Actin-binding proteinsSteven J. Winder1 and Kathryn R.Ayscough2

Departments of 1Biomedical Sciences and2Molecular Biology and Biotechnology, University ofSheffield, Sheffield, S10 2TN, UKAuthor for correspondence (e-mail:k.ayscough@sheffield.ac.uk)

Journal of Cell Science 118, 651-654 Published by TheCompany of Biologists 2005doi:10.1242/jcs.01670

Actin is an essential component of thecytoskeleton and plays a crucial role ineukaryotic cells. The actin cytoskeletonfunctions in the generation andmaintenance of cell morphology andpolarity, in endocytosis and intracellulartrafficking, in contractility, motility andcell division. In cells, the assembly anddisassembly of actin filaments, and alsotheir organisation into functional higher-

order networks, is regulated by aplethora of actin-binding proteins(ABPs) (dos Remedios et al., 2003;Maciver, 2004). The activities of theseproteins are in turn under the control ofspecific signalling pathways.

Here, we aim to highlight the mainclasses of ABP found in eukaryotic cellsand indicate their likely mechanism ofaction as far as possible on the basis ofboth in vitro and in vivo studies.

Proteins regulating F-actinassembly and disassemblyActin exists in two principal forms,globular, monomeric (G) actin, andfilamentous polymeric (F) actin.Because of the arrowhead patternobserved when myosin decorates actinfilaments, the fast-growing end of the

polarised polymer is denoted the barbedend and the slow-growing end is donotedpointed end. Actin filaments extendwhen ATP-actin monomers arepreferentially incorporated at the barbedend. As the filament matures, ATPbound in the central cleft of actin ishydrolysed, phosphate is released andthe resulting ADP-actin filament isdisassembled by loss of monomers fromthe pointed end. The released ADP-actinmonomers then undergo nucleotideexchange to generate ATP-actinmonomers that can be used for newrounds of polymerisation. This ATP-hydrolysis-driven, directional filament-growth is called actin treadmilling.

NucleationThe first stage in de novo filamentformation is nucleation. Polymerisation

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Monomer binders

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is energetically unfavourable until thereis a nucleus of three associatingmonomers and in vitro this stage offilament formation is denoted the lagphase. In vivo, ABPs are crucial toensure the rapid nucleation of filamentsand these essentially function to removethe lag period. New filaments can formde novo, from the side of existingfilaments, or by severing an existingfilament. Different proteins function topromote each of these modes ofnucleation.

The Arp2/3 complex can nucleatefilaments from the side of existingfilaments. This is important to allow thedendritic branching that is found at theleading edge of motile cells (Pollard andBorisy, 2003). Arp2 and Arp3 moleculesthemselves have a similar tertiarystructure to actin itself, and when theArp2/3 complex binds G-actin the effectis to generate a stable trimer and nucleusfor the growth of a filament. Arp2/3 thenacts as a pointed-end-capping proteinthat encourages rapid growth of thefilament from its barbed end. Whereasthe Arp2/3 complex can nucleate newfilaments in vitro, it is likely that itsactivity in vivo is enhanced byfunctional interactions with otherproteins, most importantly the WASPand SCAR/WAVE proteins. The G-actin-binding WASP homology2 (WH2)domain of WASP is important for theactin-nucleating activity of Arp2/3 andits role is probably to feed actinmonomers to the filament barbed end.Verprolin/WIP binds both monomersand F-actin and has been shownto enhance Arp2/3-mediated actinpolymerisation when bound to theArp2/3 activator cortactin (reviewed inPaavilainen et al., 2004).

Actin polymerisation is also nucleatedby the formin proteins. These proteinshave only recently come under closescrutiny and there is still much to beunderstood about their mechanism ofaction in cells. However, studies indicatethat the formin homology2 (FH2)domain dimer flexes to accommodate theprocessive addition of actin monomersto the barbed end of a filament (Xu et al.,2004). In yeast it is thought that the longactin cables that are required for cellpolarity are generated by the nucleatingaction of the formins whereas the short

branched networks in actin patches aregenerated by Arp2/3 nucleation.

Regulation of actin filament growth,stability and disassemblyOnce nucleated, actin filaments are ableto grow rapidly by addition of monomersat their barbed ends. Filaments arecontrolled by several mechanisms.Filament length is controlled by cappingproteins. Barbed end cappers such ascapping protein, gelsolin and tensinblock addition of new monomers, soacting to decrease the overall length ofthe filament. In addition, gelsolin cansever actin filaments, thereby rapidlyincreasing actin dynamics. A mechanismfor gelsolin severing has recently beenproposed at the structural level (Burtnicket al., 2004). Depending on theenvironment in the cell this can havevarious outcomes, but it is usually amechanism to disassemble F-actin-containing structures. Pointed endcappers, by contrast, reduce loss ofmonomer from the pointed end andthereby can lead to rapid filamentextension.

