Individual Actin Filaments Research Articles

How does fascin promote cancer metastasis?

Fascin is an F-actin-bundling protein that cross-links individual actin filaments into straight and stiff bundles. Fascin overexpression in cancer is strongly associated with poor prognosis and metastatic progression across different cancer types. It is well established that fascin plays a causative role in promoting metastatic progression. We will review the recent progress in our understanding of mechanisms underlying fascin-mediated cancer metastasis. This review will cover the biochemical basis for fascin-bundling activity, the mechanisms by which cancer cells upregulate fascin expression and the mechanism underlying fascin-mediated cancer cell migration, invasion, and metastatic colonization. We propose that fascin has broad roles in both metastatic dissemination and metastatic colonization. Understanding these mechanisms will be crucial to the development of anti-metastasis therapeutics targeting fascin.

Myosin II Filament Dynamics in Actin Networks Revealed with Interferometric Scattering Microscopy

The plasma membrane and the underlying cytoskeletal cortex constitute active platforms for a variety of cellular processes. Recent work has shown that the remodeling acto-myosin network modifies local membrane organization, but the molecular details are only partly understood because of difficulties with experimentally accessing the relevant time and length scales. Here, we use interferometric scattering microscopy to investigate a minimal acto-myosin network linked to a supported lipid bilayer membrane. Using the magnitude of the interferometric contrast, which is proportional to molecular mass, and fast acquisition rates, we detect and image individual membrane-attached actin filaments diffusing within the acto-myosin network and follow individual myosin II filament dynamics. We quantify myosin II filament dwell times and processivity as functions of ATP concentration, providing experimental evidence for the predicted ensemble behavior of myosin head domains. Our results show how decreasing ATP concentrations lead to both increasing dwell times of individual myosin II filaments and a global change from a remodeling to a contractile state of the acto-myosin network.

Bending of Actin Filaments into Rings by IQGAP Family of Proteins

Actin forming linear actin filaments builds up the acto-myosin cortex in cells providing mechanical stability, enabling vesicle transport, cell migration and cell division. This versality is achieved by interaction with many regulatory and actin binding proteins and the interplay with the plasma membrane to locally control actin filament length, stability and crosslinking to other filaments. Here, we report about a newly identified protein domain, Curly, within IQGAP family proteins that binds actin and causes bending of individual actin filaments into tight rings when anchored to a lipid membrane in vitro. Membrane anchored Curly induces reproducibly and efficiently F-actin ring formation of characteristic curvature (0.5-1 µm−1) and recognizes F-actin orientation. Interestingly, decoration of F-actin with the tropomyosin increases actin ring formation, while Fimbrin abrogates it. Addition of myosin II filaments results in further formation of actin rings by induced f-actin flow and, more importantly, in robust ring contraction. This constitutes, hence, a minimal (four components) contractile ring system. The discovery of Curly provides a new opportunity to address and control F-actin curvature in cells and in vitro and will open new approaches to generate synthetic cells capable of division and controlled shape changes.

Guard Cell Microfilament Analyzer Facilitates the Analysis of the Organization and Dynamics of Actin Filaments in Arabidopsis Guard Cells.

The actin cytoskeleton is involved in regulating stomatal movement, which forms distinct actin arrays within guard cells of stomata with different apertures. How those actin arrays are formed and maintained remains largely unexplored. Elucidation of the dynamic behavior of differently oriented actin filaments in guard cells will enhance our understanding in this regard. Here, we initially developed a program called ‘guard cell microfilament analyzer’ (GCMA) that enables the selection of individual actin filaments and analysis of their orientations semiautomatically in guard cells. We next traced the dynamics of individual actin filaments and performed careful quantification in open and closed stomata. We found that de novo nucleation of actin filaments occurs at both dorsal and ventral sides of guard cells from open and closed stomata. Interestingly, most of the nucleated actin filaments elongate radially and longitudinally in open and closed stomata, respectively. Strikingly, radial filaments tend to form bundles whereas longitudinal filaments tend to be removed by severing and depolymerization in open stomata. By contrast, longitudinal filaments tend to form bundles that are severed less frequently in closed stomata. These observations provide insights into the formation and maintenance of distinct actin arrays in guard cells in stomata of different apertures.

