Structural conservation between the actin monomer-binding sites of twinfilin and actin-depolymerizing factor (ADF)/cofilin

Twinfilin is an evolutionarily conserved actin monomer-binding protein that regulates cytoskeletal dynamics in organisms from yeast to mammals. It is composed of two actin-depolymerization factor homology (ADF-H) domains that show approximately 20% sequence identity to ADF/cofilin proteins. In contrast to ADF/cofilins, which bind both G-actin and F-actin and promote filament depolymerization, twinfilin interacts only with G-actin. To elucidate the molecular mechanisms of twinfilin-actin monomer interaction, we determined the crystal structure of the N-terminal ADF-H domain of twinfilin and mapped its actin-binding site by site-directed mutagenesis. This domain has similar overall structure to ADF/cofilins, and the regions important for actin monomer binding in ADF/cofilins are especially well conserved in twinfilin. Mutagenesis studies show that the N-terminal ADF-H domain of twinfilin and ADF/cofilins also interact with actin monomers through similar interfaces, although the binding surface is slightly extended in twinfilin. In contrast, the regions important for actin-filament interactions in ADF/cofilins are structurally different in twinfilin. This explains the differences in actin-interactions (monomer versus filament binding) between twinfilin and ADF/cofilins. Taken together, our data show that the ADF-H domain is a structurally conserved actin-binding motif and that relatively small structural differences at the actin interfaces of this domain are responsible for the functional variation between the different classes of ADF-H domain proteins.     

Mammalian twinfilin sequesters ADP-G-actin and caps filament barbed ends: implications in motility

Twinfilins are conserved actin-binding proteins composed of two actin depolymerizing factor homology (ADF-H) domains. Twinfilins are involved in diverse morphological and motile processes, but their mechanism of action has not been elucidated. Here, we show that mammalian twinfilin both sequesters ADP-G-actin and caps filament barbed ends with preferential affinity for ADP-bound ends. Twinfilin replaces capping protein and promotes motility of N-WASP functionalized beads in a biomimetic motility assay, indicating that the capping activity supports twinfilin's function in motility. Consistently, in vivo twinfilin localizes to actin tails of propelling endosomes. The ADP-actin-sequestering activity cooperates with the filament capping activity of twinfilin to finely regulate motility due to processive filament assembly catalyzed by formin-functionalized beads. The isolated ADF-H domains do not cap barbed ends nor promote motility, but sequester ADP-actin, the C-terminal domain showing the highest affinity. A structural model for binding of twinfilin to barbed ends is proposed based on the similar foldings of twinfilin ADF-H domains and gelsolin segments.     

Structure of the actin-depolymerizing factor homology domain in complex with actin

Actin dynamics provide the driving force for many cellular processes including motility and endocytosis. Among the central cytoskeletal regulators are actin-depolymerizing factor (ADF)/cofilin, which depolymerizes actin filaments, and twinfilin, which sequesters actin monomers and caps filament barbed ends. Both interact with actin through an ADF homology (ADF-H) domain, which is also found in several other actin-binding proteins. However, in the absence of an atomic structure for the ADF-H domain in complex with actin, the mechanism by which these proteins interact with actin has remained unknown. Here, we present the crystal structure of twinfilin's C-terminal ADF-H domain in complex with an actin monomer. This domain binds between actin subdomains 1 and 3 through an interface that is conserved among ADF-H domain proteins. Based on this structure, we suggest a mechanism by which ADF/cofilin and twinfilin inhibit nucleotide exchange of actin monomers and present a model for how ADF/cofilin induces filament depolymerization by weakening intrafilament interactions.     

Two biochemically distinct and tissue-specific twinfilin isoforms are generated from the mouse Twf2 gene by alternative promoter usage

Twf (twinfilin) is an evolutionarily conserved regulator of actin dynamics composed of two ADF-H (actin-depolymerizing factor homology) domains. Twf binds actin monomers and heterodimeric capping protein with high affinity. Previous studies have demonstrated that mammals express two Twf isoforms, Twf1 and Twf2, of which at least Twf1 also regulates cytoskeletal dynamics by capping actin filament barbed-ends. In the present study, we show that alternative promoter usage of the mouse Twf2 gene generates two isoforms, which differ from each other only at their very N-terminal region. Of these isoforms, Twf2a is predominantly expressed in non-muscle tissues, whereas expression of Twf2b is restricted to heart and skeletal muscle. Both proteins bind actin monomers and capping protein, as well as efficiently capping actin filament barbed-ends. However, the N-terminal ADF-H domain of Twf2b interacts with ADP-G-actin with a 5-fold higher affinity than with ATP-G-actin, whereas the corresponding domain of Twf2a binds ADP-G-actin and ATP-G-actin with equal affinities. Taken together, these results show that, like Twf1, mouse Twf2 is a filament barbed-end capping protein, and that two tissue-specific and biochemically distinct isoforms are generated from the Twf2 gene through alternative promoter usage.     