The best characterised proteins thatdrive depolymerisation are the actin-depolymerizing factor (ADF) and thecofilin family members. This ubiquitousprotein is highly conserved and plays acentral role in actin turnover. It binds toADP-F-actin and promotes dissociationof ADP-actin from the pointed end of thefilament. ADF/cofilin also associateswith AIP-1 (actin-interacting protein1).This interaction appears to increase thedepolymerising activity of cofilin. Thecomplex also promotes barbed endcapping, although the details of thismechanism are not yet well understood(Paavilainen et al., 2004).

Another important and highly conservedfamily of ABPs are tropomyosins. Thesebind along the filament length and, likeother proteins that bind along the side ofactin filaments, stabilise the filamentagainst spontaneous depolymerisation.Furthermore, tropomyosin has aprotective effect against gelsolinsevering and ADF/cofilin-mediateddepolymerisation. In concert withtroponins, tropomyosin also plays animportant role in regulating theinteraction of myosin with the actin

filament in striated muscle. Otherproteins that associate along the lengthof filaments act as rulers. Nebulin, forexample, is a large elongated proteinwith numerous low-affinity actin-binding sites which act together topromote, stabilise and determine thelength of actin-containing thin filamentsin striated muscle. The length of thenebulin protein in different tissues andspecies corresponds with the length ofthe thin filaments in each half sarcomere;so nebulin is thought to have a molecularruler function in determining filamentlength.

A numerous and diverse range of ABPsthat we have denoted sidebinders andsignallers are proteins that have beenshown to bind specifically to F-actin. Inaddition, these proteins have domainsthat allow them to interact with otherproteins and more specifically tofunction within signalling networks incells to allow remodelling of the actincytoskeleton at appropriate times andplaces within the cell. For example,several proteins that contain poly-prolinemotifs (e.g. VASP and vinculin) recruitcomponents of the actin polymerisationmachinery, such as profilin, whereasseveral SH3-domain-containing proteinsare involved in membrane trafficking(e.g. Abp1) and association withadhesion complexes (e.g. cortactin).

Monomer binding proteinsThe rapid growth of actin filaments thatcan be observed in motile cells and thereorganisation of actin that occurs inresponse to both intracellular andextracellular cues in other cell typesrequires the availability of actinmonomers to be tightly regulated. Thisis achieved through a number ofmechanisms by a group of highlyconserved actin-monomer-bindingproteins. Although there are a largenumber of monomer-binding proteins(>25 in mammalian cells alone), thereare six major classes that are found inorganisms from yeast to human and fourof these classes are reported in plants.

As a group, the monomer-bindingproteins are involved in binding ADP-actin as it is released from filament ends(e.g. twinfilin, ADF/cofilin), facilitatingthe nucleotide exchange of ADP for

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ATP (e.g. profilin and CAP) anddelivering the monomer to barbed ends(or to Arp2/3) to facilitate new roundsof polymerisation (e.g. twinfilin,Srv2/CAP, profilin, verprolin/WIP andWASP). As well as promotingpolymerisation of existing filaments,some monomer-binding proteins areinvolved in nucleating the formation offilaments de novo and from the side ofexisting filaments (see nucleationabove).

In motile cells it is imperative that thereis a large pool of monomer that canbe released to allow rapid filamentextension. This can be achieved bymonomer-sequestering proteins, the beststudied of which is the thymosin family.These proteins act by clamping ATP-actin top to bottom, to effectively cap atboth barbed and pointed ends andprevent incorporation into filaments(Hertzog et al., 2004; Irobi et al., 2004).Appropriate signals at the cell cortex canthen trigger activation of profilin,leading to a rapid release of thymosinbinding, and result in a massive increasein the amount of actin available forpolymerisation.

Proteins regulating higher-orderF-actin structures The organisation of actin into networksand higher-order structures is crucial toboth the form and function of cells.These structures can be responsible forthe overall shape and order in a cell, forexample, the terminal web andmicrovilli of brush border epithelial cellsor the ordered architecture of striatedmuscle – as well as more transient ordynamic structures relating to aparticular cellular function, such as theacrosomal processes of someinvertebrate spermatozoa, and filopodiaat the leading edge of locomoting cells.With the exception of dendritic branchesformed by the action of the Arp2/3complex mentioned above, all otherhigher-order F-actin structures areformed by two broad actin-bindingactivities; F-actin bundling and F-actincrosslinking.

