcomponents from eukaryotic host cells. Eukaryotic cellbiology, and potentially also medicine, has much to learnfrom the WH2-domain-based nucleation mechanisms usedby microbial pathogens to effectively abuse cytoskeletalfunctions of our body.

Spire proteinsSpire proteins have been identified in Drosophila andhigher eukaryotes. Mammals exhibit two isoforms.Spire-1 is preferentially expressed in the nervous system[7]. Spire-2 mRNA is present in the nervous system, diges-tive tract, liver and testis (E. Kerkhoff, personal communi-cation). Spire mutants were first discovered in a screen forfemale sterile Drosophila mutants. Mutations in spireresult in premature cytoplasmic streaming in oocytes,disrupting proper establishment of anterior–posteriorand dorsal–ventral body axes [8]. Premature streamingis also observed in cappuccino (fly formin) and chickadee(fly profilin) mutants [8,9]. Because treatment with cyto-chalasin D (an inhibitor of actin dynamics) causes a similarphenotype [8,9], an intact actin cytoskeleton is required to

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Box 1. Actin dynamics

Remodeling of the actin cytoskeleton is crucial for life. Reagents that

stabilize actin filaments and prevent the replenishing of the actin

monomer (G-actin) pool are toxic. However, substances such as

latrunculins or cytochalasins that disturb actin-filament formation

and thus shift the balance of monomeric and filamentous actin (F-

actin) in favor of monomers are also potent toxins.

De novo formation of actin filaments requires formation of an

actin nucleus, onto which further monomers can then sponta-

neously polymerize (Figure 1). In vitro, polymerization proceeds

until almost all G-actin is consumed. The system then reaches

steady state, a situation marked by balanced assembly and

disassembly. Assembly and disassembly are hereby asymmetrically

distributed: ATP-loaded actin monomers are preferentially added to

the so-called ‘barbed’ (plus) end because, at this end, the minimal

concentration required for net addition of monomers (the critical

concentration) is lower than at the so-called pointed (minus) end. As

filaments age, the ATPase actin hydrolyzes the bound ATP,

eventually releases the phosphate and remains as ADP-actin. As

the growing barbed end is marked by newly added ATP-actin, actin

filaments are not only polar owing to the structural asymmetry of

monomers they are built of, but also owing to the different

nucleotide status of monomers incorporated into a filament. Both

aspects contribute to the fact that the pointed end is marked by a

higher critical concentration for monomer addition. Under steady

state conditions, depolymerization at the pointed ends thus

replenishes G-actin for polymerization at the barbed ends, enabling

the filaments to perform so-called ‘treadmilling’. The dissociated

ADP-actin then needs to be recharged with ATP for a new round of

polymerization.

In vivo, all steps in actin dynamics are controlled and modulated

by actin-binding proteins. ADP-actin stretches within filaments are

preferred targets for different disassembly-promoting proteins.

Efficient exchange of ADP for ATP is ensured by the small G-actin-

binding protein profilin. Proteins, such as thymosin b4, bind to actin

monomers and thereby control the pool of non-filamentous actin in

cells. Events known as ‘capping’ can specifically stabilize the

pointed or the barbed end. Whereas at the pointed end, capping

serves as protection from depolymerization, capping proteins for

the barbed end, such as CapZ, are important for stopping actin

polymerization reactions and therefore prevent excessive actin

filament polymerization.

Figure 1. Sequence of events during spontaneous actin polymerization.

Calculations of the dissociation constants (according to Sept and McCammon

[2]) for the different steps towards formation of an actin nucleus (i.e. of a complex

of actin competent for rapid filament polymerization). Formation of dimers and

trimers is unfavorable but once a cross-filament trimer is formed, addition of a

forth actin molecule (polymerization) is accelerated by more than three orders of

magnitude. Bold arrows indicate the preferred nucleation pathway accounting for

99.7% of polymer formed upon spontaneous actin self-assembly.

Review Trends in Cell Biology Vol.19 No.6

suppress premature microtubule-based cytoplasmicstreaming. Recent examinations suggest that a cyto-plasmic actin mesh, which is assembled by Cappuccinoand Spire, helps to maintain microtubule organizationand/or to tether the vesicles, the kinesin-driven movementof which would otherwise start cytoplasmic streaming [10].A similar actin mesh that is important for spindle position-ing during asymmetric partitioning of the cytoplasm wasrecently described for vertebrate oocytes [11–13].

It is appealing to speculate that, in vertebrates too,Spire proteins are important in the female germline. Xeno-pus Spire-2 (pEg6) is a maternal gene detected only inunfertilized eggs and during oogenesis and very earlystages of embryogenesis [14]. Formin-2 (a mammalianhomologue of Cappuccino), which was identified to interactwith Spire [15,16] and seems to be part of the Spire actin-nucleation machinery (see later) [15], is important foroogenesis and female fertility as well [17]. Formin-2 iscrucial for the formation of dynamic actin filaments associ-ated with spindle and chromosome positioning duringmeiosis [11–13,17,18].

