Filament Barbed Ends Research Articles

The mode of subunit addition regulates the processive elongation of actin filaments by formin.

Formins play crucial roles in actin polymerization by nucleating filaments and regulating their elongation. Formins bind the barbed ends of filaments via their dimeric FH2 domains, which step processively onto incoming actin subunits during elongation. Actin monomers can bind formin-bound barbed ends directly or undergo diffusion-mediated delivery through interactions with formin FH1 domains and profilin. Despite its fundamental importance, a clear mechanism governing processive FH2 stepping has remained elusive. In this study, we systematically characterized the polymerization behavior of the S. cerevisiae formin Bni1p using in vitro reconstitution assays and stochastic simulations. We found that Bni1p assembles populations of filaments with lengths that depend nonlinearly on the rate of elongation. This processive behavior is dictated by a variable probability of dissociation that depends on the reaction conditions. Bni1p dissociates from barbed ends with a basal off-rate, which enables prolonged filament assembly over the course of a long lifetime at the barbed end. A bias towards FH1-mediated delivery as the dominant mechanism for polymerization curtails elongation by shortening the lifetime of the formin at the filament end. This facilitates the assembly of populations of filaments with similar average lengths, even when polymerization proceeds at different rates. Our results suggest a central role for formin FH1 domains in regulating processivity. The specific effects of FH1 domains on processivity are variable and likely tailored to the physiological function of each formin.

Reconstitution of Arp2/3-nucleated actin assembly with proteins CP, V-1, and CARMIL

Actin polymerization is often associated with membrane proteins containing capping-protein-interacting (CPI) motifs, such as capping protein, Arp2/3, myosin I linker (CARMIL), CD2AP, and WASHCAP/Fam21. CPI motifs bind directly to actin-capping protein (CP), and this interaction weakens the binding of CP to barbed ends of actin filaments, lessening the ability of CP to functionally cap those ends. The protein V-1/myotrophin binds to the F-actin-binding site on CP and sterically blocks CP from binding barbed ends. CPI-motif proteins also weaken the binding between V-1 and CP, which decreases the inhibitory effects of V-1, thereby freeing CP to cap barbed ends. Here, we address the question of whether CPI-motif proteins on a surface analogous to a membrane lead to net activation or inhibition of actin assembly nucleated by Arp2/3 complex. Using reconstitution with purified components, we discovered that CARMIL at the surface promotes and enhances actin assembly, countering the inhibitory effects of V-1 and thus activating CP. The reconstitution involves the presence of an Arp2/3 activator on the surface, along with Arp2/3 complex, V-1, CP, profilin, and actin monomers in solution, recreating key features of cell physiology.

A role for cross-linking proteins in actin filament network organization and force generation

The high turgor pressure across the plasma membrane of yeasts creates a requirement for substantial force production by actin polymerization and myosin motor activity for clathrin-mediated endocytosis (CME). Endocytic internalization is severely impeded in the absence of fimbrin, an actin filament crosslinking protein called Sac6 in budding yeast. Here, we combine live-cell imaging and mathematical modeling to gain insights into the role of actin filament crosslinking proteins in force generation. Genetic manipulation showed that CME sites with more crosslinking proteins are more effective at internalization under high load. Simulations of an experimentally constrained, agent-based mathematical model recapitulate the result that endocytic networks with more double-bound fimbrin molecules internalize the plasma membrane against elevated turgor pressure more effectively. Networks with large numbers of crosslinks also have more growing actin filament barbed ends at the plasma membrane, where the addition of new actin monomers contributes to force generation and vesicle internalization. Our results provide a richer understanding of the crucial role played by actin filament crosslinking proteins during actin network force generation, highlighting the contribution of these proteins to the self-organization of the actin filament network and force generation under increased load.

Twinfilin is a non-processive depolymerase which synergizes with formin to dramatically accelerate actin filament uncapping by 300-fold.