The dynamic instability of actin filament barbed ends

The turnover of actin filament networks in cells has long been considered to reflect the treadmilling behavior of pure actin filaments in vitro, where only the pointed ends depolymerize. Newly discovered molecular mechanisms challenge this notion, as they provide evidence of situations in which growing and depolymerizing barbed ends coexist.

IntroductionIn cells, actin assembles into filament networks with diverse architectures and lifetimes, playing key roles in functions such as endocytosis, cell motility, and cell division. These filament networks are maintained and renewed by actin turnover, which implies that assembly and disassembly must take place simultaneously and in a controlled manner within the networks. Each actin filament end has the ability to either grow or shrink, depending on the concentration of actin and regulatory proteins, but pure actin treadmills at steady state: ATP-actin is added at the barbed end at a rate matching the departure of ADP-actin from the pointed end, and ATP hydrolysis takes place within the filament. This hallmark feature of actin dynamics has been known for decades (Wegner, 1976) and has been generalized to the cell context, in which it is commonly assumed that actin polymerization takes place at the barbed end, while depolymerization takes place only at the pointed end (whether it be the ends of filaments within the network or the ends of fragments that have detached from it). This notion is reinforced by the fact that the cytoplasm contains high concentrations of monomeric actin (G-actin) in complex with profilin (Funk et al., 2019), which is unable to bind to pointed ends and should drive the elongation of all noncapped barbed ends.Recently, however, in vitro studies have identified two seemingly independent mechanisms in which, in the presence of profilin-actin, filament barbed ends alternate between phases of growth and depolymerization. This behavior, referred to as “dynamic instability,” is widely observed for microtubules but was unexpected for actin filaments. It suggests that cells could use barbed ends for both elongation and disassembly.Driving the depolymerization of barbed ends with cofilin side-decorationProteins of the actin depolymerizing factor (ADF)/cofilin family (henceforth cofilin) are composed of a single ADF-homology (ADF-H) domain and are mostly known for their actin filament–severing activity (De La Cruz, 2009). Cofilin binds cooperatively to the sides of actin filaments, forming clusters where the conformation of the filament is locally altered, leading to its severing at cofilin cluster boundaries. In addition, the barbed ends of cofilin-decorated filaments steadily depolymerize, despite the presence of G-actin and profilin-actin (Fig. 1 A) and even capping protein (CP) in solution (Wioland et al., 2017, 2019). This unexpected result likely originates from the conformational change of actin subunits at the barbed end, induced by cofilin side-binding. As a consequence, filaments exposed to G-actin (with or without profilin), CP, and cofilin alternate between phases of barbed-end elongation and barbed-end depolymerization. In these conditions, actin filament barbed ends thus exhibit a form of dynamic instability.Open in a separate windowFigure 1.Two mechanisms that give rise to barbed-end depolymerization in elongation-promoting conditions. (A) When a cofilin side-decorated region reaches the barbed end, adding a new actin or profilin-actin becomes very difficult, and the barbed end depolymerizes. Not represented: Capping by CP can lead to depolymerization, as it allows the cofilin cluster to reach the barbed end, which then has a much weaker affinity for CP and steadily depolymerizes. Also, severing events occur at cofilin cluster boundaries, creating new barbed ends, either bare or cofilin-decorated. (B) Twinfilin binds to the barbed end, preventing its elongation and causing its depolymerization. Whether twinfilin remains processively attached to the depolymerizing barbed end or departs with the actin subunits is still unknown. Twinfilin has no impact on the elongation of mDia1-bearing barbed ends.Driving the depolymerization of barbed ends with twinfilin end-targetingTwinfilin has two ADF-H domains, but unlike cofilin, it binds poorly to the sides of actin filaments. Rather, twinfilin appears to mainly sequester ADP-actin monomers and target the barbed end to modulate its elongation and capping. Recent in vitro studies have shown that the interaction of twinfilin with actin filament barbed ends could drive their depolymerization, even in the presence of G-actin and profilin-actin (Johnston et al., 2015; Hakala et al., 2021; Shekhar et al., 2021). Very interestingly, the processive barbed-end elongator formin mDia1 is able to protect barbed ends from twinfilin, allowing them to sustain elongation (Shekhar et al., 2021). This leads to a situation in which, as filaments are exposed to profilin-actin and twinfilin, mDia1-bearing barbed ends elongate while bare barbed ends depolymerize (Fig. 1 B). It is safe to assume that, if filaments were continuously exposed to this protein mix including formin in solution, they would alternate between phases of growth and shrinkage over time, as formins come on and fall off the barbed end. This mix of proteins would therefore constitute another situation causing actin filament dynamic instability.From actin treadmilling to dynamic instability, in cells?This newly identified versatile behavior of actin filaments is reminiscent of microtubules. While dynamic instability is the hallmark behavior of microtubules, they can also be made to treadmill steadily by adding 4 microtubule-associated proteins (Arpağ et al., 2020). In cells, both microtubule dynamic instability and treadmilling have been clearly observed (Wittmann et al., 2003). In contrast, the disassembly of single actin