Structural insights into actin-binding, branching and bundling proteins

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Structural insights into actin-binding, branching and bundling proteins Steven J Winder

Structural advances in our understanding of the functions of the actin cytoskeleton have come from diverse sources. On the one hand, the determination of the structure of a bacterial actin-like protein MreB reveals the prokaryotic origins of the actin cytoskeleton, whereas on the other, cryo-electron microscopy and crystallography have yielded reconstructions of many actin crosslinking, regulatory and binding proteins in complex with F-actin. Not least, a high-resolution structure of the Arp2/3 complex and a reconstruction with F-actin provides considerable insight into the eukaryotic machinery, vital for the formation of new F-actin barbed ends, a prerequisite for rapid actin polymerisation involved in cell shape change and motility. Addresses Institute of Biomedical and Life Sciences, Cell Biology Group, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK e-mail: [email protected]

the ordered nature of the muscle lattice, and with advances in electron microscopy and imaging of live muscle, we have now begun to build up a detailed structural knowledge of actomyosin contractility. However, molecular insight into the workings of the actinbased machinery of non-muscle cells has been more dif®cult to obtain. In the four years since this topic was last covered in this series [2] and elsewhere [3], many new structures of actin-binding and regulatory proteins have been solved, some also in complex with actin (Table 1). In this review, I will discuss some of the recent structures of actin-binding proteins, particularly those that have also been generated in complex with F-actin. I will also consider the implications for the regulation of actin polymerisation and modes of F-actin-binding.

Actin

Current Opinion in Cell Biology 2003, 15:14±22 0955-0674/03/$ ± see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0955-0674(02)00002-9 Abbreviations Arp actin-related protein ARPC Arp complex ABD actin-binding domain ABS actin-binding site CH calponin homology cryo-EM cryo-electron microscopy F-actin ®lamentous actin WASP Wiskott-Aldrich syndrome protein

Introduction

Actin and actin-related proteins (Arps) are major determinants of cell morphology in eukaryotic cells, and more recently it would seem, in prokaryotic cells too. In eukaryotic cells, actin also provides the building blocks required for cell motility, contractility and intracellular transport. Actin cannot achieve these various functions alone. A host of accessory proteins is required to regulate the equilibrium between monomeric G-actin and the polymeric ®lamentous F-actin, or to bundle and crosslink actin ®laments to one another, to other cytoskeletal elements or cellular structures, and to act as motor proteins producing contractile force or moving cargoes around the cell (reviewed in [1]). For some time, we have known the structures of the actinbased contractile machinery: actin and myosin. Owing to Current Opinion in Cell Biology 2003, 15:14±22

It would not be fair to continue without ®rst saying a few words about the structure of actin itself. Recently, we have seen two major advances in our knowledge of actin structure. On the one hand, we have perhaps the most signi®cant G-actin structure since G-actin was ®rst crystallised with other actin-binding proteins in the early 1990s [4±6], that of an uncomplexed G-actin monomer [7]. Not only that, this is the ®rst structure of a true ADP±actin crystallised from solution, rather than a crystal where the ATP has undergone gradual hydrolysis to ADP. The most signi®cant difference seen between the uncomplexed G-actin and the previous structure of monomeric actin is in subdomain 2 (see Figure 1) in the so-called `DNase 1 binding loop'. This loop is one of the most highly conserved regions in all of the actins, which in the free form is a helix, but when complexed with DNase 1 is a b-sheet. It has been suggested, however, that the ¯exible DNase 1 binding loop is induced to form a helix as a result of crystal contacts with other unrelated helices in the crystal [8]. There is also a shift in the relative positions of the two halves of the monomer with respect to previous structures. These changes in ADP±actin structure relative to ATP±actin might account for the observed differences in the ability of several actin-binding proteins to interact preferentially with ADP± or ATP± actin (see below). Compared with the ATP-free structure of Arp2 (see Figure 2a) the ATP binding cleft is quite tightly closed in the ADP±actin crystal structure. A comparison of all actin structures so far solved has led Sablin et al. [8] to suggest that the ADP±actin structure is in fact in a more closed ATP±actin conformation and www.current-opinion.com

F-actin-binding protein complexes Winder 15

Table 1 Structures of actin-binding and regulatory proteins Protein

Function

Method

References

G-actin F-actin a-actinin, b-spectrin Actophorin, ADF/Cofilin Arp2/3 Calponin Dystrophin, utrophin ERM Fimbrin Gelsolin, villin, severin Myosin Nebulin Profilin, Thymosin b4 VASP, WASP and others