That Spire itself is a nucleator and works closely withCappuccino (fly formin) was revealed in two studies inDrosophila [15,19], which in part were corroborated by

human Spire-1 data [15,20]. Spire proteins contain fourcentral WH2 domains (Figure 3a), small G-actin-bindingmotifs found in many proteins (Box 3). Although highconcentrations of the Drosophila Spire N-terminal half(NT) were required for considerable nucleation, and fila-ment formation rates remained far below of that of acti-vated Arp2/3 complex, the activity of Spire NT wassurprisingly resistant against various deletions of WH2domains or linkers. [19]. The highest potential for nuclea-tion seems to lay in WH2 domains 3 and 4 and the actin-binding linker L3 between them [19].

Spire proteins have the potential to form a string of fouractin monomers. Indeed, Spire complexes with four actinswere formed in efficient and cooperative association reac-tions, were stable, and had an elongated shape [19,20].These findings are in line with the linkers between theWH2 domains being so short that – for sterical reasons –

they can exclusively accommodate assembly of linear actinarrays belonging to one strand of the filament. Addition ofthe first actin monomer belonging to the second actinstrand within the filament would have to occur spon-taneously (Figure 3b). This step would resemble the secondstep in spontaneous actin nucleation, which – with acalculated Kd of 600 mM – is still very unfavorable [2](Figure 1). This could be one reason for the pronouncedlag phases observed in the kinetics of Drosophila Spire-mediated actin nucleation [19].

Bosch et al. [20] demanded that owing to a steric clash ofthe WH2 domain with the D-loop of the consecutive actin

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Figure 2. Actin nucleation by the Arp2/3 complex and formins. (a) The Arp2/3

complex contains the actin-related proteins Arp2 and Arp3, mimicking an actin

dimer when activated by members of the WASP superfamily of proteins (N-WASP

is depicted). WASP superfamily proteins bind to G-actin by means of one or two

WH2 domain(s) and recruit profilin–actin complexes associating with their proline-

rich domain (PRD). (b) Formins nucleate via their dimerized doughnut-shaped

formin-homology 2 (FH2) domain, to which profilin–actin is recruited by binding to

the neighboring proline-rich FH1 domain. During elongation, formins move

processively along with the barbed end. ‘+’ indicates plus ends and ‘�’ indicates

minus ends.

Box 2. The established nucleators Arp2/3 complex and

formins

The Arp2/3 complex was the first actin nucleator discovered. It

contains two name-giving actin-related proteins held together by

five further subunits [46] forming a template for actin nucleation and

thereby for efficient filament formation. The complex requires

activation by further components, the most prominent of which

are members of the WASP superfamily [1,47–50]. Once activated,

Arp2 and Arp3 are arranged in a manner that resembles that of actin

monomers within a filament and mimic an actin dimer onto which

the third monomer is added with the help of WASP proteins

(Figure 2a). These proteins C-terminally contain two domains

important for Arp2/3-complex activation, a G-actin-binding WH2

domain (Box 3) followed by an Arp2/3-complex-binding acidic

domain. The Arp2/3 complex furthermore interacts with the sides

of pre-existing actin filaments. This increases its nucleation activity

and generates new filament branches with a characteristic 708 angle.

The Arp2/3 complex thereby becomes integrated into the structures

it helps to form and gives rise to a rapidly expanding, branched

network of filaments, as can be observed at motile membrane

compartments and in lamellipodia of spreading or moving cells, for

example [1,47,51] (Figure 2a). Although the structure and the

mechanism of Arp2/3 complex activation has largely been unraveled

in recent years, its spatial control and the coordination of its activity

with other cellular events still require intensive further research.

Proteins of the formin family, which have only been recognized as

nucleators in recent years [52,53], catalyse de novo formation of actin

filaments in a completely different manner. They form a ‘doughnut-

shaped’ structure composed of two molecules, each of which makes

contacts to two actin molecules (Figure 2b). This doughnut-shaped

structure moves along with the barbed end as a processive cap. In this

way, formins give rise to long unbranched filaments, such as those of

the contractile actin ring formed during cell division, of yeast actin

cables or the F-actin structures at adherens junctions [23–25]. The

performance of formins is markedly increased by profilin. Profilin–

actin complexes are one of the most important sources of monomeric

actin in vivo. The functional differences between the diverse members

of the formin family and how these proteins are regulated is the focus

of intense current research.


monomer along the long pitch of the filament, the complexof Spire with four actin molecules has either to be a looselypacked, under- or even un-twisted linear actin array or theWH2 domains must be detached from actin during nuclea-tion. Bosch et al. [20] concluded that Spire–actin complexesother than the Spire–actin4 complex are the nucleatingentity. However, recent X-ray scattering analyses showedthat WH2-domain-connected actin arrays along the long-pitch of the filament can be formed without any stericalproblems [21] (Box 3).