filaments, either embedded in a network or severed from it, has not yet been directly observed in cells. Despite insights from techniques such as single-molecule speckle microscopy, it is still unclear from which end actin filaments depolymerize, even in networks that appear to globally treadmill, such as the lamellipodium. Pointed end depolymerization alone cannot account for what is observed in cells (Miyoshi et al., 2006) and alternative mechanisms have been proposed, including brutal filament-to-monomer transitions occurring in bursts, driven by cofilin, coronin, and Aip1 (Brieher, 2013; Tang et al., 2020).In cells, the high amounts of available G-actin (tens of micromolars; Funk et al., 2019) should limit barbed-end depolymerization. Based on the reported on-rate for ATP–G-actin at the barbed ends of cofilin-decorated filaments (Wioland et al., 2017, 2019), we can estimate that these barbed ends, under such conditions, would depolymerize for tens of seconds before being “rescued,” which is enough to remove tens of subunits from each filament. In contrast, twinfilin concentrations similar to those of G-actin appear necessary to drive barbed-end depolymerization (Hakala et al., 2021; Shekhar et al., 2021). As proteomics studies in HeLa cells report that twinfilin is 50-fold less abundant than actin, this may be difficult to achieve in cells (Bekker-Jensen et al., 2017). However, future studies may uncover proteins, or posttranslational modifications of actin, that enhance the ability of twinfilin to drive barbed-end depolymerization in the presence of high concentrations of profilin-actin.Molecular insights and possible synergiesWhile cofilin and twinfilin both interact with actin via ADF-H domains, they appear to drive barbed-end depolymerization through different mechanisms: twinfilin by directly targeting the barbed end, and cofilin by decorating the filament sides, thereby changing the conformation of the filament and putting its barbed end in a depolymerization-prone state.The two mechanisms, nonetheless, share clear similarities. For instance, cofilin side-binding and twinfilin end-targeting both slow down ADP-actin barbed-end depolymerization, compared with bare ADP-actin filaments (Wioland et al., 2017; Hakala et al., 2021; Shekhar et al., 2021). Strikingly, a crystal structure of the actin/twinfilin/CP complex indicates that the actin conformational change induced by twinfilin binding at the barbed end is similar to that induced by cofilin decorating the sides (Mwangangi et al., 2021). It is thus possible that the dynamic instability of actin filament barbed ends reflects the same conformation changes, triggered either by cofilin side-decoration or twinfilin end-targeting.In addition to decorating the filament sides, cofilin targets ADP-actin barbed ends. Unlike twinfilin, the direct interaction of cofilin with the barbed end cannot cause its depolymerization in the presence of ATP-actin monomers. Indeed, cofilin end-targeting accelerates the depolymerization of ADP-actin barbed ends in the absence of G-actin, but cofilin does not appear to interact with growing ATP-actin barbed ends (Wioland et al., 2017). This is in stark contrast with twinfilin end-targeting, which slows down ADP-actin depolymerization and accelerates ADP–Pi-actin depolymerization (Shekhar et al., 2021). These different behaviors regarding the nucleotide state of actin are intriguing and should be investigated further.Cofilin thus needs to decorate the filament sides in order to have an impact on barbed-end dynamics in elongation-promoting conditions. However, it is unknown whether cofilin side-decoration extends all the way to the terminal subunits and occupies sites that twinfilin would target. Thus, it is unclear whether cofilin and twinfilin would compete or synergize to drive barbed-end depolymerization.Synergies with other proteins are also worth further investigation, CP being an interesting candidate. Cofilin side-decoration drastically decreases the barbed-end affinity for CP, and capped filaments are thereby an efficient intermediate to turn growing barbed ends into depolymerizing barbed ends (Wioland et al., 2017). Twinfilin interacts with CP and the barbed end to enhance uncapping (Hakala et al., 2021; Mwangangi et al., 2021). Since CP can bind mDia1-bearing barbed ends and displace mDia1 (Bombardier et al., 2015; Shekhar et al., 2015), perhaps CP can also contribute to turn growing, mDia1-bearing barbed ends into depolymerizing barbed ends, by removing mDia1 from barbed ends and subsequently getting displaced from the barbed end by twinfilin.Finally, it is worth noting that profilin, which does not contain an ADF-H domain, also interacts with the barbed face of G-actin and with the barbed end of the filament. When profilin is in sufficient excess, it is able to promote barbed-end depolymerization in the presence of ATP–G-actin (Pernier et al., 2016). Unlike twinfilin, its depolymerization-promoting activity is not prevented by formin mDia1, and it thus does not lead to dynamic instability (bare and mDia1-bearing barbed ends all either grow or depolymerize). The coexistence of growing, mDia1-bearing barbed ends and depolymerizing, twinfilin-targeted barbed ends (Fig. 1 B) was observed in the presence of profilin (Shekhar et al., 2021), but profilin actually may not be required. Future studies should determine the exact role of profilin in this mechanism.ConclusionThe extent to which barbed-end dynamic instability contributes to actin turnover in cells is not known, but possible molecular mechanisms have now been identified. They should change the way we envision actin network dynamics, as we must now consider the possibility that cells also exploit the barbed end for disassembly. More work is needed to further document these mechanisms, but the idea of a “generalized treadmilling” has now been contradicted at its source: in vitro experiments.     