Monomer Polymer Actin crosslinker F-actin severing Actin nucleation Muscle regulation F-actin-binding F-actin-binding Actin crosslinker F-actin severing Motor protein Actin scaffold Monomer binding Actin regulation

Crystal X-ray, EM Crystal; EM Crystal/NMR; EM Crystal; EM NMR/EM Crystal; EM Crystal Crystal; EM Crystal/NMR Crystal; EM NMR;EM NMR/crystal; EM Crystal

[4-6,7,10] [49,50] [45,46,51,52] [53±57] [19,20] [28] [33,34,39,40,58] [59,60] [32,36,37] [5,61±64] [65,66] [67,68] [6,69±71] [72±75]

`Method' refers to the method used in obtaining the relevant structure. EM refers to electron microscopy reconstructions but is restricted to those including atomic models of the reconstruction. Owing to the enormous size of the actin, myosin and actin-binding protein literature, only representative references have been included. Readers are directed to the following reviews for a more comprehensive coverage [2,3,76±81]. VASP, vasodilator-stimulated phosphoprotein.

not likely to be in a true ADP±actin conformation (see [8] and elegant discussions therein). Nevertheless, this structure provides new possibilities to examine the role of ATP hydrolysis in actin polymerisation, in actin structures unconstrained by actin-binding proteins. In a similar vein is a structure of G-actin complexed with the actin depolymerising drug latrunculin-A [9]. Latrunculin-A is now widely used as a cell biological tool to study actin dynamics in living cells, replacing the more traditional cytochalasins. This structure of G-actin (co-crystallised with gelsolin) with bound latrunculin-A, provides a molecular explanation for the action of latrunculin-A. Binding of latrunculin-A within the nucleotide-binding cleft of actin results in modest changes in structure at the top of subdomains 2 and 4 of actin. It also stabilises the two domains suf®ciently to prevent the conformational changes required on actin polymerisation [9], thus effectively maintaining actin in a monomeric state. The other major ®nding was that a prokaryotic protein, MreB, had a remarkably high degree of structural similarity to actin [10]. Furthermore, the MreB protein is able to form ®laments, with similar properties to those of eukaryotic F-actin [10], that have also been proposed to be involved in cell-shape control in bacteria in a manner analogous to actin in eukaryotes [11]. Remarkably, the MreB protein crystallised in a ®lamentous form providing even more insight regarding the potential arrangement of actin monomers within a ®lament [10]. Thus, prokaryotes appear to have ancestors of both tubulin Ð FtsZ Ð and actin Ð MreB (reviewed in [12]).

The Arp2/3 complex

The discovery of the Arp2/3 complex [13] could be considered as one of the holy grails of cell motility www.current-opinion.com

research. Since actin treadmilling was ®rst put forward as a mechanism to explain ATP-dependent actin polymerisation, and it was realised that many cell types had networks of actin ®laments at the leading edge, with their barbed ends to the periphery, the search has been on for the underlying mechanisms that explain actin-based motility (reviewed in [14]). However, until the discovery of the Arp2/3 complex, the ®rst good candidate for a pointed-end nucleator, there did not appear to be a particularly compelling mechanism. The ability of the Arp2/3 complex to form new barbed ends by branching at or near the ends of existing ®laments had been observed by several groups, but precisely how this was achieved was not clear (reviewed in [15]). Did the Arps form part of an existing ®lament, effectively co-polymerising with normal actin? Did they somehow bind to the side of an existing ®lament? Or was there some other explanation for the generation of new barbed ends by Arp2/3? A series of elegant experiments from the Pollard laboratory and others suggested that Arp2/3 bound to the side of a `mother' ®lament with the new `daughter' ®lament polymerising out from it at a characteristic 708 angle (Figure 2a) [16,17,18]. But how does it really work? It has taken a crystal structure of the entire Arp2/3 complex ®tted to a cryo-electron microscopy (cryo-EM) reconstruction of F-actin with the Arp2/3 complex to resolve this [19,20], but even now the picture is not entirely complete. The whole seven-member Arp2/3 complex, was puri®ed from bovine thymus, crystallised, and solved at 2 AÊ resolution [19]. The structure comprises the tightly associated and structurally related ARPC2 (Arp complex 2) and ARPC4 proteins, which form the core of the Arp2/3 complex [21], the actin-related proteins Arp2 and Arp3, Current Opinion in Cell Biology 2003, 15:14±22

