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Published in final edited form as: Curr Opin Cell Biol. 2025 Jan 6;92:102459. doi: 10.1016/j.ceb.2024.102459

Regulation of actin dynamics by Twinfilin

Heidi Ulrichs 1, Shashank Shekhar 1,*
PMCID: PMC11769735  NIHMSID: NIHMS2040758  PMID: 39765045

Abstract

Twinfilin is an evolutionarily conserved actin-binding protein initially mischaracterized as a tyrosine kinase but later recognized as a key regulator of cellular actin dynamics. As a member of the ADF-H family, twinfilin binds both actin monomers and filaments. Its role in sequestering G-actin is well-established, but its effects on actin filaments have been debated. While early studies suggested twinfilin caps filament barbed ends, later research demonstrated its role in nucleotide-specific barbed-end depolymerization. Further, it was initially thought to be a processive depolymerase. Recent structural and single-molecule studies have however challenged this view, indicating that twinfilin binding events result in the removal of only one or two actin subunits from the barbed end. Additionally, twinfilin directly binds capping protein (CP) and facilitates uncapping of CP-bound barbed ends. Here, we summarize twinfilin’s cellular and tissue-specific localization, and examine its evolving role in regulating cellular actin dynamics in light of its known biochemical functions.

MAIN TEXT

History and overview

Twinfilin is a 40-kDa actin-binding protein conserved across eukaryotes, including fungi, insects, and mammals. It was first identified in 1994 in a human embryonic lung fibroblast cDNA screening using an anti-phospho-tyrosine antibody [1]. Despite sharing sequence similarities with ADF/Cofilins from Lilium longiflorum, Acanthamoeba castellanii, and chicken, it was initially named A6 protein tyrosine kinase [1,2]. However, subsequent studies demonstrated that mouse and yeast twinfilin lacked kinase activity and revealed that both were actin-binding proteins instead. Due to its composition containing two, or ‘twin’, ADF/cofilin -like actin-binding domains, the protein was officially named Twinfilin [3,4].

In 2002, the crystal structure of one of mouse twinfilin’s ADF domains conclusively established its structural similarities to cofilin-1, despite only ~20% amino acid sequence identity [5]. This finding further confirmed twinfilin’s membership in the ADF-H protein family, which includes other proteins such as GMF, ADF/cofilin, coactosin, and drebrin/Abp1 [6]. ADF-H proteins have been implicated in remodeling the actin cytoskeleton either by directly binding actin or through interactions with other actin-binding proteins [6,7]. The cytosolic concentrations of twinfilin in mammalian cells and Saccharomyces cerevisiae (budding yeast) have been estimated to be in the range of 0.5–1.1 μM [8–10]. Twinfilin localizes to various actin structures, including branched networks like lamellipodia, endocytic patches, and dendritic spines, as well as linear networks found in filopodia, inner ear stereocilia, and Drosophila bristles, among others [3,11–15] (Figure 1). Here, we review known biochemical functions of twinfilin, including its interactions with actin, capping protein (CP), and lipids, and how these mechanisms affect actin-related processes across cell types, model organisms, and disease.

Figure 1: Cellular localization of twinfilin.

Figure 1:

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(a) Twinfilin has been shown to localize to areas of high actin turnover in cells, like the leading edge, including filopodia and lamellipodia (left) and endocytic patches (right) [3,11,13]. Diffused pink color indicates regions with high twinfilin localization. (b) Twinfilin is also found in the smaller two tips of inner ear stereocilia (left, [13]), in post synaptic neuronal dendritic spines (middle, [12]), and generally within tips of Drosophila bristles and specifically on ends of actin bundles (right, [15]).

Model systems and domain organization

Twinfilin is evolutionarily conserved across a wide range of species, from unicellular, trypanosomatid parasites to yeasts and animals, but is notably absent in plants. Unicellular eukaryotes and invertebrate animals, including C. elegans, typically express a single twinfilin protein from a single gene [3,16]. In contrast, vertebrates have two twinfilin genes (TWF1 and TWF2) that encode three isoforms: twinfilin 1, 2a, and 2b [17,18].

Twinfilin 2a and twinfilin 2b proteins, expressed from the same gene, TWF2, through alternative promoter usage, differ by just two amino acids and share a 67% sequence identity with twinfilin 1 [18]. The three isoforms display distinct tissue-specific expression patterns: twinfilin 1 is predominantly expressed in the liver and kidney, twinfilin 2a in the lung, spleen, and brain, while twinfilin 2b is highly expressed in skeletal muscle and heart [17].