A high-affinity interaction with ADP-actin monomers underlies the mechanism and in vivo function of Srv2/cyclase-associated protein

Cyclase-associated protein (CAP), also called Srv2 in Saccharomyces cerevisiae, is a conserved actin monomer-binding protein that promotes cofilin-dependent actin turnover in vitro and in vivo. However, little is known about the mechanism underlying this function. Here, we show that S. cerevisiae CAP binds with strong preference to ADP-G-actin (Kd 0.02 microM) compared with ATP-G-actin (Kd 1.9 microM) and competes directly with cofilin for binding ADP-G-actin. Further, CAP blocks actin monomer addition specifically to barbed ends of filaments, in contrast to profilin, which blocks monomer addition to pointed ends of filaments. The actin-binding domain of CAP is more extensive than previously suggested and includes a recently solved beta-sheet structure in the C-terminus of CAP and adjacent sequences. Using site-directed mutagenesis, we define evolutionarily conserved residues that mediate binding to ADP-G-actin and demonstrate that these activities are required for CAP function in vivo in directing actin organization and polarized cell growth. Together, our data suggest that in vivo CAP competes with cofilin for binding ADP-actin monomers, allows rapid nucleotide exchange to occur on actin, and then because of its 100-fold weaker binding affinity for ATP-actin compared with ADP-actin, allows other cellular factors such as profilin to take the handoff of ATP-actin and facilitate barbed end assembly.     

A stochastic analysis of actin polymerization in the presence of twinfilin and gelsolin

We develop an efficient stochastic simulation algorithm for analyzing actin filament growth and decay in the presence of various actin-binding proteins. The evolution of nucleotide profiles of filaments can be tracked and the resulting feedback to actin-binding proteins is incorporated. The computational efficiency of the new method enables us to focus on experimentally realistic problems, and as one example we use it to analyze the experimental data of Helfer et al. [(2006). Mammalian twinfilin sequesters ADP-G-actin and caps filament barbed ends: implications in motility. EMBO J. 25, 1184-1195] on the capping and G-actin sequestering activity of twinfilin. We show that the binding specificity of twinfilin for ADP-G-actin is crucial for the observed biphasic evolution of the filament length distribution in the presence of twinfilin, and we demonstrate that twinfilin can be an essential part of the molecular machinery for regulating filament lengths after a short burst of polymerization. Significantly, our simulations indicate that the pyrenyl-actin fluorescence experiments would fail to report the emergence of large filaments under certain experimental conditions.     

