Control of actin dynamics

45

Control of actin dynamics Marie-France Carlier Actin-based motility processes are tightly linked to the rapid turnover of actin filaments. Factors that control the steady state of actin assembly, such as capping proteins and actin-depolymerizing factor/cofilin, directly affect motility. Actin-depolymerizing factor increases the treadmilling of actin filaments in vitro and in vivo. Cellular factors that are involved in linking initiation of barbed end assembly to cell signaling are being identified using Listeria monocytogenes and Saccharomyoes cerevisiae as model systems.

Addresses Laboratoire d'Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique. 91198 Gif-sur-Yvette, France; e-mail: [email protected]

Current Opinion in Cell Biology 1998, 10:45-51 http:llbiomednet.comlelecreflO955067401000045 © Current Biology Ltd ISSN 0955-0674

Abbreviations actin-depolymerizing factor ADF actin-related protein Arp capping protein CP steady-state concentration of ATP.G-actin Css Enabled/VASP homology 1 EVH1 phosphatidylinositol 4,5-bisphosphate PIP2 vasodilator-stimulated phosphoprotein VASP WASP Wiscott-Aldrich syndrome protein

Introduction Actin assembly/disassembly plays an active role in a large number of cellular functions. Recent progress in understanding the molecular mechanisms of actin-based motility in response to signaling has resulted from the synergy of different experimental approaches. First, physical models for the generation of motile force by actin polymerization have been developed. T h e generalized Brownian ratchet model [1 "°] relies on the elasticity of actin filaments and can account for the propulsion of Listena and the protrusion of lamellipodia. Second, in vitro and in vivo analyses of the control of actin dynamics by regulatory proteins delineate the fundamental concepts of actin polymerization that are used by living cells to control motility. Third, genetically tractable organisms, cell-free systems and the Listeria model provide tools with which to identify the components of the molecular scaffold that organizes the nucleation of actin filaments beneath the plasma membrane in response to stimuli. In this review, I will address the regulation of barbed (faster growing) filament end growth at steady state (treadmilling), of barbed end capping and of filament nucleation as the essential reactions that support actin-based motility.

T h e s t e a d y s t a t e o f actin a s s e m b l y : i m p l i c a t i o n s o f t r e a d m i l l i n g in a c t i n - b a s e d motility In the physiological ATP-Mg-rich cell medium, actin filaments and monomeric actin (ATP.G-actin) are maintained in a dynamic steady state. As a result of the dissipative nature of the polymerization reaction, the flux of ATP.G-actin association onto available barbed ends is balanced by the dissociation flux of actin subunits from the pointed (slower growing) ends, resulting in what is known as a treadmilling process [2] (see Figure 1). In vitro studies of pure actin showed that depolymerization from the pointed ends is very slow and imposes a rate-limiting slow treadmilling rate. As a result, the steady state concentration of ATP.G-actin, Css, that self-establishes in the medium is very close to the critical concentration of the rapidly polymerizing barbed ends (C B, so that the rate of net barbed end growth at steady B B state, k+(Css-Cc), equals the rate of depolymerization from the pointed ends (e.g. 0.5 sec -1 in the absence of any regulatory factor). Such a very slow rate of barbed end growth would generate a maximum (zero load) rate of ListeHa propulsion, or of lamellipodial protrusion, of =0.05 I.tm rain -1, two to three orders of magnitude slower than the actual velocity of actin-based motile processes. That the protusion of lamellipodia is powered by the rapid turnover of actin filaments operating by a treadmilling mechanism is, however, clearly demonstrated in many cell types ([3-6]). Even in nonmotile budding yeast, actin filaments turnover rapidly [7"']. To quantitatively account for the fast rate of actin-based foward movement of the leading edge of cells or of propulsion of Listeria (which moves at a speed of 2-20~tm min-l) in cells, the value of Css should be a few ~tM, allowing a rate of barbed end growth at steady state of 20-200 subunits sec -I. Two types of actin regulatory proteins are able to increase the value of Css through independent, potentially cumulative actions, so as to match the in vivo situation. These are capping proteins and actin-depolymerizing factors (ADFs)/cofilins.