In budding yeast, twinfilin localizes to the cytosol and cortical actin patches [3]. Overexpression of twinfilin causes these patches to shrink, whereas its deletion makes them denser. Notably, twinfilin deletion is synthetically lethal with cof1-22, a cofilin mutant with weakened F-actin binding and reduced actin disassembly activity [3,19]. Twinfilin is also synthetically lethal with pfy1-4 and pfy1-14, profilin mutants with defects in actin binding and polyproline-binding regions, respectively [20]. These observations collectively suggest that twinfilin plays a critical role in actin filament disassembly and the regulation of the actin monomer pool [3,20,21].

Like yeast, Drosophila also have a single twinfilin gene. While twinfilin knockdown is not lethal in flies, it leads to pronounced developmental defects, including prolonged larval stages, smaller adult size, reduced bristle numbers, and defects in bristle morphology. Mutant flies also exhibit reduced or complete loss of flight [15]. The complete deletion of twinfilin results in even more severe consequences, with only 1–2% of knockout flies surviving to adulthood [22]. These knockout flies exhibit excessive F-actin accumulation at neuronal postsynaptic sites, impaired border cell migration in ovaries, and reduced presynaptic endocytosis at neuromuscular junctions [22]. These findings emphasize twinfilin’s important role in cellular migration, as well as in embryonic and neuronal development.

Twinfilin isoforms and homologues are highly conserved in their domain organization (Figure 2). All isoforms contain two ADF-H domains: an N-terminal ADF domain (N-ADF or D1) and a C-terminal ADF-H domain (C-ADF or D2), connected by a flexible linker, along with a C-terminal tail [21]. Both ADF-H domains bind to actin monomers (G-actin), actin filaments (F-actin), and with lower affinities to lipids (see below). The C-terminal tail binds to capping protein, and the linker between the two ADF-H domains interacts loosely with the actin subunit bound to the N-ADF domain [23,24].

Figure 2: Domain composition and structure of twinfilin.

Figure 2:

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(a) Domain diagram of twinfilin 1 which consists of an N-terminal ADF-H domain (also called D1 or N-ADF) and a C-terminal ADF domain (also called D2 or C-ADF) that are connected by a short linker. The two ADF domains can bind actin and are trailed by a C-terminal tail region which binds CP. (b) Structures of twinfilin’s N-ADF domain (purple, left, PDB 1M4J [5]), C-ADF domain (magenta, middle, PDB 3DAW [32]), and yeast cofilin (green, right, PDB 1CFY [33]). (c) Overlapping structures of yeast cofilin and twinfilin’s D1 (left, PDB 1CFY and 1M4J [5,33], yeast cofilin and twinfilin’s D2 (middle, PDB 1CFY and 3DAW [32,33], and twinfilin’s D1 and D2 domains (right, PDB 1M4J and 3DAW [5,32]. The arrows indicate the major differences between D1 and D2, and between cofilin and D1, in their F-actin binding site. (d) Structure of the twinfilin D2 domain (magenta) bound to an actin monomer (black) (PDB 3DAW [32]). Figure panels (c) and (d) have been adapted from references [6,30]. Panel (c) Copyright (2007) National Academy of Sciences, U.S.A.

Twinfilin binds both monomeric and filamentous actin

In vitro, twinfilin binds actin monomers (G-actin) and filaments (F-actin). Twinfilin’s G-actin binding is conserved across species, from yeast to mammals, and among its three mammalian isoforms [3,4,17]. It preferentially binds ‘aged’ actin monomers, ADP-G-actin, with 10-fold higher affinity than polymerization-competent ATP-G-actin monomers. Twinfilin forms a stable 1:1 complex with G-actin and prevents nucleotide exchange on the bound actin subunit [25]. As a result, the inhibition of actin assembly by twinfilin was initially thought be caused by G-actin sequestration, similar to the effects of thymosin beta 4 [3,4,26]. When expressed individually, the two ADF-H domains can each bind an actin monomer. The C-ADF displays a 10-fold higher affinity (Kd = 0.05 μM) for G-actin than the N-ADF (Kd = 0.7 μM) [25]. Kinetic studies suggest that upon binding an actin monomer, twinfilin may undergo a conformational change followed by the transfer of the actin monomer to the high-affinity C-ADF domain [25].