Mapping the cofilin binding site on yeast G-actin by chemical cross-linking

Cofilin is a major cytoskeletal protein that binds to both monomeric actin (G-actin) and polymeric actin (F-actin) and is involved in microfilament dynamics. Although an atomic structure of the G-actin-cofilin complex does not exist, models of the complex have been built using molecular dynamics simulations, structural homology considerations, and synchrotron radiolytic footprinting data. The hydrophobic cleft between actin subdomains 1 and 3 and, alternatively, the cleft between actin subdomains 1 and 2 have been proposed as possible high-affinity cofilin binding sites. In this study, the proposed binding of cofilin to the subdomain 1/subdomain 3 region on G-actin has been probed using site-directed mutagenesis, fluorescence labeling, and chemical cross-linking, with yeast actin mutants containing single reactive cysteines in the actin hydrophobic cleft and with cofilin mutants carrying reactive cysteines in the regions predicted to bind to G-actin. Mass spectrometry analysis of the cross-linked complex revealed that cysteine 345 in subdomain 1 of mutant G-actin was cross-linked to native cysteine 62 on cofilin. A cofilin mutant that carried a cysteine substitution in the α3-helix (residue 95) formed a cross-link with residue 144 in actin subdomain 3. Distance constraints imposed by these cross-links provide experimental evidence for cofilin binding between actin subdomains 1 and 3 and fit a corresponding docking-based structure of the complex. The cross-linking of the N-terminal region of recombinant yeast cofilin to actin residues 346 and 374 with dithio-bis-maleimidoethane (12.4 Å) and via disulfide bond formation was also documented. This set of cross-linking data confirms the important role of the N-terminal segment of cofilin in interactions with G-actin.     

Mammals have two twinfilin isoforms whose subcellular localizations and tissue distributions are differentially regulated

Twinfilin is a highly conserved actin monomer-binding protein that regulates cytoskeletal dynamics in organisms from yeast to mammals. In addition to the previously characterized mammalian twinfilin-1, a second protein with approximately 65% sequence identity to twinfilin-1 exists in mouse and humans. However, previous studies failed to identify any actin binding activity in this protein (Rohwer, A., Kittstein, W., Marks, F., and Gschwendt, M. (1999) Eur. J. Biochem. 263, 518-525). Here we show that this protein, which we named twinfilin-2, is indeed an actin monomer-binding protein. Similar to twinfilin-1, mouse twinfilin-2 binds ADP-G-actin with a higher affinity (KD = 0.12 microM) than ATP-G-actin (KD = 1.96 microM) and efficiently inhibits actin filament assembly in vitro. Both mouse twinfilins inhibit the nucleotide exchange on actin monomers and directly interact with capping protein. Furthermore, the actin interactions of mouse twinfilin-1 and twinfilin-2 are inhibited by phosphatidylinositol (4,5)-bisphosphate. Although biochemically very similar, our Northern blots and in situ hybridizations show that these two proteins display distinct expression patterns. Twinfilin-1 is the major isoform in embryos and in most adult mouse non-muscle cell-types, whereas twinfilin-2 is the predominant isoform of adult heart and skeletal muscles. Studies with isoform-specific antibodies demonstrated that although the two proteins show similar localizations in unstimulated cells, they are regulated by different mechanisms. The small GTPases Rac1 and Cdc42 induce the redistribution of twinfilin-1 to membrane ruffles and cell-cell contacts, respectively, but do not affect the localization of twinfilin-2. Taken together, these data show that mammals have two twinfilin isoforms, which are differentially expressed and regulated through distinct cellular signaling pathways.     

Mammalian and Malaria Parasite Cyclase-associated Proteins Catalyze Nucleotide Exchange on G-actin through a Conserved Mechanism

Cyclase-associated proteins (CAPs) are among the most highly conserved regulators of actin dynamics, being present in organisms from mammals to apicomplexan parasites. Yeast, plant, and mammalian CAPs are large multidomain proteins, which catalyze nucleotide exchange on actin monomers from ADP to ATP and recycle actin monomers from actin-depolymerizing factor (ADF)/cofilin for new rounds of filament assembly. However, the mechanism by which CAPs promote nucleotide exchange is not known. Furthermore, how apicomplexan CAPs, which lack many domains present in yeast and mammalian CAPs, contribute to actin dynamics is not understood. We show that, like yeast Srv2/CAP, mouse CAP1 interacts with ADF/cofilin and ADP-G-actin through its N-terminal α-helical and C-terminal β-strand domains, respectively. However, in the variation to yeast Srv2/CAP, mouse CAP1 has two adjacent profilin-binding sites, and it interacts with ATP-actin monomers with high affinity through its WH2 domain. Importantly, we revealed that the C-terminal β-sheet domain of mouse CAP1 is essential and sufficient for catalyzing nucleotide exchange on actin monomers, although the adjacent WH2 domain is not required for this function. Supporting these data, we show that the malaria parasite Plasmodium falciparum CAP, which is entirely composed of the β-sheet domain, efficiently promotes nucleotide exchange on actin monomers. Collectively, this study provides evidence that catalyzing nucleotide exchange on actin monomers via the β-sheet domain is the most highly conserved function of CAPs from mammals to apicomplexan parasites. Other functions, including interactions with profilin and ADF/cofilin, evolved in more complex organisms to adjust the specific role of CAPs in actin dynamics.     