Self-Organization and Force Production by the Branched Actin Cytoskeleton during Mammalian Clathrin-Mediated Endocytosis

During clathrin-mediated endocytosis (CME), the cell's plasma membrane is deformed from a flat sheet into a round vesicle to internalize transmembrane proteins and extracellular cargo. Actin participates in mammalian CME, but the mechanism by which it ensures internalization of endocytic pits under variable physical constraints is not understood. To determine the molecular mechanism by which actin organizes to produce force in CME, we combined multi-scale mathematical modeling, cryo-electron tomography, and quantitative fluorescence imaging of diploid human cells endogenously expressing fluorescent fusion proteins. We developed a fluorescence microscopy-based calibration method that relates fluorescence intensity to numbers of molecules in live cells, to measure the number of Arp2/3 complexes at endocytic sites over time. These measurements constrained a multiscale mathematical model of actin polymerization coupled to the internalization of an endocytic pit. The model accounts for the mechanics of a coated plasma membrane, as well as individual actin filaments that diffuse, polymerize, and bend under force. Branched actin filaments are nucleated by active Arp2/3 complex at the base of the pit and attach to the coat via actin-binding proteins. Stochastic simulations of the model revealed that these branched actin networks self-organize at endocytic sites in a dendritic cone between the base and tip of the endocytic pit, which directs the growth of actin filaments toward the base of the pit. This self-organization was robust over a range of conditions, ensuring pit internalization against physiological membrane tension. Surprisingly, long actin filaments bent between their attachment sites at the coat and the base of the pit, which we confirmed with cryo-electron tomography of intact mammalian cells. We suggest that the self-organization and bending of actin filaments allows the actin network to robustly internalize cellular membranes across a range of physical constraints.

Interactions of tropomyosin Tpm1.1 on a single actin filament: A method for extraction and processing of high resolution TIRF microscopy data.

Skeletal muscle tropomyosin (Tpm1.1) is an elongated, rod-shaped, alpha-helical coiled-coil protein that forms continuous head-to-tail polymers along both sides of the actin filament. In this study we use single molecule fluorescence TIRF microscopy combined with a microfluidic device and fluorescently labelled proteins to measure Tpm1.1 association to and dissociation from single actin filaments. Our experimental setup allows us to clearly resolve Tpm1.1 interactions on both sides of the filaments. Here we provide a semi-automated method for the extraction and quantification of kymograph data for individual actin filaments bound at different Tpm1.1 concentrations. We determine boundaries on the kymograph on each side of the actin filament, based on intensity thresholding, performing fine manual editing of the boundaries (if needed) and extracting user defined kinetic properties of the system. Using our analytical tools we can determine (i) nucleation point(s) and rates, (ii) elongation rates of Tpm1.1, (iii) identify meeting points after the saturation of filament, and when dissociation occurs, (iv) initiation point(s), (v) the final dissociation point(s), as well as (vi) dissociation rates.All of these measurements can be extracted from both sides of the filament, allowing for the determination of possible differences in behaviour on the two sides of the filament, and across concentrations. The robust and repeatable nature of the method allows quantitative, semi-automated analyses to be made of large studies of acto-tropomyosin interactions, as well as for other actin binding proteins or filamentous structures, opening the way for dissection of the dynamics underlying these interactions.

Polymerization and depolymerization of actin with nucleotide states at filament ends.

Polymerization induces hydrolysis of ATP bound to actin, followed by γ-phosphate release, which helps advance the disassembly of actin filaments into ADP-G-actin. Mechanical understanding of this correlation between actin assembly and ATP hydrolysis has been an object of intensive studies in biochemistry and structural biology for many decades. Although actin polymerization and depolymerization occur only at either the barbed or pointed ends and the kinetic and equilibrium properties are substantially different from each other, characterizing their properties is difficult to do by bulk assays, as these assays report the average of all actin filaments in solution and are therefore not able to discern the properties of individual actin filaments. Biochemical studies of actin polymerization and hydrolysis were hampered by these inherent properties of actin filaments. Total internal reflection fluorescence (TIRF) microscopy overcame this problem by observing single actin filaments. With TIRF, we now know not only that each end has distinct properties, but also that the rate of γ-phosphate release is much faster from the terminals than from the interior of actin filaments. The rate of γ-phosphate release from actin filament ends is even more accelerated when latrunculin A is bound. These findings highlight the importance of resolving structural differences between actin molecules in the interior of the filament and those at either filament end. This review provides a history of observing actin filaments under light microscopy, an overview of dynamic properties of ATP hydrolysis at the end of actin filament, and structural views of γ-phosphate release.

