Drebrin-induced Stabilization of Actin Filaments

Drebrin is a mammalian neuronal protein that binds to and organizes filamentous actin (F-actin) in dendritic spines, the receptive regions of most excitatory synapses that play a crucial role in higher brain functions. Here, the structural effects of drebrin on F-actin were examined in solution. Depolymerization and differential scanning calorimetry assays show that F-actin is stabilized by the binding of drebrin. Drebrin inhibits depolymerization mainly at the barbed end of F-actin. Full-length drebrin and its C-terminal truncated constructs were used to clarify the domain requirements for these effects. The actin binding domain of drebrin decreases the intrastrand disulfide cross-linking of Cys-41 (in the DNase I binding loop) to Cys-374 (C-terminal) but increases the interstrand disulfide cross-linking of Cys-265 (hydrophobic loop) to Cys-374 in the yeast mutants Q41C and S265C, respectively. We also demonstrate, using solution biochemistry methods and EM, the rescue of filament formation by drebrin in different cases of longitudinal interprotomer contact perturbation: the T203C/C374S yeast actin mutant and grimelysin-cleaved skeletal actin (between Gly-42 and Val-43). Additionally, we show that drebrin rescues the polymerization of V266G/L267G, a hydrophobic loop yeast actin mutant with an impaired lateral interface formation between the two filament strands. Overall, our data suggest that drebrin stabilizes actin filaments through its effect on their interstrand and intrastrand contacts.     

Organelle Movements along Actin Filaments and Microtubules

Organelle movements involving microtubules and actin filaments are a conspicuous and important feature of many plant cells. Movements have recently been supported in preparations of demembranated cytoplasm and reconstituted from purified proteins. The favored mechanism involves organelles carrying a force-generating ATPase moving along a track provided by either actin filaments or microtubules. Cytoplasmic free Ca²⁺ concentration regulates at least some organelle movements.     

Human Muscle LIM Protein Dimerizes along the Actin Cytoskeleton and Cross-Links Actin Filaments

The muscle LIM protein (MLP) is a nucleocytoplasmic shuttling protein playing important roles in the regulation of myocyte remodeling and adaptation to hypertrophic stimuli. Missense mutations in human MLP or its ablation in transgenic mice promotes cardiomyopathy and heart failure. The exact function(s) of MLP in the cytoplasmic compartment and the underlying molecular mechanisms remain largely unknown. Here, we provide evidence that MLP autonomously binds to, stabilizes, and bundles actin filaments (AFs) independently of calcium and pH. Using total internal reflection fluorescence microscopy, we have shown how MLP cross-links actin filaments into both unipolar and mixed-polarity bundles. Quantitative analysis of the actin cytoskeleton configuration confirmed that MLP substantially promotes actin bundling in live myoblasts. In addition, bimolecular fluorescence complementation (BiFC) assays revealed MLP self-association. Remarkably, BiFC complexes mostly localize along actin filament-rich structures, such as stress fibers and sarcomeres, supporting a functional link between MLP self-association and actin cross-linking. Finally, we have demonstrated that MLP self-associates through its N-terminal LIM domain, whereas it binds to AFs through its C-terminal LIM domain. Together our data support that MLP contributes to the maintenance of cardiomyocyte cytoarchitecture by a mechanism involving its self-association and actin filament cross-linking.     

Stepwise Sliding of Single Actin and Myosin Filaments

Dynamics of sliding were explored in isolated actin and myosin filaments. Sliding occurs in steps. The steps are integer multiples of 2.7 nm, which is equal to the monomeric repeat along the actin filament. When filaments were forced to slide in the reverse direction, the size paradigm was the same. This size paradigm is parallel to that seen in the kinesin-microtubule system, where step size is an integer multiple of the tubulin repeat along the microtubule.     

Origin of Twist-Bend Coupling in Actin Filaments

Actin filaments are semiflexible polymers that display large-scale conformational twisting and bending motions. Modulation of filament bending and twisting dynamics has been linked to regulatory actin-binding protein function, filament assembly and fragmentation, and overall cell motility. The relationship between actin filament bending and twisting dynamics has not been evaluated. The numerical and analytical experiments presented here reveal that actin filaments have a strong intrinsic twist-bend coupling that obligates the reciprocal interconversion of bending energy and twisting stress. We developed a mesoscopic model of actin filaments that captures key documented features, including the subunit dimensions, interaction energies, helicity, and geometrical constraints coming from the double-stranded structure. The filament bending and torsional rigidities predicted by the model are comparable to experimental values, demonstrating the capacity of the model to assess the mechanical properties of actin filaments, including the coupling between twisting and bending motions. The predicted actin filament twist-bend coupling is strong, with a persistence length of 0.15-0.4 μm depending on the actin-bound nucleotide. Twist-bend coupling is an emergent property that introduces local asymmetry to actin filaments and contributes to their overall elasticity. Up to 60% of the filament subunit elastic free energy originates from twist-bend coupling, with the largest contributions resulting under relatively small deformations. A comparison of filaments with different architectures indicates that twist-bend coupling in actin filaments originates from their double protofilament and helical structure.     