While twinfilin’s interaction with G-actin is well-established, its interactions with F-actin have been debated. Twinfilin has been proposed to cap, depolymerize, and sever actin filaments depending on experimental conditions [9,27–29]. Initially, mouse twinfilin was shown to cap barbed ends in pyrene and motility assays [27]. Twinfilin exhibits a higher affinity for barbed ends under depolymerization conditions, preferring aged ADP-actin filaments (Kd = 13 nM) over ATP- or ADP-Pi-actin filaments (Kd = 0.1–0.3 μM) [25]. Since the N-ADF binds only binds G-actin, while C-ADF can also bind filament sides, it was suggested that during twinfilin’s barbed-end binding, the N-terminal ADF binds the terminal actin subunit, and the C-terminal ADF binds the same subunit on the filament side [30]. However, insights from a recent structural study have challenged this view, suggesting that both ADF domains might directly bind the terminal actin subunits, particularly in the presence of capping protein [24]. This aligns with twinfilin’s ability to inhibit profilin-mediated barbed-end depolymerization of ADP-Pi filaments, indicating that the two ADF domains sterically block profilin’s access to both terminal actin subunits [31]. At high concentrations (and in the absence of G-actin), twinfilin and cofilin can stabilize ADP-Pi filament barbed ends and inhibit depolymerization [31], reminiscent of a previously reported ternary complex between twinfilin, cofilin, and actin [25].

Recent advances using both conventional total internal reflection fluorescence (TIRF) microscopy and microfluidics-assisted TIRF (mf-TIRF) microscopy have shed new light on twinfilin’s role at filament barbed ends. Twinfilin-mediated barbed-end depolymerization depends on filament age: it accelerates depolymerization of younger ADP-Pi filaments but slows depolymerization of older ADP-actin filaments [28]. While twinfilin was initially thought to be a processive depolymerase—remaining bound to barbed ends for an average of ~70 seconds [9]—recent single-molecule studies reveal otherwise. Both mouse and yeast twinfilin bind barbed ends transiently, with an average residence time of only 0.2–0.54 seconds [34]. During this brief interaction, each twinfilin binding event likely leads to the removal of one or both terminal actin subunits, classifying twinfilin as a non-processive depolymerase (Fig. 3a). These new findings align with recent structural insights suggesting that twinfilin may bind two actin monomers simultaneously [24]. Based on a CP-twinfilin-actin structure containing one capping protein, one twinfilin, and two G-actin subunits, it was proposed that binding of the two ADF-H domains to the filament barbed end causes the terminal actin subunits to adopt a G-actin–like conformation, destabilizing them and leading to their “severing” from the filament.

Figure 3: Schematic representation of diverse interactions of twinfilin with actin filaments.

Figure 3:

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(a) Twinfilin (pink) competes with G-actin (light blue) for binding the actin filament barbed end (left). Barbed-end-bound twinfilin acts as a transient capper, preventing further addition of monomers (middle) [27]. When twinfilin dissociates from the barbed end, it departs with either one or both of the terminal actin subunits (right) [34]. (b) Twinfilin can associate with a CP-bound barbed end and cause a six-fold acceleration in dissociation of CP from the barbed end [11,24]. (c) Twinfilin can also associate with barbed ends simultaneously occupied by formin and CP (left) to form a three-protein ternary complex (middle) which promotes CP dissociation by ~300-fold and resumption of elongation by formin (right) [34,42].

Furthermore, while barbed-end depolymerization by twinfilin is conserved from yeast to mammals, yeast twinfilin (unlike mouse twinfilin) can also depolymerize filament pointed ends [9,28,35]. Yeast twinfilin synergizes with Srv2 to further enhance pointed-end depolymerization [9]. Twinfilin can also briefly associate with filament sides, causing severing, albeit only at a pH below 6.0 (and not around the physiological pH of 7.4) [29]. Additionally, twinfilin enhances cofilin-mediated severing, possibly by creating discontinuities in cofilin-decorated filament segments [36].

In summary, recent studies have helped to clarify twinfilin’s dual role in regulating actin filament assembly and disassembly. Twinfilin transiently associates with filament barbed ends, preventing filament elongation while simultaneously promoting the removal of actin monomers from the barbed end non-processively, even under conditions that favor assembly. Additionally, twinfilin sequesters actin monomers, further regulating the monomer pool available for polymerization. These biochemical activities are highly conserved across species, from unicellular parasites to animals, and are supported by complementary cell biological studies [11,16,28,34]. Together, these findings paint a clearer and unified picture of twinfilin’s essential role in maintaining proper actin filament dynamics.