Structural effects of cofilin on longitudinal contacts in F-actin

Structural effects of yeast cofilin on skeletal muscle and yeast actin were examined in solution. Cofilin binding to native actin was non-cooperative and saturated at a 1:1 molar ratio, with K(d)相似文献   

The V-ATPase subunit C binds to polymeric F-actin as well as to monomeric G-actin and induces cross-linking of actin filaments

Previously, we have shown that the V-ATPase holoenzyme as well as the V1 complex isolated from the midgut of the tobacco hornworm (Manduca sexta) exhibits the ability of binding to actin filaments via the V1 subunits B and C (Vitavska, O., Wieczorek, H., and Merzendorfer,H. (2003) J. Biol. Chem. 278, 18499-18505). Since the recombinant subunit C not only enhances actin binding of the V1 complex but also can bind separately to F-actin, we analyzed the interaction of recombinant subunit C with actin. We demonstrate that it binds not only to F-actin but also to monomeric G-actin. With dissociation constants of approximately 50 nm, the interaction exhibits a high affinity, and no difference could be observed between binding to ATP-G-actin or ADP-G-actin, respectively. Unlike other proteins such as members of the ADF/cofilin family, which also bind to G- as well as to F-actin, subunit C does not destabilize actin filaments. On the contrary, under conditions where the disassembly of F-actin into G-actin usually occurred, subunit C stabilized F-actin. In addition, it increased the initial rate of actin polymerization in a concentration-dependent manner and was shown to cross-link actin filaments to bundles of varying thickness. Apparently bundling is enabled by the existence of at least two actin-binding sites present in the N- and in the C-terminal halves of subunits C, respectively. Since subunit C has the possibility to dimerize or even to oligomerize, spacing between actin filaments could be variable in size.     

Coactosin-Like 1 Antagonizes Cofilin to Promote Lamellipodial Protrusion at the Immune Synapse

Actin depolymerizing factor-homology (ADF-H) family proteins regulate actin filament dynamics at multiple cellular locations. Herein, we have investigated the function of the ADF-H family member coactosin-like 1 (COTL1) in the regulation of actin dynamics at the T cell immune synapse (IS). We initially identified COTL1 in a genetic screen to identify novel regulators of T cell activation, and subsequently found that it associates with F-actin and localizes at the IS in response to TCR+CD28 stimulation. Live cell microscopy showed that depletion of COTL1 protein impaired T cell spreading in response to TCR ligation and abrogated lamellipodial protrusion at the T cell – B cell contact site, producing only a band of F-actin. Significantly, re-expression of wild type COTL1, but not a mutant deficient in F-actin binding could rescue these defects. In addition, COTL1 depletion reduced T cell migration. In vitro studies showed that COTL1 and cofilin compete with each other for binding to F-actin, and COTL1 protects F-actin from cofilin-mediated depolymerization. While depletion of cofilin enhanced F-actin assembly and lamellipodial protrusion at the IS, concurrent depletion of both COTL1 and cofilin restored lamellipodia formation. Taken together, our results suggest that COTL1 regulates lamellipodia dynamics in part by protecting F-actin from cofilin-mediated disassembly.     

Regulation of the Cortical Actin Cytoskeleton in Budding Yeast by Twinfilin,a Ubiquitous Actin Monomer-sequestering Protein

Here we describe the identification of a novel 37-kD actin monomer binding protein in budding yeast. This protein, which we named twinfilin, is composed of two cofilin-like regions. In our sequence database searches we also identified human, mouse, and Caenorhabditis elegans homologues of yeast twinfilin, suggesting that twinfilins form an evolutionarily conserved family of actin-binding proteins. Purified recombinant twinfilin prevents actin filament assembly by forming a 1:1 complex with actin monomers, and inhibits the nucleotide exchange reaction of actin monomers. Despite the sequence homology with the actin filament depolymerizing cofilin/actin-depolymerizing factor (ADF) proteins, our data suggests that twinfilin does not induce actin filament depolymerization. In yeast cells, a green fluorescent protein (GFP)–twinfilin fusion protein localizes primarily to cytoplasm, but also to cortical actin patches. Overexpression of the twinfilin gene (TWF1) results in depolarization of the cortical actin patches. A twf1 null mutation appears to result in increased assembly of cortical actin structures and is synthetically lethal with the yeast cofilin mutant cof1-22, shown previously to cause pronounced reduction in turnover of cortical actin filaments. Taken together, these results demonstrate that twinfilin is a novel, highly conserved actin monomer-sequestering protein involved in regulation of the cortical actin cytoskeleton.     