Cover Caption: Organization of Actin Filaments

Plant actin depolymerizing factor (ADF) binds to both monomeric and filamentous actin, and plays a key role in the organization of the actin cytoskeleton. In this issue, Dong et al. (pp. 250–261) demonstrate that charged residues Arg98 and Lys100 of ADF1 are essential for both G‐ and F‐actin binding, and that basic residues on β‐strand 5 (K82/A) and α‐helix 4 (R135/A, R137/A) form another actin binding site for F‐actin.     

Dynamin2 GTPase and Cortactin Remodel Actin Filaments

The large GTPase dynamin, best known for its activities that remodel membranes during endocytosis, also regulates F-actin-rich structures, including podosomes, phagocytic cups, actin comet tails, subcortical ruffles, and stress fibers. The mechanisms by which dynamin regulates actin filaments are not known, but an emerging view is that dynamin influences F-actin via its interactions with proteins that interact directly or indirectly with actin filaments. We show here that dynamin2 GTPase activity remodels actin filaments in vitro via a mechanism that depends on the binding partner and F-actin-binding protein, cortactin. Tightly associated actin filaments cross-linked by dynamin2 and cortactin became loosely associated after GTP addition when viewed by transmission electron microscopy. Actin filaments were dynamically unraveled and fragmented after GTP addition when viewed in real time using total internal reflection fluorescence microscopy. Cortactin stimulated the intrinsic GTPase activity of dynamin2 and maintained a stable link between actin filaments and dynamin2, even in the presence of GTP. Filaments remodeled by dynamin2 GTPase in vitro exhibit enhanced sensitivity to severing by the actin depolymerizing factor, cofilin, suggesting that GTPase-dependent remodeling influences the interactions of actin regulatory proteins and F-actin. The global organization of the actomyosin cytoskeleton was perturbed in U2-OS cells depleted of dynamin2, implicating dynamin2 in remodeling actin filaments that comprise supramolecular F-actin arrays in vivo. We conclude that dynamin2 GTPase remodels actin filaments and plays a role in orchestrating the global actomyosin cytoskeleton.Controlled assembly and disassembly of actin filaments underlies movement, shape, division, trafficking of lipids and proteins of the cell and pathogenesis by infectious bacteria and viruses. Several proteins and signaling circuits modulate actin filament dynamics, including proteins that nucleate formation of new filaments, filament cross-linking proteins that stabilize branched and bundled filament arrays, and depolymerizing factors that promote filament disassembly (1). Studies with reconstituted systems show that a single actin nucleating factor, such as the Arp2/3 complex together with a nucleation-promoting factor, a barbed end capping protein to preserve the actin monomer pool and promote nucleation, and a filament disassembly factor, such as ADF/cofilin, are sufficient to establish a dynamic dendritic actin network in vitro that mimics many properties of actin networks at the leading edge of migrating cells (2–4). However, the mechanisms for coordinating the organization and dynamics of actin filaments associated with higher-order cellular structures such as the subcortical F-actin network, F-actin at focal adhesions, and actomyosin arrays are not as well understood.Considerable evidence indicates that the large GTPase dynamin, a key mediator of membrane remodeling and fission, also influences actin filaments (reviewed in Refs. 5–7). Although the mechanisms are unknown, dynamin could influence actin filaments via its interactions with a number of proteins that directly or indirectly regulate actin filament assembly, filament stability, or filament organization. For example, several protein scaffolds biochemically link dynamin and the Arp2/3 complex activating factor, N-WASP, suggesting that the machinery for de novo actin assembly may be targeted or activated by dynamin (6, 8, 9). Dynamin2 is associated with several dynamic F-actin-containing structures in vivo, including podosomes, F-actin comet tails, phagocytic cups, dynamic cortical ruffles, and pedestal structures elaborated by enteropathogenic Escherichia coli (10–20). Cortactin, which directly binds both dynamin and actin filaments, is associated with many of the same dynamic actin structures as dynamin (5, 7) and is required for both clathrin-dependent and -independent endocytosis (21, 22). Thus, dynamin-cortactin interaction may be an important link between actin filaments and dynamin during formation or turnover of F-actin-rich structures.Considerable evidence supports the notion that GTP hydrolysis by dynamin catalyzes membrane fission activity via GTPase-dependent changes in conformation (23, 24) or via GTPase-dependent cycles of assembly and disassembly (25, 26). We hypothesize that GTPase-dependent changes in dynamin linked via its interacting proteins to actin filaments or actin regulators could similarly influence actin filaments. Overexpressed, dominant negative dynamin mutant proteins impaired in binding or hydrolyzing GTP (most often the dynamin-K44A mutation) perturb a variety of F-actin-rich cellular structures, including stress fibers and focal adhesions (27, 28), dendritic spines of neurons (29), podosomes (12, 30), actin comet tails (13, 14), phagocytic cups and bacteria-induced pedestal structures (16, 19), and dynamic cortical ruffles (15, 17). In addition, F-actin of stress fibers and overall cell morphology were perturbed in Clone9 cells expressing a mutant dynamin2 protein lacking the C-terminal proline-rich domain, the domain through which dynamin2 interacts with actin regulatory factors (11). Whereas existing data indicates that the specific effects of dynamin GTPase activity on F-actin structures are cell type- and structure-specific, a general conclusion is that dynamin GTPase activity influences the organization or turnover of a subset of actin filaments.To determine the mechanisms by which dynamin2 GTPase activity influences actin filaments, we developed biochemical and microscopic approaches to quantitatively assess and observe GTPase-dependent effects on actin filaments formed in vitro with Arp2/3 complex, cortactin, and dynamin2. The activities of dynamin2 on actin filaments in vivo were examined in cells with disrupted dynamin2 function using siRNA²-mediated suppression or pharmacologic inhibition. We report that dynamin2 GTPase, together with cortactin, functions as a dynamic actin filament remodeling complex that influences the global organization of the actomyosin cytoskeleton.     