Cellular actin networks assemble by actin filament elongation at barbed ends and are thought to disassemble primarily by depolymerization at filament pointed ends. Contrary to this conventional understanding of actin dynamics, twinfilin was recently shown to promote barbed-end depolymerization. Twinfilin has additionally been suggested to sequester monomers and cap as well as uncap filament barbed ends. As a result, the exact mechanisms by which twinfilin affects barbed-end dynamics remain controversial. Using multicolor single-molecule microscopy, we show that both mouse and yeast twinfilin are non-processive depolymerases that interact only transiently with barbed ends (~0.2-0.5 s). Each twinfilin binding event, on average, results in the removal of one or two actin subunits. At CP-capped barbed ends, twinfilin synergizes with formin to accelerate uncapping by up to ~320-fold. We find that uncapping by twinfilin, alone and together with formin, depends on the nucleotide state of the filament, with the two proteins causing a much more modest enhancement of uncapping of newly assembled filaments. Our study thus establishes twinfilin as a multifunctional barbed-end binding protein capable of non-processively depolymerizing, transiently capping, and synergizing with formin to rapidly uncap actin filament barbed ends.

Actin Filament Barbed-End Depolymerization by Combined Action of Profilin, Cofilin, and Twinfilin

Cellular actin dynamics results from the collective action of hundreds of regulatory proteins, majority of which target actin filaments at their barbed ends. Three key actin binding proteins—profilin, cofilin, and twinfilin—individually depolymerize filament barbed ends. Notwithstanding recent leaps in our understanding of their individual action, how they collectively regulate filament dynamics remains an open question. In the absence of direct and simultaneous visualization of these proteins at barbed ends, gaining mechanistic insights has been challenging. We have here investigated multicomponent dynamics of profilin, cofilin, and twinfilin using a hybrid approach that combines high-throughput single filament experiments with theory. We discovered that while twinfilin competes with profilin, it promotes binding of cofilin to filament sides. Interestingly, contrary to previous expectations, we found that profilin and cofilin can simultaneously bind the same filament barbed end, resulting in its accelerated depolymerization. Our study reveals that pairwise interactions can effectively capture depolymerization dynamics in simultaneous presence of all three proteins. We thus believe that our approach of employing a theory-experiment dialog can potentially help decipher multicomponent regulation of actin dynamics. Published by the American Physical Society 2024

Bsp1, a fungal CPI motif protein, regulates actin filament capping in endocytosis and cytokinesis.

The capping of barbed filament ends is a fundamental mechanism for actin regulation. Capping protein controls filament growth and actin turnover in cells by binding to the barbed ends of the filaments with high affinity and slow off-rate. The interaction between capping protein and actin is regulated by capping protein interaction (CPI) motif proteins. We identified a novel CPI motif protein, Bsp1, which is involved in cytokinesis and endocytosis in budding yeast. We demonstrate that Bsp1 is an actin binding protein with a high affinity for capping protein via its CPI motif. In cells, Bsp1 regulates capping protein at endocytic sites and is a major recruiter of capping protein to the cytokinetic actin ring. Lastly, we define Bsp1-related proteins as a distinct fungi-specific CPI protein group. Our results suggest that Bsp1 promotes actin filament capping by the capping protein. This study establishes Bsp1 as a new capping protein regulator and promising candidate to regulate actin networks in fungi.

Formin tails act as a switch, inhibiting or enhancing processive actin elongation