The work of Bosch et al. [20] also questioned whetherSpire is a nucleator [20], because human Spire-1 NT-mediated actin nucleation was abolished upon profilinaddition, suggesting that spire blocks barbed ends.Furthermore, Spire-1 NT seemed to have filament-sever-ing activity in vitro [20].

Questioning Spire as a nucleator, however, is hard toreconcile with several lines of in vitro and in vivo evidenceobtained for Drosophila Spire. Overexpression of greenfluorescent protein (GFP)–Spire caused F-actin formationat Spire-positive, perinuclear compartments [19]. The pre-mature cytoplasmic streaming phenotype in spiremutants[8] is similar to that caused by interfering with Cappuccinoor profilin [8,9] (i.e. components promoting filament for-

278

mation). Deficiency of a putative barbed-end cappingprotein should furthermore cause opposite but not identi-cal effects to barbed-end capping by cytochalasin D [9].Also, in vitro examinations of Drosophila Spire did notyield any hints for a barbed-end blocking activity [19].Further examinations are required to shed light onputative differences of human and Drosophila Spire andto reveal the exact function of Spire proteins in actindynamics.

Such studies, however, will have to take into accountthat Spire-mediated actin nucleation ismechanistically farmore complex than initially thought because Spire andCappuccino do not only interact with each other [15,16],but complex formation also influences the actin-nucleationproperties of both partners [15]. The kinase non-catalyticC-lobe domain (KIND) [22] of Spire binds very tightly to theFH2 domain of Cappuccino (Kd 1 � 2 nM), competes withits microtubule- and F-actin-binding activity and, further-more, inhibits its actin-nucleation activity [15]. In contrast,nucleation by Spire was enhanced by binding to the C-terminal half (FH1 + FH2) of Cappuccino [15]. Thus,association of Spire with Cappuccino would shut downthe Cappuccino-immanent functions nucleation, actinbundling and microtubule bundling, and would result inthe exclusive use of Cappuccino to boost actin nucleation bySpire.

Box 3. WH2 domains – multifunctional adapters for actin monomers

WH2 domains are found in >60 modular proteins. They are only 25–50

amino acids long and contain the consensus motif L++V/T, with ‘+’

representing basic amino acids. The LKKT variant of the motif was

first recognized as an actin-binding motif in b-thymosins. The

classification of WH2 domains and b-thymosins into one superfamily

was suggested by Paunola et al. [54] and accepted after the structures

of b-thymosins and several WH2 domains were solved in complex

with actin [55–57].

In their non-actin-bound state, WH2 domains are disordered.

During actin binding, they fold and all bind actin in basically the

same manner. The main structural element for actin binding is a

conserved N-terminal amphipathic helix of variable length. It

accounts for most of the actin-binding affinity [55] and binds in the

hydrophobic pocket between subdomains 1 and 3 of actin [55–57] – a

binding hot spot for a variety of actin-binding proteins (including

actin itself) and actin-binding drugs [58].

C-terminal of the amphipathic helix, the L++V/T motif contributes to

actin binding. The leucine of the L++V/T motif is almost invariant and

binds in a hydrophobic pocket of actin (Ile341+ 345). The basic central

residues of the L++V/T motif are probably of high importance in the

long-range recognition of an acidic patch on actin (Asp24+25),

whereas the hydrophobic contacts of the leucine dominate interaction

at close range [55]. The fourth amino acid of the motif binds in a small

pocket on the actin surface. Thus, threonine, valine and also alanine

and serine are most often found at this position.

The remaining part of the WH2 domain (a C-terminal extension

of variable sequence and length) extends along the nucleotide-

binding cleft of actin. In the case of b-thymosins, this extension is

long, contains a second helix attaching to the pointed end surface

of subdomain 2 and is important for the G-actin sequestration

activity of b-thymosins [56]. Other WH2 domains lack the C-

terminal extension completely and/or the extensions seem to

follow slightly different paths on actin. It has been proposed that

the C-terminal extension serves as main determinant of WH2-

domain function; that is, it creates proteins for actin sequestration

(e.g. b-thymosins), for promotion of actin filament elongation, or

for promotion of actin filament nucleation (e.g. Ena/VASP proteins,

WASP family proteins and WH2-domain-containing nucleators,

respectively). For excellent reviews on WH2-domain structure and

function, see Refs [54,59,60].