NMR solution structures of actin depolymerizing factor homology domains

Actin is one of the most conserved proteins in nature. Its assembly and disassembly are regulated by many proteins, including the family of actin‐depolymerizing factor homology (ADF‐H) domains. ADF‐H domains can be divided into five classes: ADF/cofilin, glia maturation factor (GMF), coactosin, twinfilin, and Abp1/drebrin. The best‐characterized class is ADF/cofilin. The other four classes have drawn much less attention and very few structures have been reported. This study presents the solution NMR structure of the ADF‐H domain of human HIP‐55‐drebrin‐like protein, the first published structure of a drebrin‐like domain (mammalian), and the first published structure of GMF β (mouse). We also determined the structures of mouse GMF γ, the mouse coactosin‐like domain and the C‐terminal ADF‐H domain of mouse twinfilin 1. Although the overall fold of the five domains is similar, some significant differences provide valuable insights into filamentous actin (F‐actin) and globular actin (G‐actin) binding, including the identification of binding residues on the long central helix. This long helix is stabilized by three or four residues. Notably, the F‐actin binding sites of mouse GMF β and GMF γ contain two additional β‐strands not seen in other ADF‐H structures. The G‐actin binding site of the ADF‐H domain of human HIP‐55‐drebrin‐like protein is absent and distorted in mouse GMF β and GMF γ.     

Mapping the interaction of cofilin with subdomain 2 on actin

Cofilin, a member of the actin-depolymerizing factor (ADF)/cofilin family of proteins, is a key regulator of actin dynamics. Cofilin binds to monomer (G-) and filamentous (F-) actin, severs the filaments, and increases their turnover rate. Electron microscopy studies suggested cofilin interactions with subdomains 2 and 1/3 on adjacent actin protomers in F-actin. To probe for the presence of a cryptic cofilin binding site in subdomain 2 in G-actin, we used transglutaminase-mediated cross-linking, which targets Gln41 in subdomain 2. The cross-linking proceeded with up to 85% efficiency with skeletal alpha-actin and WT yeast actin, yielding a single product corresponding to a 1:1 actin-cofilin complex but was strongly inhibited in Q41C yeast actin (in which Q41 was substituted with cysteine). LC-MS/MS analysis of the proteolytic fragments of this complex mapped the cross-linking to Gln41 on actin and Gly1 on recombinant yeast cofilin. The actin-cofilin (AC) heterodimer was purified on FPLC for analytical ultracentrifugation and electron microscopy analysis. Sedimentation equilibrium and velocity runs revealed oligomers of AC in G-actin buffer. In the presence of excess cofilin, the covalent AC heterodimer bound a second cofilin, forming a 2:1 cofilin/actin complex, as revealed by sedimentation results. Under polymerizing conditions the cross-linked AC formed mostly short filaments, which according to image reconstruction were similar to uncross-linked actin-cofilin filaments. Although a majority of the cross-linking occurs at Gln41, a small fraction of the AC cross-linked complex forms in the Q41C yeast actin mutant. This secondary cross-linking site was sequenced by MALDI-MS/MS as linking Gln360 in actin to Lys98 on cofilin. Overall, these results demonstrate that the region around Gln41 (subdomain 2) is involved in a weak binding of cofilin to G-actin.     

Mechanism of interaction of Acanthamoeba actophorin (ADF/Cofilin) with actin filaments.

We characterized the interaction of Acanthamoeba actophorin, a member of ADF/cofilin family, with filaments of amoeba and rabbit skeletal muscle actin. The affinity is about 10 times higher for muscle actin filaments (Kd = 0.5 microM) than amoeba actin filaments (Kd = 5 microM) even though the affinity for muscle and amoeba Mg-ADP-actin monomers (Kd = 0.1 microM) is the same (Blanchoin, L., and Pollard, T. D. (1998) J. Biol. Chem. 273, 25106-25111). Actophorin binds slowly (k+ = 0.03 microM-1 s-1) to and dissociates from amoeba actin filaments in a simple bimolecular reaction, but binding to muscle actin filaments is cooperative. Actophorin severs filaments in a concentration-dependent fashion. Phosphate or BeF3 bound to ADP-actin filaments inhibit actophorin binding. Actophorin increases the rate of phosphate release from actin filaments more than 10-fold. The time course of the interaction of actophorin with filaments measured by quenching of the fluorescence of pyrenyl-actin or fluorescence anisotropy of rhodamine-actophorin is complicated, because severing, depolymerization, and repolymerization follows binding. The 50-fold higher affinity of actophorin for Mg-ADP-actin monomers (Kd = 0.1 microM) than ADP-actin filaments provides the thermodynamic basis for driving disassembly of filaments that have hydrolyzed ATP and dissociated gamma-phosphate.     

