Twinfilin bypasses assembly conditions and actin filament aging to drive barbed end depolymerization.
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ABSTRACT:
SUBMITTER: Shekhar S
PROVIDER: S-EPMC7686915 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Twinfilin is a highly conserved member of the actin depolymerization factor homology (ADF-H) protein superfamily, which also includes ADF/Cofilin, Abp1/Drebrin, GMF, and Coactosin. Twinfilin has a unique molecular architecture consisting of two ADF-H domains joined by a linker and followed by a C-terminal tail. Yeast Twinfilin, in conjunction with yeast cyclase-associated protein (Srv2/CAP), increases the rate of depolymerization at both the barbed and pointed ends of actin filaments. However, it has remained unclear whether these activities extend to Twinfilin homologs in other species. To address this, we purified the three mouse Twinfilin isoforms (mTwf1, mTwf2a, mTwf2b) and mouse CAP1, and used total internal reflection fluorescence microscopy assays to study their effects on filament disassembly. Our results show that all three mouse Twinfilin isoforms accelerate barbed end depolymerization similar to yeast Twinfilin, suggesting that this activity is evolutionarily conserved. In striking contrast, mouse Twinfilin isoforms and CAP1 failed to induce rapid pointed end depolymerization. Using chimeras, we show that the yeast-specific pointed end depolymerization activity is specified by the C-terminal ADF-H domain of yeast Twinfilin. In addition, Tropomyosin decoration of filaments failed to impede depolymerization by yeast and mouse Twinfilin and Srv2/CAP, but inhibited Cofilin severing. Together, our results indicate that Twinfilin has conserved functions in regulating barbed end dynamics, although its ability to drive rapid pointed end depolymerization appears to be species-specific. We discuss the implications of this work, including that pointed end depolymerization may be catalyzed by different ADF-H family members in different species.
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Project description: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.
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Project description:Purified actin filaments depolymerize slowly, and cytosolic conditions strongly favour actin assembly over disassembly, which has left our understanding of how actin filaments are rapidly turned over in vivo incomplete. One mechanism for driving filament disassembly is severing by factors such as Cofilin. However, even after severing, pointed-end depolymerization remains slow and unable to fully account for observed rates of actin filament turnover in vivo. Here we describe a mechanism by which Twinfilin and Cyclase-associated protein work in concert to accelerate depolymerization of actin filaments by 3-fold and 17-fold at their barbed and pointed ends, respectively. This mechanism occurs even under assembly conditions, allowing reconstitution and direct visualization of individual filaments undergoing tunable, accelerated treadmilling. Further, we use specific mutations to demonstrate that this activity is critical for Twinfilin function in vivo. These findings fill a major gap in our knowledge of cellular disassembly mechanisms, and suggest that depolymerization and severing may be deployed separately or together to control the dynamics and architecture of distinct actin networks.
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Project description:Focal adhesions (FAs) mechanically couple the extracellular matrix to the dynamic actin cytoskeleton, via transmembrane integrins and actin-binding proteins. The molecular mechanisms by which protein machineries control force transmission along this molecular axis (i.e. modulating integrin activation and controlling actin polymerization) remain largely unknown. Talin is a major actin-binding protein that controls both the inside-out activation of integrins and actin filament anchoring and thus plays a major role in the establishment of the actin-extracellular matrix mechanical coupling. Talin contains three actin-binding domains (ABDs). The N-terminal head domain contains both the F3 integrin-activating domain and ABD1, whereas the C-terminal rod contains the actin-anchoring ABD2 and ABD3. Integrin binding is regulated by an intramolecular interaction between the N-terminal head and a C-terminal five-helix bundle (R9). Whether talin ABDs regulate actin polymerization in a constitutive or regulated manner has not been fully explored. Here, we combine kinetics assays using fluorescence spectroscopy and single actin filament observation in total internal reflection fluorescence microscopy, to examine relevant functions of the three ABDs of talin. We find that the N-terminal ABD1 blocks actin filament barbed-end elongation, whereas ABD2 and ABD3 do not show any activity. By mutating residues in ABD1, we find that this activity is mediated by a positively charged surface that is partially masked by its intramolecular interaction with R9. Our results also demonstrate that, once this intramolecular interaction is released, the integrin-bound talin head retains the ability to inhibit actin assembly.
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Project description: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 in vitro 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.