Recently, the Dominguez laboratory succeeded in visualizing the

structure of a linear actin complex held together by a chimeric

molecule (3W) composed of three WH2 domains (derived from N-

WASP) with short linkers, as they occur in Spire, for example, and a C-

terminal extension derived from thymosin-b4. Because WH2-domain-

mediated actin assembly is the focus of this review, we present here

the X-ray scattering analysis of Rebowski et al. [21], by copyright

permission of the Academy of Sciences (Figure I).

Figure I. (a) Model of 3W-actin obtained by superimposing the structure of WH2-

actin [55] onto three consecutive subunits of the long-pitch helix of the actin

filament. WH2 domains and linkers in between are in red, the three actin

molecules are in turquoise, yellow and blue and the ATP is in magenta. (b) Two

orientations of the model (rotated by 908) superimposed onto X-ray scattering

data extending to the resolution of �28 A.


Quinlan et al. [15] suggested that Spire –with the help ofCappuccino – nucleates filaments, which are then ‘handedover’ to the formin Cappuccino for barbed-end elongation.Such a model would be in line with analyses suggestingthat Spire stays associated with the pointed end of fila-ments [19], whereas formins processively move along withbarbed ends [23–25].

Owing to formin dimerization, two Spire moleculesmight cooperate during nucleation (Figure 3b). If eachSpire binds to four actin molecules of one strand, this couldbring together eight actin molecules belonging to bothstrands (i.e. could form a complete filament seed andthereby create a powerful nucleation complex;Figure 3b). Examinations of mammalian Spire and theCappuccino ortholog formin-2 showed that the aforemen-tioned findings are also observed in mammals [15]. ‘Hand-ing over’ actin seeds to formin-promoted actin filamentelongation, however, would require actin-polymerization-dependent dissociation of Spire–Cappuccino complexes,which remains to be shown. Maybe the required dis-sociation is brought about by profilin–actin complexesrecruited by the formin homology 1 domains within thecomplex (Figure 3b).

Genetic experiments indeed show that Spire and Cap-puccino do not simply operate in parallel but work inti-mately together. Whereas spire mutants can be rescuedwith a deletion mutant (aa1–488) containing the Spire

actin-nucleation domain, cappuccino mutants cannot[10]. Constitutively active Cappuccino rescues the pheno-type of spiremutants, provided that profilin is present [10].Neither Cappuccino nor Spire, however, was able to rescuechickadee (profilin) mutants [10]. In light of the tightCappuccino–Spire interaction [15], there are two possibleexplanations for this: (i) Cappuccino–profilin machineryoperating in parallel to Cappuccino–Spire-mediatednucleation might partially compensate Spire deficiencywhen overexpressed; (ii) actin nucleation by Cappuccino–

profilin as such does not even exist during oogenesis, butCappuccino exclusively works as part of Spire–Cappuccinocomplexes. Overexpression of Cappuccino would then arti-ficially create a fly Cappuccino–profilin actin-nucleationmachine, partially compensating for the loss of Spire-mediated actin nucleation. It will now be exciting to studythe emerging Cappuccino (formin-2)–Spire–profilinmachine in more detail mechanistically, to compare it withCappuccino–profilin alone and to reveal its physiologicalimportance.

That Spire represents some sort of improved componentfor actin–microtubule cross-talk is reflected by the factthat, despite the ability of an excess of Cappuccino torescue the Spire phenotype in oogenesis, the cytoplasmicactin mesh built was weaker than the wild-type mesh andpersisted for a shorter time [10]. It seems likely that thecurrently uncharacterized domains of Spire such as the

279

Figure 3. Novel WH2-domain-containing actin nucleators. (a) Domain organization of Spire, Cobl, Lmod-2, VopF, VopL and TARP. Owing to their small size, WH2 domains

and other small motifs are not drawn to scale. Abbreviations: A-h, actin-binding helix; b, basic stretch; h1 and h2, helix 1 and 2; KIND, kinase non-catalytic C-lobe domain;

NLS, nuclear localization signal; PRD, proline-rich domain; Sec, type II secretion/translocation signal; TM-h1, tropomyosin-binding helix 1. (b) Models of actin-nucleation

mechanisms for: (i) Spire; (ii) Lmod-2; and (iii) Cobl. For Spire, both the initial model [19] (left side) and a model based on newer data from Quinlan et al. [15] (right side) are

shown.


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spire-box, a potential interaction site for Rab GTPases, orthe FYVE-domain-relatedmodule (Figure 3a), which implythe existence of currently unknown functions inmembranetrafficking and which help to direct Spire proteins to theGolgi apparatus, post-Golgi vesicles and recycling endo-somes in overexpression analyses [26], are involved inthese specialized Spire functions. Coordinating vesicletrafficking by cytoskeletal structures is especially requiredin neuronal cells [27,28]. This might be a physiologicalexplanation for the striking co-expression and the highexpression levels of Spire-1 and formin-2 observed in thecentral nervous system [7].