16 Cell structure and dynamics

Figure 1

Figure 2

DNase 1

(a)

4 2

1 3

Current Opinion in Cell Biology

Structure of ADP±actin crystallised as an uncomplexed monomer. Actin is shown in its standard orientation as a red ribbon representation drawn from the PDB coordinates 1J67 [7]. Subdomains 1±4 are indicated and the ATP binding cleft at the top is arrowed. The bound ADP moiety is shown in space-filling representation and the helical DNase 1 binding loop is also marked.

(b) ARPC3 ARPC2

Arp3

ARPC1 a predicted b-propeller protein, and the two smaller ARPC3 and ARPC5 subunits (see Figure 2b). As predicted from sequence, the Arp2 and Arp3 subunits had a fold virtually identical to actin, with the exception of the insertions in Arp3. In both Arps, the nucleotidebinding cleft was more open than in actin, and devoid of bound nucleotide. The core proteins ARPC2 and ARPC4 were closely associated with each other, as predicted [21], making a pseudo coiled-coil interaction via their long carboxy-terminal extensions. On one side, ARPC2 and ARPC4 form a semi-cradle around the sides of the two Arp proteins; on the other side, they form an interface with the mother actin ®lament via ARPC2 in conjunction with ARPC1. The Arps cradle is completed by the aminoterminal extension of ARPC5. ARPC3 lies somewhat more peripherally, associated with the outer edge of Structure of the Arp2/3 complex and a filament junction. (a) Actin branches mediated by Arp2/3 complex. A two-dimensional reconstruction of Arp2/3-mediated actin branches (lower left foreground, barbed ends toward the top of figure) was derived by cryo-EM and image processing [20]. Reconstructions of the Arp2/3 complex at the branch junction (upper right foreground, green surface representation) and in the free, activated state (white surface representation) were also derived by cryo-EM and image processing [20]. The reconstruction indicates a large conformational change upon binding to actin (compare the overlay of green and white surface

Current Opinion in Cell Biology 2003, 15:14±22

ARPC4

Arp2

ARPC5 ARPC1 Current Opinion in Cell Biology

representations). A typical micrograph showing two actin branches is shown in the background. In the reconstruction, the Arp2/3 complex contacts the original actin filament at three different actin subunits and contributes to the new actin filament with the two first subunits (Arp2 and Arp3). Figure courtesy of Dorit Hanein. (b) Crystal structure of the inactive Arp2/3 complex [19] drawn from the coordinates 1K8K. Arp2 and Arp3 are shown in red and blue spacefill representation and all other components as ribbon diagrams ARPC1 yellow, ARPC2 orange, ARPC3 magenta, ARPC4 green and ARPC5 cyan. Arp2 is shown in the standard actin orientation (see Figure 1), only subdomains 3 and 4 of Arp2 were visible in the original crystal structure, subdomains 1 and 2 were modelled [19]. Note how the two Arps are cradled between ARPC1, ARPC2 and ARPC4 but do not align with filament-like geometry. Also note that the ATP-binding clefts are considerably more open than seen for actin itself (see Figure 1).

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F-actin-binding protein complexes Winder 17

Arp3. Although this cradle places Arp3 above Arp2 and their barbed ends point roughly in the same direction toward solution, they are not aligned relative to one another as subunits in an actin ®lament. The surfaces of ARPC3 and ARPC1 form two basic surface patches that might act as docking sites for the interaction of the Arp complex with the acidic regions of nucleation promoting factors such as Wiscott-Aldrich syndrome protein (WASP) and with the acidic surface of actin. Elegant as the structure is, as with many static structures it still doesn't really tell us how it works, particularly as the puri®ed bovine complex is inactive without the addition of F-actin, ATP and one of several nucleation promoting factors (reviewed in [22]). However, we can begin to see how it might work. The most compelling mechanism would be that one or both of the Arps formed part of either the mother or daughter ®laments or both. Previous data would tend to suggest that the Arp2/3 complex adds to the side of an existing ®lament [16,17,18,23], rather than integrating into it [24]. If we accept that one or other Arp is going to be part of either mother or daughter ®laments or both, then intuitively it looks extremely unlikely that the complex is going to undergo massive rearrangements in order for one Arp to be in mother and one in daughter ®laments. Furthermore, the sequence inserts in the Arps relative to actin would be predicted to form a cap and terminate a ®lament. It is more likely, therefore, that the two Arps undergo a modest rearrangement to bring them into a conformation consistent with normal actin ®lament geometry. This is precisely what Robinson et al. [19] propose, suggesting a 208 rigid-body rotation of Arp3, ARPC2 and ARPC3 relative to the other subunits to bring Arp3 into a position such that it would be equivalent to a short-pitch dimer in an actin ®lament. Arp2 and Arp3 would then form the nucleus of the new daughter ®lament.