Capping proteins enhance actin-based motility Living cells contain a variety of barbed end capping proteins. Gelsolin and its relatives (severin, adseverin/scinderin, villin and fragmin) bind very tightly to barbed ends (KA=1011-1012M-I) and sever filaments [8]. Other 'weaker', nonsevering capping proteins (which bind with an affinity of 108-109M -1) have recently received much attention. These are capping protein 132 (CP), which is the homolog of muscle Cap Z in non-muscle cells, and Cap G [9].

46

Cytoskeleton

Figure 1

~ I > I >

> Fila>'ment ] ,

ATP ADP.Pi ADP Polymerization

~

> i >~

ADP ADP ADP Depolymerization

ADP

ATP CurrentOpinionin CellBiology

Treadmilling cycle of F-actin at the steady state. At the steady state. of actin filament assembly the net rate of filament growth is zero. The steady-state concentration of ATP.G-actin established in the medium (Css) is such that the rate of depolymerizationfrom the pointed ends, k+ P (C~-Css) balances the rate of polymerizationfrom the barbed [] (Css-CC). [] CC [] and C C P are the critical concentrations for ends, k.~ assembly at the barbed and pointed ends, respectively, and are defined as the concentrations of monomeric ATP.G-actin at which the rate of growth of the considered end is zero. The value of Css lies between C B and C P, but is very close to G~ because the rate constant for G-actin association to barbed ends is much higher than that for association to pointed ends (k.~.~C(~).

cascade leads to phosphatidylinositol 4,5-bisphosphate (PIPz)-elicited dissociation of the capping proteins from barbed ends, making them available for massive actin assembly [21]. In vitro, PIP z does cause dissociation of CP from barbed ends [22"], but the situation in vivo is less clear. The amount of CP bound to F-actin unexpectedly increases upon platelet stimulation [23°]; this result may indicate that newly created barbed ends are eventually capped by CP. In a cell-free system in which actin polymerization is induced by GTPTS-loaded cdc42, the stimulation of actin assembly does not correlate with PIP 2 synthesis [24°], in agreement with previous in vivo studies. However Ca 2+- and pH-mediated modulation of PIPz's affinity for different actin-binding proteins may help to reconcile these apparently conflicting data [25°]. In addition, the high intracellular concentration of CP (1-5~tM) compared with the concentration of filament ends [14,23 °] seems incompatible with the known high affinity o f - 1 0 9 M -1 of CP for barbed ends and the partial capping of barbed ends in the cell medium. T h e possibility that the affinity of CP for barbed ends is lowered by interaction with some regulatory factor may be invoked to explain these data. T h e regulation of CP and Cap G function is an important focus for future research.

ADF/cofilin enhances filament treadmilling and actin-based motility Although strong and weaker cappers both block the dynamics of actin at the barbed end, their precise functions in vivo may differ somewhat. Indeed, the absence of severin does not affect motility of Dictyostelium [10]; the absence of gelsolin has mild effects in multicellular organisms [11] and on actin-based motility of ListeHa in cell free extracts [12"]; while mutations in CP are lethal in Drosophila at the early stage of development [13°]. T h e fact that CP is necessary for the proper organization of actin bundles in the morphogenesis of Drosophila bristles [13 °] adds to the large body of evidence in support of an active role of CP [14], of gelsolin in some cases [15-17] and of CapG [18] in actin-based motility in response to signaling. These effects were initially considered as puzzling within the conventional view that capping proteins abolish actin dynamics at the barbed ends. However, they can be accounted for if one considers that capping proteins establish a high steady state concentration of ATP-G-actin in the cell medium by capping a large proportion of barbed ends, which thus cannot incorporate actin subunits. T h e few barbed ends that are generated at the plasma membrane in response to signaling therefore grow very quickly, consistent with a 'funneled' treadmilling flux of subunits from the pointed ends of a large number of capped filaments to a small number of localized uncapped barbed ends [19",20"]. In other words, in vivo data on the function of CP substantiate the view that treadmilling supports actin-based motility.