Binding of [3H]Serotonin to Skeletal Muscle Actin

We previously observed that the neurotransmitter 5-hydroxytryptamine (5-HT, serotonin) binds with high- and low-affinity interactions to an actin-like protein prepared from rat brain synaptosomes. In this study, we examined its binding to highly purified actin obtained from rabbit skeletal muscle. Monomeric G-actin bound serotonin with high and low affinities, exhibiting equilibrium dissociation constants (KD values) of 5 X 10(-5) M and 4 X 10(-3) M, respectively. The serotonin binding site on actin was distinct from those sites previously characterized for divalent cations, nucleotides, and cytochalasin alkaloids. The binding of serotonin (1 microM) to G-actin was increased as much as 26-fold by divalent cations. Potassium iodine (KI) increased the affinity of G-actin for serotonin, KD values for this binding being 3 X 10(-7) M and X 10(-5) M. Serotonin bound with even higher affinity to polymerized F-actin, with KD values of 2 X 10(-8) M and 2 X 10(-5) M. However, the total number of binding sites on F-actin was only about 4% of the number of G-actin. The binding of serotonin (0.1 microM) to G-actin could be inhibited by phenothiazines (1 microM) or reserpine (10 microM), but not by classical antagonists of serotonin receptors or by drugs that release serotonin or inhibit its uptake. The binding of serotonin to actin in vivo may participate in a contractile process related to neurotransmitter release.     

Mapping the ADF/cofilin binding site on monomeric actin by competitive cross-linking and peptide array: evidence for a second binding site on monomeric actin

The binding sites for actin depolymerising factor (ADF) and cofilin on G-actin have been mapped by competitive chemical cross-linking using deoxyribonuclease I (DNase I), gelsolin segment 1 (G1), thymosin beta4 (Tbeta4), and vitamin D-binding protein (DbP). To reduce ADF/cofilin induced actin oligomerisation we used ADP-ribosylated actin. Both vitamin D-binding protein and thymosin beta4 inhibit binding by ADF or cofilin, while cofilin or ADF and DNase I bind simultaneously. Competition was observed between ADF or cofilin and G1, supporting the hypothesis that cofilin preferentially binds in the cleft between sub-domains 1 and 3, similar to or overlapping the binding site of G1. Because the affinity of G1 is much higher than that of ADF or cofilin, even at a 20-fold excess of the latter, the complexes contained predominantly G1. Nevertheless, cross-linking studies using actin:G1 complexes and ADF or cofilin showed the presence of low concentrations of ternary complexes containing both ADF or cofilin and G1. Thus, even with monomeric actin, it is shown for the first time that binding sites for both G1 and ADF or cofilin can be occupied simultaneously, confirming the existence of two separate binding sites. Employing a peptide array with overlapping sequences of actin overlaid by cofilin, we have identified five sequence stretches of actin able to bind cofilin. These sequences are located within the regions of F-actin predicted to bind cofilin in the model derived from image reconstructions of electron microscopical images of cofilin-decorated filaments. Three of the peptides map to the cleft region between sub-domains 1 and 3 of the upper actin along the two-start long-pitch helix, while the other two are in the DNase I loop corresponding to the site of the lower actin in the helix. In the absence of any crystal structures of ADF or cofilin in complex with actin, these studies provide further information about the binding sites on F-actin for these important actin regulatory proteins.     

Cytochalasin D acts as an inhibitor of the actin-cofilin interaction

Cofilin, a key regulator of actin filament dynamics, binds to G- and F-actin and promotes actin filament turnover by stimulating depolymerization and severance of actin filaments. In this study, cytochalasin D (CytoD), a widely used inhibitor of actin dynamics, was found to act as an inhibitor of the G-actin-cofilin interaction by binding to G-actin. CytoD also inhibited the binding of cofilin to F-actin and decreased the rate of both actin polymerization and depolymerization in living cells. CytoD altered cellular F-actin organization but did not induce net actin polymerization or depolymerization. These results suggest that CytoD inhibits actin filament dynamics in cells via multiple mechanisms, including the well-known barbed-end capping mechanism and as shown in this study, the inhibition of G- and F-actin binding to cofilin.