Mechanical Regulation of the WAVE Complex by Actin Elongation in the Lamellipodium

Actin filaments generate forces driving membrane movements during trafficking, endocytosis and cell migration. Reciprocally, the lamellipodium of migrating cells adapts to forces by geometrical reorganization of branched actin networks. But, local forces generated by growing actin filaments might also act on individual actin regulators. We demonstrate that piconewton forces exerted by actin filament elongation on WAVE regulatory complex (WRC), the lamellipodial Arp2/3 activator, control its dissociation from the lamellipodium tip. Using single protein tracking, we reveal WRC lateral movements along the lamellipodium tip which are driven by actin elongation and correlate with WRC turnover. Using optical tweezers to mechanically manipulate functional trans-membrane WRC, we show that WRC dissociation from the lamellipodium tip requires forces (>1 pN) larger than its dissociation from the flowing branched actin network. Thus forces generated by individual actin filaments at sites of actin assembly can foster mechano-transduction events tuning the function of actin regulatory proteins.

Modulation of formin processivity by profilin and mechanical tension.

Formins are major regulators of actin networks. They enhance actin filament dynamics by remaining processively bound to filament barbed ends. How biochemical and mechanical factors affect formin processivity are open questions. Monitoring individual actin filaments in a microfluidic flow, we report that formins mDia1 and mDia2 dissociate faster under higher ionic strength and when actin concentration is increased. Profilin, known to increase the elongation rate of formin-associated filaments, surprisingly decreases the formin dissociation rate, by bringing formin FH1 domains in transient contact with the barbed end. In contrast, piconewton tensile forces applied to actin filaments accelerate formin dissociation by orders of magnitude, largely overcoming profilin-mediated stabilization. We developed a model of formin conformations showing that our data indicates the existence of two different dissociation pathways, with force favoring one over the other. How cells limit formin dissociation under tension is now a key question for future studies.

Large Scale Imaging by Fine Spatial Alignment of Multi-Scanning Data with Gel Cube Device

In vitro three-dimensional (3D) culturing is considered essential in many biological fields. However, the imaging of developed 3D formations is often difficult, especially if the size of the sample is relatively large. The z-resolution of fluorescent imaging is low using low magnification lenses (4× and 10×) due to large focal depths. This paper describes 3D culture platform enabling large scale 3D imaging by fine spatial alignment of the image dataset obtained from multiple directions. A gel cube device was employed to conduct the multi-scanning and then a self-fluorescent microstructure in a cubic frame allows us spatially align image dataset within a few pixels. By synthesizing data from multiple scans, the platform enables us to visualize millimeter-sized 3D sample structure and individual cellular actin filaments at the same time. Millimeter depth imaging of a developed bronchial tree was achieved with high z-resolution. The device, which is applicable to most microscopy systems, can enhance the image quality without modifying current systems.

Fascin in tooth germs: an immunohistochemical analysis

Development of a tooth is a complex process that involves three stages namely bud, cap and bell which results from the interaction of cells of oral mucosa and neural crest. During this process, oral and neural crest cells undergo various changes resulting in proliferation, apoptosis, morphological, and cyto-differentiation, which in turn would have changes in the cytoskeletal framework of the cell that includes actin and tubulin. Fascin is an actin-bundling protein that cross-links individual actin filaments into straight, compact, stiff bundles, which are crucial for the formation of finger-like membrane protrusions and aids in cell migration but is less explored in tooth germs. The aim of this study was analyze the expression and distribution of fascin in various stages of human tooth germs. Twenty human tooth germs were stained with hematoxylin and eosin and with immunohistochemical staining for fascin. Strong membranous expression for fascin was observed in various stages with decreased expression in stellate reticulum from cap stage to bell stage. Also, outer enamel epithelial cells had a weak fascin expression. Although cells of dental papilla did not express fascin in early cap stage of tooth development, strong expression of fascin was observed in later bell stages. It was also observed the inner enamel epithelium close to areas of calcification showed a decreased expression. Thus, the expression of fascin in various stages of tooth development being site and time specific may play an important role in cell remodulation.

Biphasic Effect of Profilin Impacts the Formin mDia1 Force-Sensing Mechanism in Actin Polymerization

Formins are force-sensing proteins that regulate actin polymerization dynamics. Here, we applied stretching tension to individual actin filaments under the regulation of formin mDia1 to investigate the mechanical responses in actin polymerization dynamics. We found that the elongation of an actin filament was accelerated to a greater degree by stretching tension for ADP-G-actin than that for ATP-G-actin. An apparent decrease in the critical concentration of G-actin was observed, especially in ADP-G-actin. These results on two types of G-actin were reproduced by a simple kinetic model, assuming the rapid equilibrium between pre- and posttranslocated states of the formin homology domain two dimer. In addition, profilin concentration dramatically altered the force-dependent acceleration of actin filament elongation, which ranged from twofold to an all-or-none response. Even under conditions in which actin depolymerization occurred, applications of a several-piconewton stretching tension triggered rapid actin filament elongation. This extremely high force-sensing mechanism of mDia1 and profilin could be explained by the force-dependent coordination of the biphasic effect of profilin; i.e., an acceleration effect masked by a depolymerization effect became dominant under stretching tension, negating the latter to rapidly enhance the elongation rate. Our findings demonstrate that the biphasic effect of profilin is controlled by mechanical force, thus expanding the function of mDia1 as a mechanosensitive regulator of actin polymerization.