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Project description:Capping protein, a heterodimeric protein composed of alpha and beta subunits, is a key cellular component regulating actin filament assembly and organization. It binds to the barbed ends of the filaments and works as a 'cap' by preventing the addition and loss of actin monomers at the end. Here we describe the crystal structure of the chicken sarcomeric capping protein CapZ at 2.1 A resolution. The structure shows a striking resemblance between the alpha and beta subunits, so that the entire molecule has a pseudo 2-fold rotational symmetry. CapZ has a pair of mobile extensions for actin binding, one of which also provides concomitant binding to another protein for the actin filament targeting. The mobile extensions probably form flexible links to the end of the actin filament with a pseudo 2(1) helical symmetry, enabling the docking of the two in a symmetry mismatch.
| S-EPMC152911 | biostudies-literature
Project description:The interaction of capping protein (CP) with actin filaments is an essential element of actin assembly and actin-based motility in nearly all eukaryotes. The dendritic nucleation model for Arp2/3-based lamellipodial assembly features capping of barbed ends by CP, and the formation of filopodia is proposed to involve inhibition of capping by formins and other proteins. To understand the molecular basis for how CP binds the barbed end of the actin filament, we have used a combination of computational and experimental approaches, primarily involving molecular docking and site-directed mutagenesis. We arrive at a model that supports all of our biochemical data and agrees very well with a cryo-electron microscopy structure of the capped filament. CP interacts with both actin protomers at the barbed end of the filament, and the amphipathic helix at the C-terminus of the ?-subunit binds to the hydrophobic cleft on actin, in a manner similar to that of WH2 domains. These studies provide us with new molecular insight into how CP binds to the actin filament.
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Project description:Cell motility and actin homeostasis depend on the control of polarized growth of actin filaments. Profilin, an abundant regulator of actin dynamics, supports filament assembly at barbed ends by binding G-actin. Here, we demonstrate how, by binding and destabilizing filament barbed ends at physiological concentrations, profilin also controls motility, cell migration, and actin homeostasis. Profilin enhances filament length fluctuations. Profilin competes with Capping Protein at barbed ends, which generates a lower amount of profilin-actin than expected if barbed ends were tightly capped. Profilin competes with barbed end polymerases, such as formins and VopF, and inhibits filament branching by WASP-Arp2/3 complex by competition for filament barbed ends, accounting for its as-yet-unknown effects on motility and metastatic cell migration observed in this concentration range. In conclusion, profilin is a major coordinator of polarized growth of actin filaments, controlled by competition between barbed end cappers, trackers, destabilizers, and filament branching machineries.
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Project description:We investigate, using molecular dynamics, how the severing protein, actin depolymerization factor (ADF)/cofilin, modulates the structure, conformational dynamics, and mechanical properties of actin filaments. The actin and cofilactin filament bending stiffness and corresponding persistence lengths obtained from all-atom simulations are comparable to values obtained from analysis of thermal fluctuations in filament shape. Filament flexibility is strongly affected by the nucleotide-linked conformation of the actin subdomain 2 DNase-I binding loop and the filament radial mass density distribution. ADF/cofilin binding between subdomains 1 and 3 of a filament subunit triggers reorganization of subdomain 2 of the neighboring subunit such that the DNase-I binding loop (DB-loop) moves radially away from the filament. Repositioning of the neighboring subunit DB-loop significantly weakens subunit interactions along the long-pitch helix and lowers the filament bending rigidity. Lateral filament contacts between the hydrophobic loop and neighboring short-pitch helix monomers in native filaments are also compromised with cofilin binding. These works provide a molecular interpretation of biochemical solution studies documenting the disruption of filament subunit interactions and also reveal the molecular basis of actin filament allostery and its linkage to ADF/cofilin binding.
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Project description:The focal adhesion protein vinculin is an actin-binding protein involved in the mechanical coupling between the actin cytoskeleton and the extracellular matrix. An autoinhibitory interaction between the N-terminal head (Vh) and the C-terminal tail (Vt) of vinculin masks an actin filament side-binding domain in Vt. The binding of several proteins to Vh disrupts this intramolecular interaction and exposes the actin filament side-binding domain. Here, by combining kinetic assays and microscopy observations, we show that Vt inhibits actin polymerization by blocking the barbed ends of actin filaments. In low salt conditions, Vt nucleates actin filaments capped at their barbed ends. We determined that the interaction between vinculin and the barbed end is characterized by slow association and dissociation rate constants. This barbed end capping activity requires C-terminal amino acids of Vt that are dispensable for actin filament side binding. Like the side-binding domain, the capping domain of vinculin is masked by an autoinhibitory interaction between Vh and Vt. In contrast to the side-binding domain, the capping domain is not unmasked by the binding of a talin domain to Vh and requires the dissociation of an additional autoinhibitory interaction. Finally, we show that vinculin and the formin mDia1, which is involved in the processive elongation of actin filaments in focal adhesions, compete for actin filament barbed ends.
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