ADF/cofilin proteins are ubiquitous, conserved actin-binding proteins [26], whose three-dimensional structures have been solved (see Almo, this issue, pp 23-34). Genetic studies pointed out a role for ADF/cofilin in stimulating cell movement [27]; however, the biochemical in vitro properties of ADF that could support this function have only recently been elucidated [28°° 1 (see [29] for a review). T h e biological function of ADF is to enhance the treadmilling rate of actin filaments. This effect is mediated by the ability of ADF to bind preferentially to the ADP-bound forms of G-actin and F-actin, thereby participating in and changing the rate parameters of actin assembly. ADF increases by 25-fold the rate of depolymerization of ADP.F-actin from the pointed filament ends specifically. As a result of the greater flux of depolymerizing ADP.G-actin, the steady-state concentration of ATP.G-actin (Css) increases up to a value C'ss such that the steady-state rate of barbed end growth, k B (C'ss--C~), balances the high rate of pointed end disassembly. By this mechanism, ADF increases the rate of barbed end growth at steady state, thus enhancing actin-based motility. T h e ADF-induced increase in the rate of propulsion of ListeHa in platelet extracts [28 °°] corroborates the enhancement of treadmilling measured in vitro. A shortening of the actin tails is also observed [12°,28°']. In budding yeast, cofilin was shown to enhance filament turnover [30°]. Rapid turnover of cortical actin also appears essential in endocytosis [7°°,30°].

T h e regulation of barbed end capping is not understood yet. It is currently hypothesized that the signaling

Whether ADF/cofilin is a filament severing factor has been questioned. T h e localization of ADF in the lamellipodium,

Control of actin dynamics earlier

where filaments are long [6], argues against a severing function in vivo. In vitro, the end-specific effects of ADF on filament assembly leave little room for a severing activity. Severing by itself cannot increase the value of Css. T h e large drops in fluorescence and viscosity of F-actin upon addition of ADF, thought to be due to severing, both result from a major change in structure and flexibility of the filaments, visualized by light scattering and electron microscopy. Recent cryoelectron microscopy and image reconstruction studies of ADF-F-actin confirm this view and demonstrate a large change in twist of the filament linked to ADF binding [31°]. Such structural alterations, which parallel the decreased thermodynamic stability of ADF-F-actin, may increase somewhat the probability of spontaneous breakage of the filament and account for the weak severing observed in optical microscopy. Systematic mutagenesis of yeast cofilin [32"'] has provided evidence for distinct regions of the protein being involved in G- and F-actin binding; further, cofilin binds actin in a manner different from that of gelsolin segment-l, despite the structural similarity between the two proteins, but consistent with their difference in function. What are the functional consequences of the effect of ADF on filament turnover? We anticipate that the increase in Css will correlate with an increase in the amount of actin sequestered by proteins such as T]34, according to the law of mass action. The ADF-induced increase in Css will also favor spontaneous nucleation of filaments from ATP.G-actin at specific sites. Since the value of the pointed end critical concentration (C P) is increased by ADF, capping proteins and ADF are expected to synergize to increase Css and the rate of barbed end growth [20"] (Figure 2). Hence, overexpression of CP and ADF may have cumulative effects in enhancing cell motility. Whether defects in CP may be rescued by overexpression of ADF may be worth examining. Similarly, as profilin is known to actively participate in barbed end growth, we anticipate that the effects of ADF on filament turnover will be amplified by profilin. The effects of a combination of these different regulatory proteins can easily be tested in reconstituted in vitro systems as well as in genetically manipulable organisms, and will open promising perspectives for future experiments. The activity of ADF/cofilins is regulated by reversible phosphorylation of a serine residue in the amino-terminal region (Set3 in vertebrate ADF [33]). Dephosphorylation, leading to ADF activation, is induced by a variety of stimuli [26], leading to morphological changes involved in macropinocytotic uptake of hormones, or secretion of granules. The associated change in the cytoplasm, from a gel to a sol, may correlate with the large drop in viscosity of F-actin that is linked to ADF binding. Therefore, ADF may play a dual role in the cell motile response, by affecting both the kinetics of assembly and the mechanical properties of actin. T h e control of ADF dephosphorylation