Distinct VASP tetramers synergize in the processive elongation of individual actin filaments from clustered arrays

Ena/VASP proteins act as actin polymerases that drive the processive elongation of filament barbed ends in membrane protrusions or at the surface of bacterial pathogens. Based on previous analyses of fast and slow elongating VASP proteins by in vitro total internal reflection fluorescence microscopy (TIRFM) and kinetic and thermodynamic measurements, we established a kinetic model of Ena/VASP-mediated actin filament elongation. At steady state, it entails that tetrameric VASP uses one of its arms to processively track growing filament barbed ends while three G-actin-binding sites (GABs) on other arms are available to recruit and deliver monomers to the filament tip, suggesting that VASP operates as a single tetramer in solution or when clustered on a surface, albeit processivity and resistance toward capping protein (CP) differ dramatically between both conditions. Here, we tested the model by variation of the oligomerization state and by increase of the number of GABs on individual polypeptide chains. In excellent agreement with model predictions, we show that in solution the rates of filament elongation directly correlate with the number of free GABs. Strikingly, however, irrespective of the oligomerization state or presence of additional GABs, filament elongation on a surface invariably proceeded with the same rate as with the VASP tetramer, demonstrating that adjacent VASP molecules synergize in the elongation of a single filament. Additionally, we reveal that actin ATP hydrolysis is not required for VASP-mediated filament assembly. Finally, we show evidence for the requirement of VASP to form tetramers and provide an amended model of processive VASP-mediated actin assembly in clustered arrays.

Competition between Tropomyosin, Fimbrin, and ADF/Cofilin drives their sorting to distinct actin filament networks.

The fission yeast actin cytoskeleton is an ideal, simplified system to investigate fundamental mechanisms behind cellular self-organization. By focusing on the stabilizing protein tropomyosin Cdc8, bundling protein fimbrin Fim1, and severing protein coffin Adf1, we examined how their pairwise and collective interactions with actin filaments regulate their activity and segregation to functionally diverse F-actin networks. Utilizing multi-color TIRF microscopy of in vitro reconstituted F-actin networks, we observed and characterized two distinct Cdc8 cables loading and spreading cooperatively on individual actin filaments. Furthermore, Cdc8, Fim1, and Adf1 all compete for association with F-actin by different mechanisms, and their cooperative association with actin filaments affects their ability to compete. Finally, competition between Fim1 and Adf1 for F-actin synergizes their activities, promoting rapid displacement of Cdc8 from a dense F-actin network. Our findings reveal that competitive and cooperative interactions between actin binding proteins help define their associations with different F-actin networks.

Formin' bridges between microtubules and actin filaments in axonal growth cones.

Formin' bridges between microtubules and actin filaments in axonal growth cones.

Monoubiquitination Inhibits the Actin Bundling Activity of Fascin

Fascin is an actin bundling protein that cross-links individual actin filaments into straight, compact, and stiff bundles, which are crucial for the formation of filopodia, stereocillia, and other finger-like membrane protrusions. The dysregulation of fascin has been implicated in cancer metastasis, hearing loss, and blindness. Here we identified monoubiquitination as a novel mechanism that regulates fascin bundling activity and dynamics. The monoubiquitination sites were identified to be Lys247 and Lys250, two residues located in a positive charge patch at the actin binding site 2 of fascin. Using a chemical ubiquitination method, we synthesized chemically monoubiquitinated fascin and determined the effects of monoubiquitination on fascin bundling activity and dynamics. Our data demonstrated that monoubiquitination decreased the fascin bundling EC50, delayed the initiation of bundle assembly, and accelerated the disassembly of existing bundles. By analyzing the electrostatic properties on the solvent-accessible surface of fascin, we proposed that monoubiquitination introduced steric hindrance to interfere with the interaction between actin filaments and the positively charged patch at actin binding site 2. We also identified Smurf1 as a E3 ligase regulating the monoubiquitination of fascin. Our findings revealed a previously unidentified regulatory mechanism for fascin, which will have important implications for the understanding of actin bundle regulation under physiological and pathological conditions.