that make it a good activator of the Arp2/3 complex: it is able to bind several components of the Arp2/3 complex and actin, as well as inducing conformational change in the Arp2/3 complex upon ®lament binding [25]. This serves to bring Arp2 and Arp3 into a more `®lament-like' orientation. WASP could also act to stabilise the addition of the ®rst true actin monomer onto the daughter ®lament, thus promoting nucleation by preventing the dissociation of the unstable Arp2±Arp3±actin trimer [26].

Calponin homology domain-containing actin-binding proteins

Compared with the Arp2/3 complex, discovered only nine years ago, the calponin homology (CH) domain-containing family of actin-binding proteins have been studied for considerably longer. It is only recently, however, that we have gained structural insight into the molecular interactions between these proteins and actin. The CHdomain family is divided into three main subfamilies, based on how their CH domains group: single CH domains; pairs of CH domains; and tandem pairs of CH domains (reviewed in [27]). Almost all the structural information has come from only six different proteins: calponin, which has a single CH domain; members of the spectrin family of proteins, including a-actinin, b-spectrin, dystrophin and utrophin, all of which have pairs of CH domains; and ®mbrin, which has tandem pairs of CH domains. The six proteins representing all three subfamilies not only have quite different cellular functions with respect to their organisation of the actin cytoskeleton, but also they appear to use the CH domain to interact with F-actin in different ways. The core CH-domain folds of calponin, ®mbrin and utrophin are extremely similar (Figure 3a) [28], despite low sequence similarity and diverse functional differences [29±31].

The cryo-EM reconstruction of the activated Arp2/3 complex from Acanthamoeba with F-actin branch junctions would tend to support all of these hypotheses [20]. Thus, the Arp2/3 complex binds to the side of a pre-existing ®lament, preferring newly polymerised ATP-bound F-actin over older ADP-bound ®laments. The complex would appear to contact the side of the mother ®lament via ARPC1, ARPC2 and ARPC5, with Arp3 and Arp2 forming the ®rst two subunits of the new daughter ®lament. It is thought that Arp3 is located at the pointed end of the new ®lament (for a discussion, see [19,20]).

Given the similarity in the individual CH-domain fold, the utrophin and ®mbrin actin-binding domains (ABDs), each containing two CH domains, crystallised in very different conformations. Fimbrin crystallised as a compact monomer [32], whereas utrophin and its close homologue dystrophin crystallised as extended dimers [33,34], despite all being monomers in solution. Moreover, the presence of a conserved interface between CH domains in both ®mbrin and dystrophin/utrophin crystals was indicative of a three-dimensional domain swap [33,35]. Cryo-EM reconstructions of ®mbrin or utrophin with Factin also revealed a structural difference in how these CH domain pairs interacted with actin, which in part recapitulated their crystallographic conformations.

WASP is one of many related nucleation-promoting factors required for activation of the Arp2/3 complex [22]. Gold labelling of the WASP activation domain placed WASP close to the interface between the mother ®lament and the Arp2/3 complex [20]. WASP has several activities

Using the amino-terminal half of ®mbrin, comprising a pair of EF-hands and two CH domains, Hanein et al. [36] determined a cryo-EM reconstruction with F-actin. Fimbrin bound to F-actin in a conformation consistent with that observed in the crystal structure: a compact monomer

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Current Opinion in Cell Biology 2003, 15:14±22

18 Cell structure and dynamics

Figure 3

(a)

(b)

(c)

(d)

(e)

(f)