Myosin and Tropomyosin Stabilize the Conformation of Formin-nucleated Actin Filaments

The conformational elasticity of the actin cytoskeleton is essential for its versatile biological functions. Increasing evidence supports that the interplay between the structural and functional properties of actin filaments is finely regulated by actin-binding proteins; however, the underlying mechanisms and biological consequences are not completely understood. Previous studies showed that the binding of formins to the barbed end induces conformational transitions in actin filaments by making them more flexible through long range allosteric interactions. These conformational changes are accompanied by altered functional properties of the filaments. To get insight into the conformational regulation of formin-nucleated actin structures, in the present work we investigated in detail how binding partners of formin-generated actin structures, myosin and tropomyosin, affect the conformation of the formin-nucleated actin filaments using fluorescence spectroscopic approaches. Time-dependent fluorescence anisotropy and temperature-dependent Förster-type resonance energy transfer measurements revealed that heavy meromyosin, similarly to tropomyosin, restores the formin-induced effects and stabilizes the conformation of actin filaments. The stabilizing effect of heavy meromyosin is cooperative. The kinetic analysis revealed that despite the qualitatively similar effects of heavy meromyosin and tropomyosin on the conformational dynamics of actin filaments the mechanisms of the conformational transition are different for the two proteins. Heavy meromyosin stabilizes the formin-nucleated actin filaments in an apparently single step reaction upon binding, whereas the stabilization by tropomyosin occurs after complex formation. These observations support the idea that actin-binding proteins are key elements of the molecular mechanisms that regulate the conformational and functional diversity of actin filaments in living cells.     

Coarse-Graining Provides Insights on the Essential Nature of Heterogeneity in Actin Filaments

Experiments have shown that actin is structurally polymorphic, but knowledge of the details of molecular level heterogeneity in both the dynamics of a single subunit and the interactions between subunits is still lacking. Here, using atomistic molecular dynamics simulations of the actin filament, we identify domains of atoms that move in a correlated fashion, quantify interactions between these domains using coarse-grained (CG) analysis methods, and perform CG simulations to explore the importance of filament heterogeneity. The persistence length and torsional stiffness calculated from molecular dynamics simulation data agree with experimental values. We additionally observe that distinct actin conformations coexist in actin filaments. The filaments also exhibit random twist angles that are broadly distributed. CG analysis reveals that interactions between equivalent CG pairs vary from one subunit to another. To explore the importance of heterogeneity on filament dynamics, we perform CG simulations using different methods of parameterization to show that only by including heterogeneous interactions can we reproduce the twist angles and related properties. Free energy calculations further suggest that in general the actin filament is best represented as a set of subunits with differing CG sites and interactions, and the incorporating heterogeneity into the CG interactions is more important than including that in the CG sites. Our work therefore presents a systematic method to explore molecular level detail in this large and complex biopolymer.     