47

is a crucial yet unresolved issue. The regulation of the unknown phosphatase is complex, as the phosphatase inhibitors okadaic acid and calyculin A both promote dephosphorylation of cofilin in vivo [34,35]. Control of actin nucleation The view that actin filaments undergo treadmilling in a biased fashion in vivo raises a number of issues. If the growth of a small number of uncapped filaments, at the leading edge, is fed by the flux of subunits depolymerizing from the pointed ends of a large number of capped filaments, how are steady concentrations of capped and uncapped filaments, and of free capping proteins, maintained? Presumably, capping proteins become free when depolymerizing capped filaments eventually vanish, and are recycled by capping the growing barbed ends (Figure 3). The slow kinetics of the association of capping protein with barbed ends may by itself provide a means to regulate the length of actin filaments [22"]. The long axial and short peripheral filaments in Listena comet tails in protrusions [36"] seem to illustrate the variable probability of capping. To balance the effect of capping, some site-directed nucleation mechanism must operate, in conjunction with membrane-linked signaling pathways, to maintain a constant number of uncapped, steadily growing barbed ends. Two model systems have been used to identify cellular components responsible for initiation of barbed end growth. Budding yeast, in which cortical actin assembly is required for polarized growth, is a genetically suitable model that integrates all the components of the signaling cascade from the membrane to the cytokeleton; intracellular pathogens such as Listelia are simpler models of the leading edge of motile cells--these pathogens bypass the signaling pathway and use a single protein (ActA in Listeria) to initiate actin assembly and move in biochemically manipulable cell extracts [37,38]. Progress has been made using these two systems, as follows.

Extensive genetic studies have led to the definition of two functional entities in the protein ActA. Deletion studies show that amino acids 21-158 in the amino-terminal domain contain all the necessary information for actin tail formation [39"]. The central domain consists of four proline-rich repeats which form the binding site of VASP (vasodilator-stimulated phosphoprotein), the only protein thus far known to interact with ActA [40]. This region controls the rate of Listena movement [40,41]. VASP appears to belong to the Enabled/mammalian Enabled (Mena)/VASP family of proteins which is involved in signaling pathways mediating axonal outgrowth [42"']. These proteins are targetted to proline-rich ligands in focal adhesions (possibly to zyxin or vinculin) via their EVH1 (Enabled/VASP homology 1) domain, which is also found in WASP (Wiscott-Aldrich syndrome protein, [43]). VASP/Enabled/WASP proteins act as 'molecular connectors', recruiting other ligands via a Gly-(Gly-Pros)3-Gly sequence. Profilin is currently thought to be a candidate for binding to the proline-rich sequence of VASP [44].