Current Opinion in Cell Biology

Fimbrin, utrophin and calponin in complex with F-actin. (a) An overlay of the Ca backbones of the CH domains from fimbrin in white (1A0A, [32]) utrophin in grey (1BHD, [58]) and calponin in black (1H67, [28]) using LOCK (http://gene.stanford.edu/LOCK/). The core helices of the CH domains all align well, with most differences being in the interhelical loops. (b) Reconstruction of the single calponin CH domain bound to F-actin [28]. (c) Reconstruction of fimbrin ABD, comprising two CH domains, bound to F-actin [36]. (d) Reconstruction of utrophin ABD bound to F-actin at a density of 1 utrophin per actin [40]. (e) Reconstruction of utrophin ABD bound to F-actin `half-decorated' Ð one utrophin per two actins [39]. (f) Reconstruction of utrophin ABD bound to F-actin `single-decorated' Ð one utrophin per actin [39]. In all cases (b±f) two actin monomers from one strand of the F-actin filament are shown in spacefilling representation coloured red and blue, from pointed to barbed end, respectively. The CH-domain-containing ABD is shown as a yellow and gold ribbon representation with the amino-terminal CH domain (or single CH domain in the case of calponin) in yellow in each case.

with the two CH domains in close apposition (Figure 3c). Furthermore, in an extension of this work, using twodimensional arrays of actin ®laments bundled by ®mbrin, Volkmann et al. [37] were also able to determine the geometry of a complete actin crosslink. Using a combination of EM and homology modelling, it was possible to build an atomic model of a polar threedimensional actin±®mbrin bundle. The two pairs of CH domains in ®mbrin constitute two distinct ABDs through which ®mbrin is able functionally to crosslink actin ®laments, inducing conformational changes in the actin ®lament on ®mbrin binding [38]. Slight differences in the two ABDs of ®mbrin in their af®nities for F-actin, and coupling of ®mbrin binding to F-actin, crossbridge formation and actin polymerisation, suggest a dynamic feedback mechanism for the assembly of actin±®mbrin Current Opinion in Cell Biology 2003, 15:14±22

arrays [37]. Unlike the ®mbrin ABD, the utrophin ABD associated with F-actin in an open conformation similar to that observed for the utrophin ABD monomer within the crystallographic dimer [39,40] (see Figure 3d±f). These reconstructions are not in complete agreement, however. Moores et al. [40] proposed an induced ®t to account for utrophin binding to actin (Figure 3d), whereas Egelman and colleagues [39] were able to ®t the crystallographic monomer without signi®cant rearrangement, other than rotation of one the CH domains, but found two different modes of F-actin-binding (Figure 3e,f). The half-decorated ®t provides an alternative interpretation from [40] of how utrophin might bind to F-actin. The single-decorated ®t is more dif®cult to understand. In this ®t, the carboxy-terminal CH domain does not appear to contact F-actin (Figure 3f), but biochemical analyses www.current-opinion.com

F-actin-binding protein complexes Winder 19

indicate that the carboxy-terminal CH domain, while having little or no intrinsic actin-binding capacity on its own, contributes signi®cantly to the overall actin-binding properties of the whole domain, increasing its af®nity for F-actin by an order of magnitude [31,41]. The precise functional signi®cance of these different modes of binding is not clear, some may be an artefact of the way in which the EM reconstructions are necessarily achieved (i.e. under saturation binding conditions). It is unlikely that a protein with such low abundance as utrophin would ever reach a concentration at the membrane suf®cient to saturate F-actin. But these models do give us pointers from which to take a more rational approach to other analyses that will determine the precise molecular details of how utrophin and relatives interact with F-actin. Despite the different modes of binding exhibited by the different CH domains, the conserved actin-binding sites (ABS1±3 [31]) within the ABDs of ®mbrin and utrophin are still brought into close proximity with actin, and probably function as the main interface between these ABDs and F-actin [36,37,39,40]. Interestingly, a-actinin, which shares 60% homology with utrophin, crystallised as a compact monomer similar in overall conformation to ®mbrin (K Djinovic, personal communication). Moreover, in cryo-EM reconstructions a-actinin appears as a single globular density [42], consistent with it also binding to actin in a compact conformation, like ®mbrin. The globular ABD conformation may be a constraint on F-actin bundling proteins that produce links between parallel actin ®laments, in order that ABSs 1±3 can all be presented to the actin surface ef®ciently, as in ®mbrin and a-actinin. Whereas in utrophin and dystrophin, which are simple side-binding proteins that also extend along the length of the actin ®lament [43,44], an open conformation is preferred. In a recent reconstruction of whole a-actinin, obtained by cryo-EM and homology modelling, however, the best ®t was achieved to the ABD using an open conformation [45]. But this ®t did allow for the possibility of Ca2‡dependent regulation of a-actinin binding to F-actin, achieved through the interaction of the EF-hand region in one a-actinin molecule with the exposed helical linker between the two CH domains of the ABD in the apposing a-actinin molecule in the dimer [45]. The single CH domain of calponin does not have any of the conserved ABSs 1±3 found in the other CH domains and does not have intrinsic actin-binding properties [41]. Nevertheless, the cryo-EM reconstruction of the calponin CH domain NMR structure with F-actin [28] placed the calponin CH domain density in a very similar position and orientation to the utrophin CH domain (Figure 3b), where it is likely to serve as a locator for the true actin-binding residues of calponin that lie carboxy-terminal of the CH domain [28]. www.current-opinion.com