Formins are large, multidomain proteins that nucleate new actin filaments and accelerate elongation through a processive interaction with the barbed ends of filaments. Their actin assembly activity is generally attributed to their eponymous formin homology (FH) 1 and 2 domains; however, evidence is mounting that regions outside of the FH1FH2 stretch also tune actin assembly. Here, we explore the underlying contributions of the tail domain, which spans the sequence between the FH2 domain and the C terminus of formins. Tails vary in length from ∼0 to >200 residues and contain a number of recognizable motifs. The most common and well-studied motif is the ∼15-residue-long diaphanous autoregulatory domain. This domain mediates all or nothing regulation of actin assembly through an intramolecular interaction with the diaphanous inhibitory domain in the N-terminal half of the protein. Multiple reports demonstrate that the tail can enhance both nucleation and processivity. In this study, we provide a high-resolution view of the alternative splicing encompassing the tail in the formin homology domain (Fhod) family of formins during development. While four distinct tails are predicted, we found significant levels of only two of these. We characterized the biochemical effects of the different tails. Surprisingly, the two highly expressed Fhod-tails inhibit processive elongation and diminish nucleation, while a third supports activity. These findings demonstrate a new mechanism of modulating actin assembly by formins and support a model in which splice variants are specialized to build distinct actin structures during development.

Mechanism of actin capping protein recruitment and turnover during clathrin-mediated endocytosis.

Clathrin-mediated endocytosis depends on polymerization of a branched actin network to provide force for membrane invagination. A key regulator in branched actin network formation is actin capping protein (CP), which binds to the barbed end of actin filaments to prevent the addition or loss of actin subunits. CP was thought to stochastically bind actin filaments, but recent evidence shows CP is regulated by a group of proteins containing CP-interacting (CPI) motifs. Importantly, how CPI motif proteins function together to regulate CP is poorly understood. Here, we show Aim21 and Bsp1 work synergistically to recruit CP to the endocytic actin network in budding yeast through their CPI motifs, which also allosterically modulate capping strength. In contrast, twinfilin works downstream of CP recruitment, regulating the turnover of CP through its CPI motif and a non-allosteric mechanism. Collectively, our findings reveal how three CPI motif proteins work together to regulate CP in a stepwise fashion during endocytosis.

Gelsolin from mussel's catch muscle

Proteins of the gelsolin family are Ca2+-dependent, multifunctional, actin-binding proteins containing three (S1–S3, about 40 kDa) or six (S1–S6, about 80 kDa) highly conserved repeats in the amino acid sequence. The pattern of interaction of these proteins with actin is complex: they can sever actin filaments; promote polymer nucleation after binding to two actin monomers; and cap the growing barbed end of actin filaments. In the present study, an actin polymerizing factor (46 kDa) from the adductor muscle of a bivalve mollusc has been discovered and identified for the first time. This protein has turned out to belong to the gelsolin family of actin regulatory proteins. The expression of gelsolin-like proteins in the tissues of bivalves was predicted after analyzing their proteome, but this is the first study where an actually expressed protein has been found. A primary determination of its physicochemical properties such as molecular weight, charge, resistance to urea, influence on actin polymerization by viscosity, and light scattering is carried out and the molecular structure analyzed.

Cyclase-associated protein interacts with actin filament barbed ends to promote depolymerization and formin displacement

Cyclase-associated protein (CAP) has emerged as a central player in cellular actin turnover, but its molecular mechanisms of action are not yet fully understood. Recent studies revealed that the N terminus of CAP interacts with the pointed ends of actin filaments to accelerate depolymerization in conjunction with cofilin. Here, we use invitro microfluidics-assisted TIRF microscopy to show that the C terminus of CAP promotes depolymerization at the opposite (barbed) ends of actin filaments. In the absence of actin monomers, full-length mouse CAP1 and C-terminal halves of CAP1 (C-CAP1) and CAP2 (C-CAP2) accelerate barbed end depolymerization. Using mutagenesis and structural modeling, we show that these activities are mediated by the WH2 and CARP domains of CAP. In addition, we observe that CAP collaborates with profilin to accelerate barbed end depolymerization and that these effects depend on their direct interaction, providing the first known example of CAP-profilin collaborative effects in regulating actin. In the presence of actin monomers, CAP1 attenuates barbed end growth and promotes formin dissociation. Overall, these findings demonstrate that CAP uses distinct domains and mechanisms to interact with opposite ends of actin filaments and drive turnover. Further, they contribute to the emerging view of actin barbed ends as sites of dynamic molecular regulation, where numerous proteins compete and cooperate with each other to tune polymer dynamics, similar to the rich complexity seen at microtubule ends.