48

Cytoskeleton

Figure 2 (a)

(b)

+ capping protein

~>

+ ADF

+ ADF

(dl

(c)

c _ _

+ capping protein

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Current Opinion in Cell Biology

Capping proteins and ADF/cofilin act together to enhance barbed (fast-growing) end assembly and thus cause lamellipodial protrusion (or Listeria movement). Actin filaments in the lamellipodium are schematically arranged in a radial array with their barbed ends growing at the leading edge. The position of the leading edge at time zero is indicated by a vertical dashed line. A curved thin gray line represents the leading edge at time zero and a thicker line the leading edge at a later time point. Straight black lines represent more recently grown actin filaments than do straight gray lines. The size of the ATP.G-actin pool is also schematized. (a) Treadmilling in the absence of ADF or capping proteins (very slow forward movement occurs). (b) The effect of capping proteins alone (represented as closed circles at the barbed ends) is to block a large number of barbed ends in the cell medium. Depolymerization of actin from the pointed ends of these filaments feeds the growth of the few uncapped barbed ends at the leading edge. (¢) ADF alone increases the steady-state rate of barbed end growth. (d) Cumulative effects of ADF and capping proteins.

Yet movement of Listena is observed in profilin-depleted extracts [451, while it is arrested by the Gly-(Gly-Pro5)3 peptide [44].

for the integrity and motility of actin patches [50"] and for endocytosis [51] in yeast altogether suggest that it plays a general role in actin-based motility. However, its function as a nucleator remains elusive.

In searching for factors that initiate actin assembly at the surface of ListeHa, Welch et al. [46"] isolated a stable complex containing the actin-related proteins Arp2 and Arp3. T h e Arp2-Arp3 complex contains five additional polypeptides, now identified [47]. This complex, first isolated from Acanthamoeba [48"], was demonstrated to bind to the sides of actin filaments in vitro. Its high conservation among eukaryotes, its intracellular distribution in the cortex of amoebae [48"] and yeast [49,50"] and in the lamellipodia of higher eukaryotes [47], and its requirement

T h e cytoplasmic factors responsible for cortical actin assembly can also be isolated from yeast. Biochemical fractionation and reconstitution assays led to the identification of a minimum of two factors that act sequentially to nucleate actin assembly, namely, Bee-1 (also known as las-17) and a new protein, pca-1 [52"']. T h e surprise is that Bee-1 is a yeast homolog of the members of the VASP/Enabled/WASP family [53"]; hence, it appears as a common factor to the yeast and Listeria systems.

Control of actin dynamics Carlier

49

Figure 3

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CurrOpi entninioCel nBilology Actin-based motility at the leading edge. Cycles of actin filament nucleation, steady-state barbed end growth, capping and release generate cell movement. ADF (A) binds only to the ADP-bound subunits of F-actin (D), not to the ATP-bound or ADP-Pi-bound subunits (T) which form the stiffer growing barbed ends [28"]. The view that the nucleating machinery preferentially binds ADP-Pi terminal subunits at the barbed ends, while capping proteins bind preferentially ADP-bound subunits, has a mechanistic interest, but is purely speculative. The effect of ADF is to increase the size of the steady-state pool of ATP.G-actin (T). Propulsive force is generated by elongation of anchored filaments through cycles of attachment-detachment of the barbed ends at the nucleating site. Capping of the barbed ends is coupled to the detachment of the filament from the nucleating site. Cross-linking proteins provide the fulcrum necessary for propulsion.

In addition, genetic evidence suggests some functional relationship between Arp2-Arp3 and Bee-1 [50e].

Conclusions T h e analysis of the roles of different actin-binding proteins in the regulation of actin dynamics in vivo and in vitro has led to the emerging concept that the control of the steady-state parameters of actin assembly (the concentration of of monomeric ATP.actin, turnover rate of F-actin etc.) plays a key role in cell motility. Regulatory proteins may act in synergy with this control mechanism.

Many pieces of the puzzle remain unconnected, however. In the future, progress is expected in several areas: the search for synthetically-developed lethal mutations in genetically-tractable organisms; more refined in vitro reconstituted sytems combining several actin-binding proteins; and systematic analyses of cellular factors involved in motility, using biochemically/immunochemically-manipulatable acellular extracts. Outstanding questions address the mechanism of actin nucleation, the structure of the nucleating machinery and its interface with actin, and the nature of the physical link between the Arp2/3 complex, Bee-1/VASP and the cell membrane.