The Interaction of Mip-90 with Microtubules and Actin Filaments in Human Fibroblasts

The novel microtubule-interacting protein Mip-90 was originally isolated from HeLa cells by using affinity columns of agarose derivatized with peptides from the C-terminal regulatory domain on β-tubulin. Biochemical and immunocytochemical data have suggested that the association of Mip-90 with the microtubule system contributes to its cellular organization. Here we report the interaction patterns of Mip-90 with microtubules and actin filaments in interphase human fibroblasts. A polyclonal monospecific antibody against Mip-90 was used for immunofluorescence microscopy analysis to compare the distribution patterns of this protein with tubulin and actin. A detailed observation of fibroblasts revealed the colocalization of Mip-90 with microtubules and actin filaments. These studies were complemented with experiments using cytoskeleton-disrupting drugs which showed that colocalization patterns of Mip-90 with microtubules and actin filaments requires the integrity of these cytoskeletal components. Interestingly, a colocalization of Mip-90 with actin at the leading edge of fibroblasts grown under subconfluency was observed, suggesting that Mip-90 could play a role in actin organization, particularly at this cellular domain. Mip-90 interaction with actin polymers was further supportedin vitroby cosedimentation and immunoprecipitation experiments. The cosedimentation analysis indicated that Mip-90 bound to actin filaments with an association constantK_a= 1 × 10⁶M⁻¹, while an stoichiometry Mip-90/actin of 1:12 mol/mol was calculated. Western blots of the immunoprecipitates revealed that Mip-90 associated to both actin and tubulin in fibroblasts extracts. These studies indicate that Mip-90, described as a microtubule-interacting protein, also bears the capacity to interact with the microfilament network, suggesting that it may play a role in modulating the interactions between these cytoskeletal filaments in nonneuronal cells.     

Electrical Propagation of Condensed and Diffuse Ions Along Actin Filaments

Journal of Computational Neuroscience - In this article, we elucidate the roles of divalent ion condensation and highly polarized immobile water molecules on the propagation of ionic calcium waves...     

Chlamydia trachomatis Tarp Harbors Distinct G and F Actin Binding Domains That Bundle Actin Filaments

All species of Chlamydia undergo a unique developmental cycle that transitions between extracellular and intracellular environments and requires the capacity to invade new cells for dissemination. A chlamydial protein called Tarp has been shown to nucleate actin in vitro and is implicated in bacterial entry into human cells. Colocalization studies of ectopically expressed enhanced green fluorescent protein (EGFP)-Tarp indicate that actin filament recruitment is restricted to the C-terminal half of the effector protein. Actin filaments are presumably associated with Tarp via an actin binding alpha helix that is also required for actin nucleation in vitro, but this has not been investigated. Tarp orthologs from C. pneumoniae, C. muridarum, and C. caviae harbor between 1 and 4 actin binding domains located in the C-terminal half of the protein, but C. trachomatis serovar L2 has only one characterized domain. In this work, we examined the effects of domain-specific mutations on actin filament colocalization with EGFP-Tarp. We now demonstrate that actin filament colocalization with Tarp is dependent on two novel F-actin binding domains that endow the Tarp effector with actin-bundling activity. Furthermore, Tarp-mediated actin bundling did not require actin nucleation, as the ability to bundle actin filaments was observed in mutant Tarp proteins deficient in actin nucleation. These data shed molecular insight on the complex cytoskeletal rearrangements required for C. trachomatis entry into host cells.     

The More the Tubular: Dynamic Bundling of Actin Filaments for Membrane Tube Formation

Tubular protrusions are a common feature of living cells, arising from polymerization of stiff protein filaments against a comparably soft membrane. Although this process involves many accessory proteins in cells, in vitro experiments indicate that similar tube-like structures can emerge without them, through spontaneous bundling of filaments mediated by the membrane. Using theory and simulation of physical models, we have elaborated how nonequilibrium fluctuations in growth kinetics and membrane shape can yield such protrusions. Enabled by a new grand canonical Monte Carlo method for membrane simulation, our work reveals a cascade of dynamical transitions from individually polymerizing filaments to highly cooperatively growing bundles as a dynamical bottleneck to tube formation. Filament network organization as well as adhesion points to the membrane, which bias filament bending and constrain membrane height fluctuations, screen the effective attractive interactions between filaments, significantly delaying bundling and tube formation.     