A-Band assembly in avian skeletal muscles observed with super-resolution microscopy.

Myofibrils in vertebrate skeletal muscle are organized in aligned arrays of filaments formed from multiple protein components. Despite considerable information describing individual proteins, how they assemble de novo into mature myofibrils is still a challenge. Studies in our lab of sarcomeric protein localization during myofibril assembly led us to propose a three-step progression: premyofibrils to nascent myofibrils, culminating in mature myofibrils. Premyofibrils, forming at the spreading edges of muscle cells, are composed of minisarcomeres containing small bands of non-muscle myosin II filaments alternating with muscle-specific α-actinin Z-Bodies attached to barbed ends of actin filaments, establishing bipolar F-actin arrangements in sarcomeres. Assembly of nascent myofibrils occurs with addition of muscle-specific myosin II, F-actin, titin, and the alignment of Z-Bodies in adjacent fibrils to form beaded Z-Bands. Muscle-specific myosin II filaments in nascent myofibrils appear in an overlapping arrangement when viewed with wide-field and confocal microscopes. In mature myofibrils, non-muscle myosin II is absent, and M-Band proteins localize to the muscle myosin II filaments, aiding their alignment by cross-linking them into A-Bands. Super-resolution microscopy (SIM and STED) revealed muscle myosin II in mini-A-Bands in nascent myofibrils. In contrast to previous reports that vertebrate muscle myosin thick filaments form at their final 1.6 μm lengths, mini-A-Bands are first detected at a length of about 0.4 μm, and gradually increase four-fold in length to 1.6 μm in mature myofibrils. These new discoveries in avian skeletal muscle cells share a common characteristic with invertebrate muscles where some A-Bands can grow to lengths reaching 25 μm.

Molecular mechanisms of inorganic-phosphate release from the core and barbed end of actin filaments.

The release of inorganic phosphate (Pi) from actin filaments constitutes a key step in their regulated turnover, which is fundamental to many cellular functions. The mechanisms underlying Pi release from the core and barbed end of actin filaments remain unclear. Here, using human and bovine actin isoforms, we combine cryo-EM with molecular-dynamics simulations and in vitro reconstitution to demonstrate how actin releases Pi through a 'molecular backdoor'. While constantly open at the barbed end, the backdoor is predominantly closed in filament-core subunits and opens only transiently through concerted amino acid rearrangements. This explains why Pi escapes rapidly from the filament end but slowly from internal subunits. In a nemaline-myopathy-associated actin variant, the backdoor is predominantly open in filament-core subunits, resulting in accelerated Pi release and filaments with drastically shortened ADP-Pi caps. Our results provide the molecular basis for Pi release from actin and exemplify how a disease-linked mutation distorts the nucleotide-state distribution and atomic structure of the filament.

The intrinsically disordered cytoplasmic tail of a dendrite branching receptor uses two distinct mechanisms to regulate the actin cytoskeleton.

Dendrite morphogenesis is essential for neural circuit formation, yet the molecular mechanisms underlying complex dendrite branching remain elusive. Previous studies on the highly branched Caenorhabditis elegans PVD sensory neuron identified a membrane co-receptor complex that links extracellular signals to intracellular actin remodeling machinery, promoting high-order dendrite branching. In this complex, the claudin-like transmembrane protein HPO-30 recruits the WAVE regulatory complex (WRC) to dendrite branching sites, stimulating the Arp2/3 complex to polymerize actin. We report here our biochemical and structural analysis of this interaction, revealing that the intracellular domain (ICD) of HPO-30 is intrinsically disordered and employs two distinct mechanisms to regulate the actin cytoskeleton. First, HPO-30 ICD binding to the WRC requires dimerization and involves the entire ICD sequence, rather than a short linear peptide motif. This interaction enhances WRC activation by the GTPase Rac1. Second, HPO-30 ICD directly binds to the sides and barbed end of actin filaments. Binding to the barbed end requires ICD dimerization and inhibits both actin polymerization and depolymerization, resembling the actin capping protein CapZ. These dual functions provide an intriguing model of how membrane proteins can integrate distinct mechanisms to fine-tune local actin dynamics.