Conclusions

Until it is possible to crystallise a complex that models F-actin well, we will continue to rely on cryo-EM reconstructions, homology modelling and biochemical approaches to make any advance in our understanding of F-actin-binding protein complexes. Methodology is improving all the time and recent reconstructions are testimony to the success of the technique. Advances in our understanding of nucleotide exchange in actin will contribute greatly to deciphering the mechanistic complexities of actin dynamics and the interactions of a myriad of actin-binding proteins: the `free' G-actin structure [7] will undoubtedly help. So far, it has not been possible to crystallise free ATP±actin or ADP±Pi±actin. Perhaps proteins of this size will soon be within the range of NMR techniques; but once solved, we should have most of the necessary information to fully understand actin polymerisation. Current NMR technology is unlikely to yield structural information on the whole Arp2/3 complex. But a crystal structure of an activated complex will provide immense insight into precisely how the formation of branch junctions are regulated and precisely what orientation Arp2 and Arp3 adopt to act as a nucleus for new ®lament growth. Furthermore, the expression of fully functional Arp2/3 complex in the baculovirus system [21] paves the way for comprehensive rational mutational analysis of the Arp2/3 complex and its regulation. A model for a ®mbrin±actin crosslink has already been determined [37], and a-actinin may be soon to follow. Recent structures of the entire a-actinin rod [46], part of the a-actinin EF-hand region [47] and the a-actinin actinbinding domain (K Djinovic, personal communication), coupled with the a-actinin reconstructions carried out by the Taylor laboratory [45,48], will no doubt soon also yield information on the a-actinin crosslink.

Acknowledgements

As with any short review, it is impossible to cite all deserving works, my apologies. I am grateful to Ed Egelman, Dorit Hanein, Carolyn Moores and Tom Pollard for providing coordinates and material for ®gures, and to Kathryn Ayscough, Sutherland Maciver, Paul McLaughlin and members of my laboratory for critical reading of the manuscript and helpful comments.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:  of special interest  of outstanding interest 1.

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2.

Puius YA, Mahoney NM, Almo SC: The modular structure of actin-regulatory proteins. Curr Opin Cell Biol 1998, 10:23-34.

3.

Troys MV, Vandekerckhove J, Ampe C: Structural modules in actin binding proteins: towards a new classification. Biochim Biophys Acta 1999, 1448:323-348.

4.

Kabsch W, Mannherz HG, Suck D, Pai EF, Holmes KC: Atomic structure of the actin:DNase 1 complex. Nature 1990, 347:37-44. Current Opinion in Cell Biology 2003, 15:14±22

20 Cell structure and dynamics

5.

McLaughlin PJ, Gooch J, Mannherz H-G, Weeds AG: Atomic structure of Gelsolin Segment1 in complex with Actin and the mechanism of filament severing. Nature 1993, 364:685-692.

accuracy of a large number of previous studies investigating the molecular associations between Arp2/3 subunits and how they might interact to generate new actin filaments.

6.

Schutt CE, Myslik JC, Rozycki MD, Goonesekere NCW, Lindberg U: The structure of crystalline profilin beta-actin. Nature 1993, 364:810-816.

20. Volkmann N, Amann KJ, Stoilova-McPhie S, Egile C, Winter DC,  Hazelwood L, Heuser JE, Li R, Pollard TD, Hanein D: Structure of Arp2/3 complex in its activated state and in actin filament branch junctions. Science 2001, 293:2456-2459. This is a cryo-electron microscopy reconstruction of the Arp2/3 complex in its activated state showing significant rearrangements compared with the inactive Arp2/3 complex. The active Arp2/3 complex reconstruction was also fitted to actin branches, showing for the first time that the Arp2 and Arp3 subunits were likely to form the first two subunits of the new daughter filament.