Transportation of Nanoscale Cargoes by Myosin Propelled Actin Filaments

Myosin II propelled actin filaments move ten times faster than kinesin driven microtubules and are thus attractive candidates as cargo-transporting shuttles in motor driven lab-on-a-chip devices. In addition, actomyosin-based transportation of nanoparticles is useful in various fundamental studies. However, it is poorly understood how actomyosin function is affected by different number of nanoscale cargoes, by cargo size, and by the mode of cargo-attachment to the actin filament. This is studied here using biotin/fluorophores, streptavidin, streptavidin-coated quantum dots, and liposomes as model cargoes attached to monomers along the actin filaments (“side-attached”) or to the trailing filament end via the plus end capping protein CapZ. Long-distance transportation (>100 µm) could be seen for all cargoes independently of attachment mode but the fraction of motile filaments decreased with increasing number of side-attached cargoes, a reduction that occurred within a range of 10–50 streptavidin molecules, 1–10 quantum dots or with just 1 liposome. However, as observed by monitoring these motile filaments with the attached cargo, the velocity was little affected. This also applied for end-attached cargoes where the attachment was mediated by CapZ. The results with side-attached cargoes argue against certain models for chemomechanical energy transduction in actomyosin and give important insights of relevance for effective exploitation of actomyosin-based cargo-transportation in molecular diagnostics and other nanotechnological applications. The attachment of quantum dots via CapZ, without appreciable modulation of actomyosin function, is useful in fundamental studies as exemplified here by tracking with nanometer accuracy.     

Global and Local Regulation of Clathrin-Coated Pit Dynamics Detected on Patterned Substrates

Live-cell imaging of individual clathrin-coated pit (CCP) dynamics has revealed a broad variation in their internalization kinetics, but the functional significance and mechanistic underpinnings of this heterogeneity remain unknown. One contributing factor may be the spatial variations in the underlying actin cortex. To test this, we cultured cells on fibronectin (Fn) micropatterned substrates to vary the cortical actin mechanics in a defined manner. Under these conditions, stress fibers became organized to bridge adhesive islands, creating spatial heterogeneity in the cortical actin architecture. CCP lifetimes within the Fn-coated islands were selectively prolonged. This differential effect was not due to adherence to Fn-coated surfaces, and was not observed in cells grown on patterned surfaces that did not induce organized stress fiber assembly. Pharmacological agents that lower cortical tension selectively lowered CCP lifetimes within Fn islands, thus abolishing the spatial heterogeneity in the CCP dynamics. Although we cannot rule out the possibility that other factors might locally affect CCP dynamics at Fn islands, our data suggest that localized modulation in cortical tension may spatially regulate clathrin-mediated endocytosis.     

肌动蛋白体外自组织复合纤维结构的观察

利用原子力显微镜(atomic force microscope,AFM)和透射电子显微镜(transmission electron microscope,WEM)技术,研究了低浓度肌动蛋白在体外简单热力学体系中,形成的自组织复合纤维结构。肌动蛋白在体外通过自组织过程能够聚合形成大尺度的、离散的、复杂的聚集纤维体系,分散的单根微丝较少;在微丝稳定剂鬼笔环肽干预下,肌动蛋白通过受调控的自装配过程,主要形成分散的单根微丝,以及少量由单根微丝组成的微丝束和纤维分支等简单微丝聚集结构。     

Actin Filament Severing by Cofilin Dismantles Actin Patches and Produces Mother Filaments for New Patches

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Structure and Dynamics of the Actin Filament

We used all-atom molecular dynamics simulations to investigate the structure and properties of the actin filament, starting with either the recent Oda model or the older Holmes model. Simulations of monomeric and polymerized actin show that polymerization changes the nucleotide-binding cleft, bringing together the Q137 side chain and bound ATP in a way that may enhance the ATP hydrolysis rate in the filament. Simulations with different bound nucleotides and conformations of the DNase I binding loop show that the persistence length of the filament depends only on loop conformation. Computational modeling reveals how bound phalloidin stiffens actin filaments and inhibits the release of γ-phosphate from ADP-P_i actin.