CoblCobl contains three C-terminalWH2 domains and seems tobe a vertebrate-specific nucleator. Cobl-mediated actinnucleation is very efficient. Already at low nanomolarconcentrations, it gives rise to unbranched filaments andreaches the performance of N-WASP–WA–Arp2/3-com-plex-mediated actin nucleation [3]. Like Spire-1, expres-sion of Cobl is mainly restricted to the brain. Much weakerexpression was detected in other tissues [3].

The cobl gene was originally identified in lacZ-gene-trapexperiments [29]. Cobl mRNA was detected as early as atday 7.5 post-coitum (E7.5) in the gastrula organizer andextended towards the axial midline at E8. The organizer isa small group of embryonal cells that organizes the entirebody plan because it gives rise to the axial midline – animportant source of patterning and morphogenesis cues.The striking accumulation and restriction ofCoblmRNA tothe organizer and axial midline structures led to the ideathat Cobl is involved in vertebrate axis formation [29].Unfortunately, the CoblC101 allele obtained by the gene-trap experiments [29] is only a weakly hypomorphic allele[30] and mice homozygous for CoblC101 did not show anyobvious phenotype [29]. A genetic interaction with Vangl2suggested that Cobl has some role in midbrain neural tubeclosure [30]. Cobl knockout mice are needed to clarify therole of Cobl during early development.

Studies in rat primary hippocampal neurons revealed arole for Cobl in neuromorphogenesis. Overexpression ofCobl or theWH2-domain-containing C terminus (Cobl CT),but not of mutants lacking the actin-nucleation activity,led to a striking induction and branching of neurites [3].Mechanistically, this might reflect an identical cell bio-logical problem: breaking a sphere and a tube, respectively,to initiate a protrusion. Because the plasma membrane iseffectively deformed by excessive cortical actin assemblyand similar phenotypes can, for example, be observed uponaberrant activation of Arp2/3-complex-mediated actinnucleation at the cell cortex of primary developing neurons[31], the Cobl-induced phenotype suggests that Cobl pro-motes actin-filament formation at the cell cortex. Consist-ently, RNAi experiments and accompanying rescueexperiments have demonstrated that Cobl is a crucialcomponent for proper neuritogenesis and dendritic branchinduction of vertebrate neurons [3].

Interestingly, in early neuronal development, the Arp2/3 complex is exclusively crucial for proper formation of theaxon [31,32] but not of dendrites. This suggests thatneuronal cells use different actin nucleators to steer the

complex cytoskeletal reorganizations underlying neuronalshape modulation and formation of neuronal networks –

prerequisites for proper brain development and neuronalcell–cell communication.

How Cobl exerts its cell biological functions is far fromclear because, thus far, solely its mechanism of actinnucleation has been studied. In contrast to Spire, Coblonly has three WH2 domains. By a carefully orchestratedaction of these three individual G-actin-binding modules,Cobl assembles actin monomers into actin nuclei. All threeWH2 domains, which bind to G-actin with different affi-nities, are crucial for actin nucleation in vitro and forinduction of excessive arborization in neurons. Deletionof single WH2 domains resulted in almost complete loss ofnucleation activity [3]. This can be viewed as strong exper-imental support for the old hypothesis that a minimum ofthree actin molecules needs to be assembled to form apolymerization-competent nucleus.

The arrangement of its WH2 domains with two con-secutive WH2 domains and a third spaced away a littlefurther and the requirement for all three domains suggestthat Cobl assembles a trimeric cross-filament nucleus. Thelow dissociation constants for WH2 domains 1 and 2 (Kd

values �40 nM) and their short spacing enable efficientformation of a linear dimer, which is not yet an effectiveactin nucleus, as demonstrated in vitro and in vivo [3]. Theaddition of a cross-filament of a third monomer is crucial.Whereas such an addition to linear arrays of actin mono-mers along the long pitch of a filament would have to occurspontaneously, Cobl seems to be able to also promote thissecond crucial step. Because the linker between Cobl WH2domains 2 and 3 (L2) is long enough to reach around theforming nucleus, a third actin monomer could be added tothe back side of the dimer, creating a cross-filament trimer(i.e. a complete barbed end), which can then rapidlyelongate spontaneously. Indeed, shortening the linkerL2 abolished Cobl-mediated actin nucleation. Strikingly,at least in vitro, it was not the sequence of the poorlyconserved linker L2 but its length that is of outmostimportance for Cobl-mediated actin nucleation [3].

It will be exciting to address the hypothetical Coblmechanism by resolving the structure of actin nucleiformed by Cobl, an endeavor complicated by the need toarrest Cobl-mediated actin nucleation at the short-livedintermediate step of a trimer without affecting complexcomposition or topology. In vitro reconstitutions and colo-calization studies showed that Cobl resides on actin fila-ments, but the presence of Cobl did not shield the pointedend from depolymerization [3]. Further experiments arerequired to show where Cobl resides on filaments and tocompare its nucleationmechanismwith that of otherWH2-domain-containing nucleators. The molecular compositionof Cobl-based actin-nucleation machineries and how thenucleation power of Cobl is controlled in time and spacealso remain open questions.