50

Cytoskeleton

Acknowledgements This work was funded in part by the Association pour la Recherche contre le Cancer (ARC), the Association Franqaise contre les Myopathies (AFM), the European Community (grant #CH RX-CT94-0652) and the Ligue Nationale Fran~:aise contre le Cancer.

sembled in bundles may be consistent with a relatively low concentration of stady-state ATP.G-actin which results from incomplete capping. 14.

Hug C, Jay P, Reddy I, McNally J, Bridgman P, Elson E, Cooper J: Capping protein levels influence actin assembly and cell motility in Dictyostelium. Cell 1995, 81:591-600.

15.

Cunningham C, Stossel T, Kwiatkowski D: Enhanced motility in NIH 3T3 fibroblasts that overexpress gelsolin. Science 1991, 251:1233-1236. Kwiatkowski DJ: Predominant induction of gelsolin and actinbinding protein during myeloid differentiation. J Biol Chem 1988, 263:13857-13862.

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

Mogilner A, Oster G: Cell motility driven by actin polymerization. Biophys J 1996, 71:3030-3045. he 'Brownian ratchet' model [54] is extended to include the elastic properties of actin filaments in generating propulsive force. The velocity of a polymerizing filament tip is calculated as a function of the load and of the angle of the filament to the load. The calculated value matches the measured rate of Listeria movement, assuming that the steady-state concentration of ATP.G-Actin is -10pM. The calculated value predicts a 'critical angle' of 48", for fast rate of lamellipodium protusion, which is close to the observed orthogonal arrangements of filaments in lamellipodia [6]. 1.

2. 3.

4.

5.

Wegner A: Head to tail polymerization of actin. J Mol Biol 1976, 108:139-150. Wang Y: Exchange of actin subunits at the leading edge of living fibroblasts: possible role of treadmilling. J Cell Bio/1985, 101:597-602. Okabe S, Hirokawa N: Incorporation and turnover of biotinlabeled actin microinjected into fibroblastic cells: an immunoelectron microscopic study. J Cell Bio/1989, 109:1581 1595. Symons MH, Mitchison TJ: Control of actin polymerization in live and permeabilized fibroblasts. J Cell Biol 1991, 114:503-513.

Small JV: Getting the actin filaments straight: nucleationrelease or treadmilling. Trends Cell Bio/1995, 5:52-55. 7. Ayscough KR, Stryker J, Pokala N, Sanders M, Crews P, Drubin •. DG: High rates of actin filament turnover in budding yeast and roles for actin in establishment and maintenance of cell polarity revealed using the actin inhibitor latrunculin-A. J Cell Bio11997, 137:399-416. These authors are pioneering in showing that a nonmotile organism such as yeast has a dynamic actin cytoskeleton. Latrunculin-A, an actin-depolymerizing drug, causes rapid disassembly of the actin cytoskeleton. Interestingly, the drug enables the probing of cellular functions in which actin is involved, such as endocytosis, bud formation, cell polarity and polarized location of some regulatory proteins (Abpl p, cofilin, sec4p, sec8p and Smyl p). 8. Weber A, Pring M, Lin SL, Bryan, J: Role of the N- and Cterminal actin-binding domains of gelsolin in barbed end capping. Biochemistry 1991, 30:9327-9334. 9. Schafer DA, Cooper JA: Control of actin assembly at filament ends. Annu Rev Cell Dev Biol 1995, 11:497-518. 6.

10.

11.

12. •

Andre E, Brink M, Gerisch G, Isenberg G, Noegel A, Schleicher M, Segall JE, Wallraff E: A Dictyostelium mutant deficient in severin, an F-actin fragmenting protein, shows normal motility and chemotaxis. J Cell Bio/1989, 108:985-995. Witke W, Sharpe A, Hartwig J, Azuma T, Stossel T, Kwiatkowski D: Hemostatic, inflammatory, and fibroblast responses are blunted in mice lacking gelsolin. Cell 1995, 81:41-51.