Cappin' or formin': Formin and capping protein competition for filament ends shapes actin networks.

How cells assemble distinct actin networks from shared cytoplasmic components remains an important unresolved question. In this issue, Wirshing et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202209105) demonstrate how capping protein and formin competition for actin filament barbed ends controls the assembly of branched and linear actin networks.

Acetylation of fission yeast tropomyosin does not promote differential association with cognate formins.

It was proposed from cellular studies that S. pombe tropomyosin Cdc8 (Tpm) segregates into two populations due to the presence or absence of an amino-terminal acetylation that specifies which formin-mediated F-actin networks it binds, but with no supporting biochemistry. To address this mechanism in vitro, we developed methods for S. pombe actin expression in Sf9 cells. We then employed 3-color TIRF microscopy using all recombinant S. pombe proteins to probe in vitro multicomponent mechanisms involving actin, acetylated and unacetylated Tpm, formins, and myosins. Acetyl-Tpm exhibits tight binding to actin in contrast to weaker binding by unacetylated Tpm. In disagreement with the differential recruitment model, Tpm showed no preferential binding to filaments assembled by the FH1-FH2-domains of two S. pombe formins, nor did Tpm binding have any bias towards the growing formin-bound actin filament barbed end. Although our in vitro findings do not support a direct formin-tropomyosin interaction, it is possible that formins bias differential tropomyosin isoform recruitment through undiscovered mechanisms. Importantly, despite a 12% sequence divergence between skeletal and S. pombe actin, S. pombe myosins Myo2 and Myo51 exhibited similar motile behavior with these two actins, validating key prior findings with these myosins that used skeletal actin.

Evolutionary tuning of barbed end competition allows simultaneous construction of architecturally distinct actin structures.

How cells simultaneously assemble actin structures of distinct sizes, shapes, and filamentous architectures is still not well understood. Here, we used budding yeast as a model to investigate how competition for the barbed ends of actin filaments might influence this process. We found that while vertebrate capping protein (CapZ) and formins can simultaneously associate with barbed ends and catalyze each other's displacement, yeast capping protein (Cap1/2) poorly displaces both yeast and vertebrate formins. Consistent with these biochemical differences, in vivo formin-mediated actin cable assembly was strongly attenuated by the overexpression of CapZ but not Cap1/2. Multiwavelength live cell imaging further revealed that actin patches in cap2∆ cells acquire cable-like features over time, including recruitment of formins and tropomyosin. Together, our results suggest that the activities of S. cerevisiae Cap1/2 have been tuned across evolution to allow robust cable assembly by formins in the presence of high cytosolic levels of Cap1/2, which conversely limit patch growth and shield patches from formins.

Profilin enhances cofilin induced actin dynamics.