LeiomodinLeiomodin-2 (Lmod-2; cardiac leiomodin; C-Lmod) adds tothe expanding catalog of recently identified WH2-domain-containing actin nucleators [4]. Besides Lmod-2, twofurther highly similar leiomodin isoforms can be identified,

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Lmod-1 (smooth-muscle leiomodin; SM-Lmod) and theuncharacterized Lmod-3 (fetal leiomodin). The domainorganization of leiomodins is closely related to that oftropomodulins (Tmods) [33]. The N-terminal half of Tmodsconsists of an actin-binding and two tropomyosin-bindinghelices. This region of Tmods caps the pointed end of actinfilaments in a tropomyosin-dependent manner [34]. Asecond, tropomyosin-independent actin-binding and -cap-ping site is the extended leucine-rich repeat representingthe C-terminal half of Tmods [35]. Leiomodins resembleTmods, although they have only one N-terminal tropomyo-sin-binding helix and, C-terminally, extend �150 aminoacids (Figure 3). In Lmod-2, this extra stretch contains asingleWH2 domain. Similar to Spire-1 and Cobl, Lmod-2 isnot ubiquitously expressed but seems to be a ratherspecialized nucleator. Lmod-2 is exclusively present infetal and adult heart and in adult skeletal muscles [32],that is, in tissues apparently lacking Spire [7] and Cobl [3].

Already, low nanomolar concentrations of Lmod-2nucleate actin. Deletion analyses have suggested that allrecognized domains of Lmod-2 are required for actinnucleation [4]. Of the mutants tested, an N-terminaldeletion mutant starting with the leucine-rich repeat (Lm-od-2 [162–495]) still had the highest nucleation activity(�30% of wild type). Deletion of any singlemotif within thisfragment led to further dramatic reductions in its residualnucleation activity in in vitro examinations.

Lmod-2 [162–495] binds to two actin monomers. Assuggested from Tmod analyses, the Lmod-2 N terminushas another actin-binding site [4]. Thus, provided that theN-terminal domain does not bind to the actin dimeralready bound by the Lmod-2 C-terminal half but makescontact to another actin molecule, the three actin-bindingsites make contact with three actin molecules. Deducedfrom the pointed-end capping activity of Tmods, which bindto the two adjacent actin molecules of the pointed end, itwas proposed that Lmod-2-mediated nucleation might bebrought about by assembly of three actin monomers withthe third being recruited by the C-terminal WH2 domain[4] (Figure 3b). This would result in a cross-filament actintrimer similar to that proposed for Cobl-mediated nuclea-tion [3]. Similarly to Spire [7] and Cobl [3], the actinmonomer at the barbed end would be WH2-domain-bound.Also similarly to Spire [7] and Cobl [3], Leiomodin givesrise to unbranched actin filaments in in vitro reconstitu-tions [4]. These filaments might then be stabilized bytropomyosin binding to Lmod-2 (Figure 3b).

Profilin had inhibitory effects on filament formationirrespective of whether Lmod-2 [162–495] or a mutant inwhich a C-terminal poly-proline motif had been replacedwas used. This might suggest that profilin does not bind tothis proline-rich motif in Lmod C termini and is not part ofthe Lmod actin-nucleation machinery [4].

Compelling evidence for Lmod-2 being an actin nuclea-tor in vivowas obtained by overexpression of GFP–Lmod-2[162–495]. This fragment triggered the formation of abnor-mal actin bundles in the nucleus, to which it aberrantlylocalized [4].

Endogenous Lmod-2 showed some colocalization withthe M-line protein myomesin in cardiomyocytes. Thismight suggest that Lmod-2 resides at pointed ends of actin

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filaments, which are oriented towards the M-line, but invitro examinations showed that Lmod-2 affects filamentelongation neither at the barbed nor at the pointed end.Overexpression studies with deletion mutants revealedthat the Lmod-2N terminus is crucial for targeting tothe middle of sarcomeres [4].

Strong overexpression of GFP-fusion proteins, whichprobably act as dominant-negative constructs attackingLmod interaction partners because they either localizesomewhere to themiddle of sarcomeres but lack nucleationactivity (GFP–Lmod2 [1–342]) or exhibit actin-nucleationactivity but mislocalize (GFP–Lmod-2 [162–495]); GFP-Lmod full-length), were mentioned to disrupt sarcomerorganization [4]. That Lmod-2 itself has a role in propersarcomer organization is suggested from effects obtainedwith siRNA, sarcomeres had somewhat disorganized F-actin structures and less striated and smaller actinin-positive areas reflecting the Z-discs [4].