Twinfilin’s other binding partners: PIP2 and CP

In addition to its direct effects on actin dynamics, twinfilin can also indirectly influence actin dynamics through its interactions with lipids and other proteins. Twinfilin binds PIP2 through a two-step process: an initial electrostatic interaction between twinfilin’s C-terminal tail and the negatively charged lipid headgroup, followed by binding of its ADF domains [21,37]. Since the C-ADF domain is also the primary site for G-actin binding, this interaction inhibits twinfilin’s ability to bind actin [21,23,37]. Phosphoinositides and CP share a binding site in twinfilin’s C-terminal tail, suggesting that PIP2 can also disrupt CP-twinfilin interaction (see below) [37]. Other actin binding proteins including cofilin [38], profilin [39], and gelsolin [40,41] are also similarly regulated by phosphoinositides at the plasma membrane.

Twinfilin directly binds CP via its ‘capping protein interaction’ (CPI) motif present in its C-terminal tail [23]. In mammalian cells, rapid actin dynamics and proper localization of CP depend upon its ability to bind twinfilin [12], and twinfilin depletion slows turnover of both actin and CP at the leading edge [11]. In vitro, twinfilin promotes CP uncapping from barbed ends by a modest six-fold increase [11] (Fig. 3b), with single-molecule analysis showing that it takes about 30 twinfilin binding events to dissociate CP [42]. Structural studies reveal that twinfilin binds CP’s “stalk” via its C-terminal tail [24,43], but its uncapping function requires interaction with actin rather than CP [11], suggesting that twinfilin and CP interact with terminal actin subunits simultaneously at barbed ends. Twinfilin also collaborates with formin to dramatically accelerate uncapping [34,42]. Although twinfilin and formin compete for barbed-end binding, the two proteins can simultaneously occupy a barbed end when CP is present [42] (Fig. 3c). The barbed-end, trimeric, CP-formin-twinfilin complex is short-lived (~1 s), and results in dissociation of CP, promoting formin-based elongation. Uncapping by twinfilin and formin (alone and together) depends on filament age. While twinfilin alone uncaps ADP filaments fourfold faster than ADP-Pi filaments, [34], when present together with formin, the two proteins accelerate uncapping of ADP and ADP-Pi filaments by ~320-fold and 11-fold respectively [34]. Thus, in vitro analysis suggests that when formin, CP, and twinfilin are all present simultaneously (e.g., in filopodia, stereocilia and lamellipodia), twinfilin could conditionally act as a pro-polymerization factor [14,17,28,44–46].

Other CP-binding proteins, such as V1/myotrophin and CARMIL, also influence CP-twinfilin interactions. While V-1 alone cannot displace CP, in combination with twinfilin, it enhances uncapping by 48-fold, eight times faster than twinfilin alone [11]. Though twinfilin protects CP from CARMIL-mediated uncapping in vitro [10], studies in mammalian cells and budding yeast suggest that twinfilin’s primary role in vivo might be to catalyze uncapping and disassemble filaments rather than shielding CP from other CPI-motif proteins like CARMIL [11,47].

Regulation of Twinfilin in vivo and its role in disease

Twinfilin is tightly regulated in vivo, with its disruption linked to various diseases. In neurons, twinfilin-1’s binding to CP is crucial for localizing CP to dendritic spine protrusions and for neuronal maturation [12]. This interaction has also been implicated in opioid addiction and withdrawal in rat models, where decreased twinfilin expression results in reduced behavioral plasticity [48]. In mice, depletion of twinfilin 2a causes blood platelet dysfunction, increased F-actin, hyperreactivity, and accelerated platelet clearance, implicating twinfilin-2a in macrothrombocytopenia, a disorder associated with low platelet counts [49].

A key regulatory mechanism of twinfilin involves microRNA that bind twinfilin-1 mRNA, acting as negative regulators of twinfilin expression. Reduced levels of these microRNAs lead to upregulation of twinfilin-1, which has been observed in non-small cell lung cancer [50], cardiac hypertrophy [51], lungs with pulmonary fibrosis from microplastics [52], and human pancreatic cancer tissue [53]. In both pancreatic and non-small cell lung cancer, this upregulation is associated with increased tumor growth, metastasis, and reduced patient survival [50,53,54].