In eukaryotic cells, spatiotemporal control of actin dynamics is crucial to regulate essential functions. Cellular structure, behavior and response to stimuli are directly regulated by the economy of actin monomers within different cellular networks. This is obtained by tightly regulating the rates of polymerization, depolymerization, and regeneration of actin monomers. Unfortunately, treadmilling of actin alone cannot sustain the volume of polymerization necessary to maintain cellular functions, since the rate of subunit loss becomes prohibitive to generating a constant pool of polymerizable actin monomers. Faster depolymerization can be achieved by a) manipulating the number of filament ends, and b) increasing the availability of monomers. Combination of Profilin and Cofilin leads to a synergistic rise in ATP consumption during bulk actin polymerization. This presents us with two possibilities where either the monomer becomes limiting or the nucleotide. From pyrene-actin traces we concluded that presence of Cofilin and Profilin together suppresses the initial rate of polymerization of the filament but does not change the critical concentration. To observe the effect of this cocktail on individual filaments, we reconstituted actin filaments in vitro and measured rates of filament severing and monomer dissociation. Despite no evidence of binding to filament sides, Profilin enhances Cofilin mediated filament severing in a dose dependent manner. Addition of Profilin also accelerates Cofilin mediated subunit loss from the barbed end of filaments. Using wildtype and mutant variants of Cofilin we also demonstrate that Profilin's effect on monomer dissociation in presence of Cofilin is distinct from its effect on severing by Cofilin. We present macroscopic evidence of direct interplay between Cofilin and Profilin to enhance barbed end monomer dissociation at Profilin concentrations much lower than previously reported.

Apical constriction during embryogenesis is driven by self-assembled actomyosin networks that spontaneously develop radial polarity and pulsing.

During embryogenesis, cells exert coordinated forces to sculpt tissues into complex shapes that are precursors of organs and other specialized structures. Apical constriction is a conserved deformation mode that contracts and folds tissues. In many organisms, apical constriction is driven by contractile pulses generated by apical actomyosin cellular networks (Martin and Goldstein, 2014) with radial polarity: formins and actin filament barbed ends localize to peripheral cell-cell junctions, while actin filaments point inwards with myosin II in the medial apical zone (Mason et al., 2013).

Direct observation of the conformational states of formin mDia1 at actin filament barbed ends and along the filament.

The fine regulation of actin polymerization is essential to control cell motility and architecture and to perform essential cellular functions. Formins are key regulators of actin filament assembly, known to processively elongate filament barbed ends and increase their polymerization rate. Different models have been extrapolated to describe the molecular mechanism governing the processive motion of formin FH2 domains at polymerizing barbed ends. Using negative stain electron microscopy, we directly identified for the first time two conformations of the mDia1 formin FH2 domains in interaction with the barbed ends of actin filaments. These conformations agree with the speculated open and closed conformations of the "stair-stepping" model. We observed the FH2 dimers to be in the open conformation for 79% of the data, interacting with the two terminal actin subunits of the barbed end while they interact with three actin subunits in the closed conformation. In addition, we identified and characterized the structure of single FH2 dimers encircling the core of actin filaments, and reveal their ability to spontaneously depart from barbed ends.

Actin capping protein regulates postsynaptic spine development through CPI-motif interactions.

Dendritic spines are small actin-rich protrusions essential for the formation of functional circuits in the mammalian brain. During development, spines begin as dynamic filopodia-like protrusions that are then replaced by relatively stable spines containing an expanded head. Remodeling of the actin cytoskeleton plays a key role in the formation and modification of spine morphology, however many of the underlying regulatory mechanisms remain unclear. Capping protein (CP) is a major actin regulating protein that caps the barbed ends of actin filaments, and promotes the formation of dense branched actin networks. Knockdown of CP impairs the formation of mature spines, leading to an increase in the number of filopodia-like protrusions and defects in synaptic transmission. Here, we show that CP promotes the stabilization of dendritic protrusions, leading to the formation of stable mature spines. However, the localization and function of CP in dendritic spines requires interactions with proteins containing a capping protein interaction (CPI) motif. We found that the CPI motif-containing protein Twinfilin-1 (Twf1) also localizes to spines where it plays a role in CP spine enrichment. The knockdown of Twf1 leads to an increase in the density of filopodia-like protrusions and a decrease in the stability of dendritic protrusions, similar to CP knockdown. Finally, we show that CP directly interacts with Shank and regulates its spine accumulation. These results suggest that spatiotemporal regulation of CP in spines not only controls the actin dynamics underlying the formation of stable postsynaptic spine structures, but also plays an important role in the assembly of the postsynaptic apparatus underlying synaptic function.