Rosenblatt J, Agnew BJ, Abe H, Bamburg JR, Mitchison TJ: Xenopus actin depolymerizing factor/cofilin (XAC) is responsible for the turnover of actin filaments in Listeria monocytogenes tails. J Cell Biol 1997, 136:1323-1332. Immunodepletion of actin-depolymerizing factor (ADF)/cofilin from Xenopus egg extracts causes an increase in the length of actin tails at the rear of Listeria. Immunodepletion of gelsolin does not affect movement of Listeria. Adding back ADF, but not the Ser3-~Glu ADF mutant, restores the control tail length. The authors conclude that ADF, but not gelsolin, increases the rate of depolymerization of actin filaments in the actin tails. 13. Hopmann R, Cooper J, Miller K: Actin organization, bristle • morphology, and viability are affected by actin capping protein mutations in Drosophila. J Cell Biol 1996, 133:1293-1305. This is the first report that capping protein (CP) has an essential function in a multicellular organism. Reduced levels of CP in viable transheterozygous adults lead to defects in the organization of actin bundles in the bristles, causing abnormal morphology. The apparent increased amount of actin as-

16.

17.

Arora P, McCulloch C: Dependence of fibroblast migration on actin severing activity of gelsolin. J Biol Chem 1996, 271:20516-20523.

18,

Sun H, Kwiatkowska K, Wooten D, Yin H: Effects of CapG overexpression on agonist-induced motility and second messenger generation. J Cell Biol 1995, 129:147-156.

19. •

Dufort PA, Lumsden C J: How profilin/barbed-end synergy controls actin polymerization: a kinetic model of the ATP hydrolysis circuit. Cell Motil Cytoskeleton 1996, 35:309-330. This computer simulation study reveals the interesting point that if barbed ends are suddenly made available in a population of capped filaments, they grow at a fast rate. 20. Carlier MF, Pantaloni D: Control of actin dynamics in cell • motility. J Mol Biol 1997, 269:459. Shows how the treadmilling of actin filaments can be biased towards uncapped barbed ends by capping proteins and enhanced by actin-depolymerizing factor/cofilin, which act in a cumulative fashion to quantitatively account for the fast rates of barbed end growth observed in vivo in actin-based motility processes (lamellipodium protusion or Listeria movement). 21. Hartwig J, Bokoch G, Carpenter C, Janmey P, Taylor L, Toker A, Stossel T: Thrombin receptor ligation and activated Rac uncap actin filament barbed ends through phosphoinositide synthesis in permeabilized human platelets. Cell 1995, 82:643653. 22. •

Schafer DA, Jennings PB, Cooper JA: Dynamics of capping protein and actin assembly in vitro: uncapping barbed ends by polyphosphoinositides. J Cell Biol 1996, 135:169-179. The kinetics of capping protein (CP) association with/dissociation from barbed actin filament ends in vitro are relatively slow. The interesting consequence is that the kinetics of association of CP with growing barbed ends may by themselves control the length of actin filaments in vivo. Phosphatidylinositol 4,5-bisphosphate efficiently and rapidly dissociates CP from barbed ends. Possible models for the regulation of capping/uncapping/nucleation in actin-based motility are discussed in this paper. 23. Barkalow K, Witke W, Kwiatkowski DJ, Hartwig JH: Coordinated • regulation of platelet actin filament barbed ends by gelsolin and capping protein. J Cell Biol 1996, 134:389-399. The respective roles of gelsolin and capping protein (CP) in the regulation of actin assembly upon platelet stimulation are examined. CP is the major capping protein in platelets. The association of CP with the actin cytoskeleton increases upon platelet stimulation, which is unexpected in the context of the view that uncapping of barbed ends occurs following cell stimulation. 24. •