Actin-bundling proteinsActin bundling is the parallel orantiparallel alignment of F-actin into

linear arrays and is generally achievedby proteins that either contain twodiscrete actin-binding domains withintheir sequence or by multimeric proteinsthat contain only a single bindingdomain per subunit. Actin bundles arealso usually further subdivided intoloose or tight bundles, the topography ofwhich is more or less dependent on thearchitecture of the bundling protein. Thepresence of two actin-binding domainsin close proximity, such as in the proteinfimbrin, leads to the formation of tightactin bundles – as found in microvilli.The more loosely ordered structures ofactin stress fibres, however, areorganised by the dimeric and antiparallelprotein α-actinin, which has a singleactin-binding site per subunit. These areseparated by a helical spacer region,placing the two actin-binding domains ofthe dimer some distance apart, whichresults in a looser association of actinfilaments.

Actin crosslinking proteinsThe arrangement of actin filaments intoorthogonal arrays is also mediated byproteins or protein complexes containingmultiple actin-binding domains, but ingeneral the two domains are separatedby longer, more flexible spacer regions,which allows a more perpendiculararrangement of actin filaments.Examples of this type of protein are thelarge flexible dimeric filamin ortetrameric spectrin complexes, butcrosslinking is also achieved by smallmonomeric proteins, such as transgelin,which under certain conditions organisesactin filaments into dense meshworks.Interestingly, the yeast homologue oftransgelin, known as Scp1p, inducestight actin bundles rather thanmeshworks (Winder et al., 2003).

Actin as a mechanical frameworkRather than affecting actin dynamics orregulating actin structure per se, anenormous number of ABPs use actin asa scaffold, physical support or track.Whilst some of these proteins mayindirectly alter actin dynamics andstructure, this is not their primary role inthe cell. We have classified theseproteins into three main categories;myosins, anchors to membrane

complexes, and linkers between actinand other cytoskeletal elements.

MyosinsMyosins are actin-dependent molecularmotors that produce movement (andforce) through the hydrolysis of ATP. Assuch, myosins simply use actin as a trackalong which to move. Most people arefamiliar with the two-headed myosin IIinvolved in contractility and tensiongeneration, but there are now over 17different classes of myosins with variousdiverse functions (Hodge and Cope,2000). Nonetheless, where tested, theyall use actin as a track to move theirspecific cargo; whether it be membranesor vesicles, actin filaments or a host ofother proteins – and mostly but notexclusively from the pointed to thebarbed end of the filament.

Cytoskeletal linkers and membraneanchorsThe utility of F-actin as a structuralframework within cells necessitates itsconnection to other cellular elements.Most of these individual proteins havequite specific functions relating to their(sub)cellular and organismal context. Butas alluded to above they can besubdivided into two broad groups:proteins that connect actin to membranesor membrane proteins and those thatinterconnect different cytoskeletalelements. In the former category areproteins such as dystrophin and utrophinor talin and vinculin, which connect theactin cytoskeleton to the cell adhesionreceptors dystroglycan or integrin,respectively; and proteins that can binddirectly to membranes and also interactwith actin such as annexins. The lattercategory comprises a small but importantgroup of proteins that can link actin tomicrotubules, actin to intermediatefilaments or, in the case of plectin, actinto both microtubules and intermediatefilaments. Clearly such proteins are ofgreat importance to the cell in theintegration of structure and signallingbetween the cytoskeletal elements andthe maintenance of cell integrity.

Work in the authors labs is supported by theMedical Research Council (MRC): an MRC SeniorResearch Fellowship to K.R.A. (G117/394),MRC Career Establishment Grant to S.J.W.(G104/51660).

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Hodge, T. and Cope, M. J. T. V. (2000). AMyosin Family Tree. J. Cell Sci. 113, 3353-3354.Irobi, E., Aguda, A. H., Larsson, M., Guerin, C.,Yin, H. L., Burtnick, L. D., Blanchoin, L. andRobinson, R. C. (2004). Structural basis of actinsequestration by thymosin-beta4: implications forWH2 proteins. EMBO J. 23, 3599-3608.Maciver, S. K. (2004). http://www.bms.ed.ac.uk/research/others/smaciver/Encyclop/encycloABP.htm.Paavilainen, V. O., Bertling, E., Falck, S. andLappalainen, P. (2004). Regulation ofcytoskeletal dynamics by actin-monomer-bindingproteins. Trends Cell Biol. 14, 386-394.Pollard, T. D. and Borisy, G. G. (2003). Cellularmotility driven by assembly and disassembly ofactin filaments. Cell 112, 453-465 (Erratum in Cell2003 113, 549).

Winder, S. J., Jess, T. and Ayscough, K. R.(2003). SCP1 encodes an actin bundling protein inyeast. Biochem. J. 375, 287-295.Xu, Y., Moseley, J. B., Sagot, I., Poy, F.,Pellman, D., Goode, B. L. and Eck, M. J. (2004).Crystal structures of a Formin Homology-2domain reveal a tethered dimer architecture. Cell116, 711-723.

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