7. 

Otterbein LR, Graceffa P, Dominguez R: The crystal structure of uncomplexed actin in the ADP state. Science 2001, 293:708-711. Rhodamine-labelled ADP G-actin was crystallised in the absence of other actin-binding proteins, for the first time providing an unhindered, but not completely unconstrained, view of G-actin (see Sablin et al. (2002) [8]). 8. 

Sablin EP, Dawson JF, VanLoock MS, Spudich JA, Egelman EH, Fletterick RJ: How does ATP hydrolysis control actin's associations? Proc Natl Acad Sci USA 2002, 99:10945-10947. An elegant discussion of the structural changes that may take place in actin upon ATP binding and hydrolysis. The authors compare all crystallographic structures of actin and define closed ATP-like and open ADP-like structures. Interestingly, the ADP G-actin structure in Otterbein et al. (2001) [7] falls into the former category. 9. 

Morton WM, Ayscough KR, McLaughlin PJ: Latrunculin alters the actin-monomer subunit interface to prevent polymerisation. Nat Cell Biol 2000, 2:375-378. The first structure of actin in complex with a compound that alters actin dynamics ± latrunculin A Ð is described. The co-crystallisation of an actin±gelsolin complex with latrunculin A provides structural evidence for how latrunculin A binds in the ATP-binding cleft of actin, thus stabilising the actin monomer and preventing actin polymerisation. 10. van den Ent F, Amos LA, Lowe J: Prokaryotic origin of the actin  cytoskeleton. Nature 2001, 413:39-44. The structure of MreB, an actin-related protein from prokaryotes, reveals an evolutionary conserved actin. Furthermore, the MreB protein crystallised as single-stranded filaments, giving further insight into how actin filaments might form. 11. Jones LJF, Carballido-Lopez R, Errington J: Control of cell shape  in bacteria: helical, actin-like filaments in Bacillus subtilis. Cell 2001, 104:913-922. Biological evidence of a role for MreB, a prokaryotic actin-like protein, in the control of cell shape in bacteria. 12. van den Ent F, Amos LA, Lowe J: Bacterial ancestry of actin and tubulin. Curr Opin Microbiol 2001, 4:634-638. 13. Machesky LM, Atkinson SJ, Ampe C, Vanderkerckhove J, Pollard TD: Purification of a cortical complex containing two unconventional actins from Acanthamoeba by affinity chromatography and profilin-agarose. J Cell Biol 1994, 127:107-115. 14. Mitchison TJ, Cramer LP: Actin-based cell motility and cell locomotion. Cell 1996, 84:371-379. 15. Borisy GG, Svitkina TM: Actin machinery: pushing the envelope. Curr Opin Cell Biol 2000, 12:104-112. 16. Amann KJ, Pollard TD: The Arp2/3 complex nucleates actin  filament branches from the sides of pre-existing filaments. Nat Cell Biol 2001, 3:306-310. Two elegant studies (see also Blanchoin et al. (2000) [17]) demonstrating the ability of the Arp 2/3 complex to nucleate new branches from the side of pre-existing actin filaments. 17. Blanchoin L, Amann KJ, Higgs HN, Marchand JB, Kaiser DA, Pollard TD: Direct observation of dendritic actin filament networks nucleated by Arp2/3 complex and WASP/Scar proteins. Nature 2000, 404:1007-1011. 18. Mullins RD, Heuser JA, Pollard TD: The interaction of Arp2/3 complex with actin: nucleation, high affinity pointed end capping, and formation of branching networks of filaments. Proc Natl Acad Sci USA 1998, 95:6181-6186. 19. Robinson RC, Turbedsky K, Kaiser DA, Marchand J-B, Higgs HN,  Choe S, Pollard TD: Crystal structure of Arp2/3 complex. Science 2001, 294:1679-1684. The 2 AÊ crystal structure of the whole Arp2/3 complex purified from bovine thymus is described. The structure of the inactive Arp2/3 complex provides a wealth of molecular detail on how the complex might act to nucleate actin filaments. It also confirmed the surprising Current Opinion in Cell Biology 2003, 15:14±22

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22 Cell structure and dynamics

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