The study by Chereau et al. [4] has introduced Lmod-2as an exciting new player in actin nucleation. Further workis now required to study the mechanism of this specializedmachine for actin nucleation in skeletal muscles in moredetail. It will also be exciting to unravel how an M-line-associated component, such as Lmod-2, affects Z-discorganization; this will provide important insight intoassembly and function of the entire sarcomer structure.

It seems very likely that all leiomodin isoforms are actinnucleators because they exhibit a high similarity to eachother (e.g. Lmod-1 also binds to tropomyosin) [36]. It will beespecially important to know whether the entire Lmodfamily is restricted to muscle cells of different types orwhether certain non-muscle cells also exhibit Lmods.Given that Lmod-2 expression is restricted to skeletaland heart muscles and that Lmod-1 seems restricted tosmooth muscle cells, consistent with its occurrence insmooth-muscle-containing organs and tissues such asaorta, trachea, colon, small intestine, bladder, uterusand stomach [33], the analysis of the expression profileof the still uncharacterized Lmod-3 will determinewhetherLmods are a specialty of muscle cells or are more widelyused actin nucleators.

Abusing the powerful mechanisms of WH2-domain-mediated actin nucleationThe studies on Spire, Cobl and Lmod-2 have revealed thatWH2-domain-mediated actin nucleation is a powerful wayofmaking filaments. Therefore, it is not a surprise that thisstrategy has also been adopted by pathogens.

Microbial pathogens use a plethora of different mech-anisms to manipulate the host cell actin cytoskeletonduring infection to prevent or induce their own phagocy-tosis and to facilitate their motility within and on thesurface of infected host cells. They do so differently: atthe level of Rho-type GTPases, bacterial factors mimicGTPase exchange factors (GEFs), GTPase-activatingproteins (GAPs) or even GTPases themselves. Other bac-terial pathogens use effectors (such as Listeria ActA, Rick-ettsia RickA and Shigella IscA) that can hijack theeukaryotic actin nucleator Arp2/3 complex [37,38].

Recently, bacterial effectors have also been shown todirectly nucleate actin filaments. Thus far, three such


proteins were identified. All make use of multiple WH2domains. These WH2 domains either occur as multiplecopies in one protein (Vibrio outer protein [Vop]F andVopL) or are brought together by oligomerization ofproteins encompassing a single WH2 domain (Chlamydiatranslocated actin-recruiting phosphoprotein [TARP])(Figure 3a). The fact that both the VopF protein fromVibrio cholerae and the related VopL protein from Vibrioparahaemolyticus exhibit actin-nucleation activity inde-pendent of any eukaryotic factors [39,40] establishes thatnucleation of actin filaments bymultipleWH2 domains is ageneralized molecular mechanism.

VopF, VopL and TARP are secreted into host cells bytype III secretion systems – a specialized apparatusenabling pathogens to translocate virulence factors oreffector proteins into host cells where they function to alterand/or abuse the eukaryotic cellular machinery [41]. Con-sidering the usually very efficient ways in which patho-genic bacteria exploit their host cells and the fact that thebacterial system has to be able to override existingendogenous mechanisms of the host, it is not surprisingthat the newly discovered bacterial actin nucleators seemat least as powerful as their eukaryotic counterparts andthe fully activated Arp2/3 complex [39].

Upon injection or expression, both VopL and VopF alterthe eukaryotic actin cytoskeleton, albeit in a distinct man-ner. VopL induced stress fibers [39], whereas VopF inducedaberrant actin-rich protrusions [40]. Interestingly, VopFlocalizes to the tips of these protrusions, suggesting that itmight associate with barbed ends of actin filaments,whereas VopL was observed to bind along actin stressfibers, suggesting a side-binding activity [39,40].

In contrast to the eukaryotic nucleator Cobl, which alsocontains three WH2 domains [3], neither the effects ofVopL nor those of its relative VopF were completely de-pendent on all three WH2 domains. VopL mutants withonly two or even only one functionalWH2 domainwere stillable to induce actin stress fiber formation, albeit to some-what reduced extents [39]. These results are somewhatreminiscent of the nucleation domain analysis reported forSpire [19]. Currently, two explanations seem plausible andwould be interesting to test. First, VopL self-associationmight form complexes with multiple WH2 domains. Sec-ond, one WH2 domain would be sufficient for adding anactin monomer, if the other part of the actin seed formed isheld together by the currently uncharacterized actin side-binding activity of VopL [39], which could very well beindependent from the WH2 domains.