Zigmond S, Joyce M, Borleis J, Bokoch G, Devreotes P: Regulation of actin polymerization in cell-free systems by GTPgammaS and Cdc42. J Cell Biol 1997, 138:363-374. Actin polymerization is elicited by cdc42 in cell lysates of D. discoideum or neutrophils, and by GTP'y S-loaded cdc42 in supernatants resulting from high speed centrification of these lysates. These cell free systems are promising models for biochemical identification of the links between small G proteins and the response of actin. 25. Lin KM, Wenegieme E, Lu PJ, Chen CS, Yin HL: Gelsolin binding • to phosphatidylinositol 4,5-bisphosphate is modulated by calcium and pH. J Biol Chem 1997, 272:20443-20450. A careful study shows that although a decrease in phosphatidylinositol 4,5bisphosphate (PIP2) concentration correlates with the burst in actin assembly that occurs when cells are stimulated by agonists, the rise in cytosolic Ca2+ levels increases PIP2's affinity for gelsolin and Cap G, leading to their dissociation from barbed actin filament ends (uncapping). 26. Moon A, Drubin D: The ADF/cofilin proteins: stimulusresponsive modulators of actin dynamics. Mol Biol Cell 1995, 6:1423-1431. 27. Aizawa H, Sutoh K, Yahara I: Overexpression of cofilin stimulates bundling of actin filaments, membrane ruffling, and cell movement in Dictyostelium. J Cell Biol 1996, 132:335-344. 28. ••

Carlier MF, Laurent V, Santolini J, Melki R, Didry D, Xia GX, Hong Y, Chua NH, Pantaloni D: Actin depolymerizing factor (ADF/cofilin) enhances the rate of filament turnover:

Control of actin dynamics Carlier

implication in actin-based motility. J Cell Bio/1997, 136:13071322. Actin-depolymerizing factor (ADF) increases the rate of treadmilling of actin filaments in vitro by 25-fold. It does this by increasing the rate of filament disassembly from the pointed end specifically. ADF consistently increases the rate of Listeria propulsion in platelet extracts. Barbed end growth, which also powers lameliipodium protusion or filopodium extension, results from the ADF-regulated treadmilling of actin filaments. 29.

Theriot JA: Accelerating on a treadmill: ADF/cofilin promotes rapid actin filament turnover in the dynamic cytoskeleton. J Cell Biol 1997, 136:1165-1168.

30. he

Lappalainen P, Drubin DG: Cofilin promotes rapid actin filament turnover in vivo. Nature 1997, 388:78-82. function of cofilin in budding yeast is adressed by a genetic approach. Cofilin mutants display a slower rate of F-actin depolymerization by latrunculin-A, and harbor defects in endocytosis, while the motility of cortical actin patches is unaffected. 31.

McGough A, Pope B, Chiu W, Weeds A: Cofilin changes the

•

twist of F-actin. Implications for actin filament dynamics and

cellular function. J Cell Bio/1997, 138:771-781. Cryoelectron microscopic observations and image reconstructions show that, in binding to F-actin in a 1:1 ratio, cofilin makes contacts with two F-actin subunits along the long pitch helix, thereby increasing the twist of the filament. Interestingly, the cofilin-binding site overlaps with interfaces of myosin, profilin and gelsolin with actin, although the resulting structural features of the complexes are different. 32. ••

Lappalalnen P, Fedorov EV, Fedorov AA, Almo SC, Drubin DG: Essential functions and acUn-binding surfaces of yeast cofilin revealed by systematic mutagenesis. EMBO J 1997, 16:55205530. This mutagenesis study delineates the regions of ADF that interact with both G- and F-actin, and demonstrates the existence of other regions more specifically involved in F-actin binding. The amino-terminal region of ADF, which contains the phosphorylatable serine residue, plays a role in actin binding. Despite the similar folds in ADF and gelsolin segment-I, the two proteins bind actin differently, consistent with their different functions. 33.

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