Engineering Circular Gliding of Actin Filaments Along Myosin-Patterned DNA Nanotube Rings To Study Long-Term Actin-Myosin Behaviors.

Nature has evolved molecular motors that are critical in cellular processes occurring over broad time scales, ranging from seconds to years. Despite the importance of the long-term behavior of molecular machines, topics such as enzymatic lifetime are underexplored due to the lack of a suitable approach for monitoring motor activity over long time periods. Here, we developed an "O"-shaped Myosin Empowered Gliding Assay (OMEGA) that utilizes engineered micron-scale DNA nanotube rings with precise arrangements of myosin VI to trap gliding actin filaments. This circular gliding assay platform allows the same individual actin filament to glide over the same myosin ensemble (50-1000 motors per ring) multiple times. First, we systematically characterized the formation of DNA nanotubes rings with 4, 6, 8, and 10 helix circumferences. Individual actin filaments glide along the nanotube rings with high processivity for up to 12.8 revolutions or 11 min in run time. We then show actin gliding speed is robust to variation in motor number and independent of ring curvature within our sample space (ring diameter of 0.5-4 μm). As a model application of OMEGA, we then analyze motor-based mechanical influence on "stop-and-go" gliding behavior of actin filaments, revealing that the stop-to-go transition probability is dependent on motor flexibility. Our circular gliding assay may provide a closed-loop platform for monitoring long-term behavior of broad classes of molecular motors and enable characterization of motor robustness and long time scale nanomechanical processes.

Graphene-Enabled Electron Microscopy and Correlated Super-Resolution Microscopy of Wet Cells

The application of electron microscopy (EM) to hydrated biological samples has been limited by the harsh conditions found in EM chambers. Conventional methods require challenging and laborious sample dehydration procedures, often resulting in structural artifacts and causing difficulties in correlating results with those obtained from fluorescence microscopy (FM). We here present our recent results utilizing graphene, a single-atom-thick carbon sheet, as the thinnest possible impermeable and conductive membrane to protect whole cells from vacuum, thus enabling high-resolution EM of hydrated and untreated cells with unprecedented ease. Our approach further allows for facile correlative super-resolution FM and EM of wet, fixed cells directly on the culturing substrate. This simple, one-step procedure requires no special materials or equipment, and can be readily performed by biologists with basic experience using FM and SEM. Excellent results are obtained with conventional scanning electron microscopes. As an example of the power of this technique, individual cytoskeletal actin filaments are resolved in hydrated samples through electron microscopy and are well correlated with super-resolution fluorescence results. Our approach thus offers an accessible method by which researchers can obtain multimodal information of biological specimens in their native, hydrated state.

Trapping, entrainment and synchronization of semiflexible polymers in narrow, asymmetric confinements.

The physical properties of polymeric actin facilitate many mechanical processes within the cell, including cellular deformation and locomotion, whereby the polymers can be confined to a range of different geometries. As actin polymers often form entangled solutions in the cell, we have investigated the effect of confinement on the evolution of entangled semiflexible polymer solutions. Using a microfluidic platform, we examined the physical dynamics of actin polymers confined within narrow (2-4 μm) rectangular channels. Focusing on the entanglement process of two actin polymers, we found that their prolonged entrainment leads to synchronized horizontal undulations and decreased translational diffusion. In the absence of cross-linking molecules or proteins, the long-range entrainment interactions are predominantly controlled by the geometric boundaries. We directly measure the deflection length Λ for an individual polymer, either solitarily confined within a channel or confined in the presence of a second filament, enabling the determination of the change in free energy associated with polymer entanglement. Our results indicate that geometrical confinement can serve as a solitary variable influencing the physical dynamics of entangled semiflexible polymers.

DeFiNe: an optimisation-based method for robust disentangling of filamentous networks.

Thread-like structures are pervasive across scales, from polymeric proteins to root systems to galaxy filaments, and their characteristics can be readily investigated in the network formalism. Yet, network links usually represent only parts of filaments, which, when neglected, may lead to erroneous conclusions from network-based analyses. The existing alternatives to detect filaments in network representations require tuning of parameters over a large range of values and treat all filaments equally, thus, precluding automated analysis of diverse filamentous systems. Here, we propose a fully automated and robust optimisation-based approach to detect filaments of consistent intensities and angles in a given network. We test and demonstrate the accuracy of our solution with contrived, biological, and cosmic filamentous structures. In particular, we show that the proposed approach provides powerful automated means to study properties of individual actin filaments in their network context. Our solution is made publicly available as an open-source tool, “DeFiNe”, facilitating decomposition of any given network into individual filaments.