Similarly, VopF still retained some nucleation activityin vitro when individual WH2 domains were deleted [40].Despite the reduced in vitro nucleation performance of twoWH2-domain-containing mutants, they did not, however,affect the ability of V. cholerae to colonize the infant mousesmall intestine. By contrast, mutants lacking all WH2domains did exhibit strong colonization defects. The samewas observed for a mutant lacking the proline-rich domainin front of the WH2 domain cluster of VopF. In vitroreconstitutions showed that both types of mutants lacksignificant actin-nucleation activity [40]. This stronglysuggests that VopF-mediated actin nucleation is importantfor intestinal colonization.

The type III secreted effector TARP from Chlamydiatrachomatis, which also promoted actin nucleation in vitro,contains only a single WH2 domain per molecule [42].Because TARP oligomerization is essential for the nuclea-tion activity of a small TARP fragment encompassing theWH2 domain and a proline-rich sequenceN-terminal of theWH2 domain [42], TARP seems to form a multi-WH2-domain complex via oligomerization. Further studiesshould help to unravel whether full-length TARP alsomultiplies its WH2 domain by oligomerization andwhether self-assembly is still required for nucleation byfull-length TARP.

An indication of the physiological relevance of the actin-nucleation activity of TARP specifically is also urgentlyawaited because TARP additionally interfaces with actin-nucleation machineries of host cells by binding to two RacGEFs, resulting in WAVE2–Arp2/3-complex-mediatedrecruitment of TARP to the side of Chlamydia attachment[43]. These interactions are steered by tyrosine phos-phorylations – post-translational modifications that seemnot to be required for TARP-mediated actin nucleation,because non-phosphorylated Chlamydophila caviae TARPalso accumulated F-actin [44]. It will be interesting to seewhether and how the Arp2/3-dependent and -independentactin-nucleation mechanisms might cooperate for Chlamy-dia-induced actin recruitment and invasion.

PerspectivesWH2-domain-mediated actin nucleation seems to be a verypowerful way of making filaments and adds a prominentfunction to the stunning versatile properties of WH2domains (Box 3). Complementary efforts including struc-tural work, biochemical analyses, cell biological and phys-iological studies and genetic examinations, some of whichwere outlined in this review, are now required to revealhow and why nature uses WH2 domains for so manypurposes and to further elucidate the different mechan-isms employed within the group of WH2-domain-contain-ing actin nucleators.

Because unraveling the mechanisms by which patho-gens interface with the Arp2/3 complex has been a pro-ductive route towards gaining deeper insights into howcells steer and execute Arp2/3-complex-mediated actinnucleation, it is very attractive to examine the molecularmechanisms used by the bacterial WH2-domain-contain-ing nucleators TARP, VopF and VopL in more detail. Thevery efficient ways in which pathogenic bacteria hijackhost cell components might serve as a fruitful researchavenue towards identification of further components ofWH2-domain-based nucleation machines and towards bet-ter understanding of WH2-domain-mediated actin nuclea-tion in vivo. Insight into how pathogens interface with ourbody cells will also be of outmost interest for pharmaco-logical research helping to fight the frightening currenttrend of bacteria that are increasingly counteracting ouranti-bacterial strategies.

Although the required biochemical, biophysicaland structural work and the insight gained from compari-sons with mechanisms used by pathogens will advanceour understanding of WH2-domain-based actin nucleationconsiderably, particular genetic and cell biological exam-

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inations will additionally reveal the nature of the distinctcellular and physiological processes that the differentnucleators are crucial for. Such studies will finally helpus to understand why the more complex body plans devel-oped during evolution of vertebrates require an increasingcomplexity within the group of actin-nucleating machines.Important steps towards these aims have been made forSpire, Cobl and Lmod-2 but we are convinced that theindividual functions unraveled thus far – oogenesis forSpire, neuromorphogenesis and neuronal network for-mation for Cobl and sarcomer organization for Lmod-2 –

only represent parts of the full picture.Furthermore, the picture of actin nucleation drawn

during the past decade has been grossly oversimplifiedby concentrating solely on distinguished functions of thedifferent molecular pathways towards filament formation.As also highlighted by a recent review by Chesarone andGoode [45], nature does not bring about the different typesof actin structures by the exclusive use of very specializednucleators, but there is plenty of collaborative work per-formed by these molecular machines and scaffold proteins,which interconnect them. It will thus be very exciting toreveal the complete set of players for actin-filament for-mation, to unravel how they work in vitro and in vivo andfinally to try to figure out how they are coordinated andfine-tuned in vivo. Together, this will provide a detailedunderstanding of how one of the crucial components ofeukaryotic life, the actin cytoskeleton with its astonishingcomplexity and dynamics, works and malfunctions in ageand disease, respectively.

AcknowledgementsWe thank Eugen Kerkhoff for communicating unpublished work. Thiswork was supported by the Deutsche Forschungsgemeinschaft.

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new players in actin polymerization – wh2-domain-containing actin nucleators

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