Twinfilin-1 expression regulated via the mTOR signaling pathway has been linked to cellular proliferation [55]. Additionally, it plays a crucial role in the myogenic differentiation of progenitor cells through the YAP protein in the Hippo signaling pathway [56]. Small GTPases like Rac1 and Cdc42 induce the redistribution of twinfilin-1 (but not twinfilin-2) to cell-cell contacts and membrane ruffles, suggesting that twinfilin-1 and twinfilin-2 may be differentially regulated [17]. Post-translational modifications, such as phosphorylation, may also regulate twinfilin, although the specific residues and functional implications remain unclear. In summary, these diverse regulatory mechanisms highlight the critical role of twinfilin in various cellular processes, and its dysregulation is increasingly associated with a wide range of diseases, making it a potential target for therapeutic intervention.

Conclusions and future directions

Over the past three decades, twinfilin has transitioned from being mistakenly identified as a tyrosine kinase to being recognized as a versatile regulator of actin dynamics. It plays critical roles in actin filament turnover by sequestering actin monomers, uncapping, depolymerizing barbed ends, and working together with other proteins like capping protein (CP) and formins to regulate filament assembly and turnover. At the same time, twinfilin is also emerging as a key player in various diseases, highlighting its broader physiological significance.

Despite substantial progress in elucidating twinfilin’s biochemical functions, several questions still remain unanswered. Future research should focus on clarifying the precise molecular mechanisms by which twinfilin interacts with its partners and regulates actin dynamics in cellular and organismal contexts. The synergy between twinfilin and cyclase-associated protein (CAP) is of particular interest, given the species-specific nature of their interactions [35]. It would be crucial to investigate whether mouse CAP, like S. cerevisiae Srv2, can stabilize twinfilin’s association with filament barbed ends. This has become especially important in light of recent findings that CAP can depolymerize and uncap barbed ends [57,58]. Further research is needed to determine whether these effects occur in vivo and how they might potentially affect cellular actin dynamics.

Additionally, it will be important to investigate whether twinfilin can influence the activities of other barbed-end polymerases, such as Ena/VASP proteins, similar to its effects on formins. The recent focus has been on competition between twinfilin and CARMIL for CP binding, but with the presence of many CPI proteins, the next step should be to explore how these proteins work together in a concerted fashion, both in vitro and in vivo. Other key questions include the impact of post-translational modifications on twinfilin function, the regulation of twinfilin isoforms in vivo, and how their dysfunction may contribute to disease. Advances in high-resolution imaging and structural biology techniques will be instrumental in addressing these questions, ultimately providing a more comprehensive understanding of twinfilin’s multifunctional roles.

Acknowledgements:

SS thanks Bruce Goode for extensive discussions on twinfilin over the years. We thank the anonymous reviewers for their constructive comments that have greatly improved this review. This work was supported by NIH NIGMS grant R35GM143050 to SS.

Glossary

Capping Protein (CP)

A heterodimeric protein that binds to the barbed end of actin filaments, preventing both polymerization and depolymerization at this site

CARMIL

A protein containing a “capping protein interaction (CPI)” motif that potently uncaps capping protein (CP) from actin filament barbed ends

V1/myotrophin

A small protein that binds CP and sequesters it in an inactive state

Profilin

An actin monomer-binding protein that suppresses filament nucleation and sterically blocks actin monomer addition to the pointed end while promoting addition at the barbed end. It also accelerates barbed-end elongation by formin

Formin

An actin-binding protein that binds to the barbed end of filaments and accelerates their elongation

Cofilin

An essential actin-binding protein that binds to the sides of actin filaments, promoting severing and enhancing depolymerization at the pointed end

Cyclase-associated protein (CAP)

A protein that promotes filament depolymerization at both barbed and pointed ends. While depolymerizing, it works in conjunction with cofilin and twinfilin. It also facilitates G-actin recycling by accelerating nucleotide exchange. S. cerevisiae homolog is called Srv2

Ena/VASP

A family of tetrameric proteins that promote filament elongation by binding and tracking the barbed ends of actin filaments

PIP2

A phospholipid found in the inner leaflet of the plasma membrane. It binds and regulates several actin-binding proteins incl., cofilin, profilin, and twinfilin

Thymosin beta 4

Actin monomer-binding protein that sequesters G-actin, preventing it from polymerizing into filaments

Footnotes

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Declarations of interest: none

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