The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis

Dynamic remodeling of the actin cytoskeleton is required for both development and tissue homeostasis. While fixed image analysis has provided significant insight into such events, a complete understanding of cytoskeletal dynamics requires live imaging. Numerous tools for the live imaging of actin have been generated by fusing the actin-binding domain from an actin-interacting protein to a fluorescent protein. Here we comparatively assess the utility of three such tools – Utrophin, Lifeact, and F-tractin – for characterizing the actin remodeling events occurring within the germline-derived nurse cells during Drosophila mid-oogenesis or follicle development. Specifically, we used the UAS/GAL4 system to express these tools at different levels and in different cells, and analyzed these tools for effects on fertility, alterations in the actin cytoskeleton, and ability to label filamentous actin (F-actin) structures by both fixed and live imaging. While both Utrophin and Lifeact robustly label F-actin structures within the Drosophila germline, when strongly expressed they cause sterility and severe actin defects including cortical actin breakdown resulting in multi-nucleate nurse cells, early F-actin filament and aggregate formation during stage 9 (S9), and disorganized parallel actin filament bundles during stage 10B (S10B). However, by using a weaker germline GAL4 driver in combination with a higher temperature, Utrophin can label F-actin with minimal defects. Additionally, strong Utrophin expression within the germline causes F-actin formation in the nurse cell nuclei and germinal vesicle during mid-oogenesis. Similarly, Lifeact expression results in nuclear F-actin only within the germinal vesicle. F-tractin expresses at a lower level than the other two labeling tools, but labels cytoplasmic F-actin structures well without causing sterility or striking actin defects. Together these studies reveal how critical it is to evaluate the utility of each actin labeling tool within the tissue and cell type of interest in order to identify the tool that represents the best compromise between acceptable labeling and minimal disruption of the phenomenon being observed. In this case, we find that F-tractin, and perhaps Utrophin, when Utrophin expression levels are optimized to label efficiently without causing actin defects, can be used to study F-actin dynamics within the Drosophila nurse cells.     

Zebrafish lines expressing UAS‐driven red probes for monitoring cytoskeletal dynamics

Actin filaments and microtubules are principal components of the cytoskeleton that regulate the basic cellular phenomena underlying many fundamental cellular processes. Therefore, analyzing their dynamics in living cells is important for understanding cellular events more precisely. In this article, we report two novel transgenic zebrafish lines expressing red fluorescent proteins tagged with Lifeact or EB1 that interact with actin filaments and microtubule plus ends, respectively, under the control of the GAL4‐UAS system. Using these transgenic lines, we could detect F‐actin and microtubule plus end dynamics in specific tissues of living zebrafish embryos by crossing with GAL4 driver lines. In addition, we could achieve multi‐color imaging using these transgenic lines with GFP‐expressing transgenic lines. Therefore, our transgenic lines that carry UAS‐driven red fluorescent cytoskeletal probes are useful tools for analyzing spatiotemporal changes of the cytoskeletal elements using multicolor live imaging.     

Lifeact: a versatile marker to visualize F-actin

Live imaging of the actin cytoskeleton is crucial for the study of many fundamental biological processes, but current approaches to visualize actin have several limitations. Here we describe Lifeact, a 17-amino-acid peptide, which stained filamentous actin (F-actin) structures in eukaryotic cells and tissues. Lifeact did not interfere with actin dynamics in vitro and in vivo and in its chemically modified peptide form allowed visualization of actin dynamics in nontransfectable cells.     

Actin turnover-dependent fast dissociation of capping protein in the dendritic nucleation actin network: evidence of frequent filament severing

Actin forms the dendritic nucleation network and undergoes rapid polymerization-depolymerization cycles in lamellipodia. To elucidate the mechanism of actin disassembly, we characterized molecular kinetics of the major filament end-binding proteins Arp2/3 complex and capping protein (CP) using single-molecule speckle microscopy. We have determined the dissociation rates of Arp2/3 and CP as 0.048 and 0.58 s(-1), respectively, in lamellipodia of live XTC fibroblasts. This CP dissociation rate is three orders of magnitude faster than in vitro. CP dissociates slower from actin stress fibers than from the lamellipodial actin network, suggesting that CP dissociation correlates with actin filament dynamics. We found that jasplakinolide, an actin depolymerization inhibitor, rapidly blocked the fast CP dissociation in cells. Consistently, the coexpression of LIM kinase prolonged CP speckle lifetime in lamellipodia. These results suggest that cofilin-mediated actin disassembly triggers CP dissociation from actin filaments. We predict that filament severing and end-to-end annealing might take place fairly frequently in the dendritic nucleation actin arrays.     

Five-parameter fluorescence imaging: wound healing of living Swiss 3T3 cells

Cellular functions involve the temporal and spatial interplay of ions, metabolites, macromolecules, and organelles. To define the mechanisms responsible for completing cellular functions, we used methods that can yield both temporal and spatial information on multiple physiological parameters and chemical components in the same cell. We demonstrated that the combined use of selected fluorescent probes, fluorescence microscopy, and imaging methods can yield information on at least five separate cellular parameters and components in the same living cell. Furthermore, the temporal and spatial dynamics of each of the parameters and/or components can be correlated with one or more of the others. Five parameters were investigated by spectrally isolating defined regions of the ultraviolet, visible, and near-infrared spectrum based on five distinct fluorescent probes. The parameters included nuclei (Hoechst 33342), mitochondria (diIC1-[5] ), endosomes (lissamine rhodamine B-dextran), actin (fluorescein), and the cell volume Cy7-dextran). Nonmotile, confluent Swiss 3T3 cells did not show any detectable polarity of cell shape, or distribution of nuclei, endosomes, or mitochondria. These cells also organized a large percentage of the actin into stress fibers. In contrast, cells migrating into an in vitro wound exhibited at least two stages of reorganization of organelles and cytoplasm. During the first 3 h after wounding, the cells along the edge of the wound assumed a polarized shape, carried the nuclei in the rear of the cells, excluded endosomes and mitochondria from the lamellipodia, and lost most of the highly organized stress fibers. The cell showed a dramatic change between 3 and 7 h after producing the wound. The cells became highly elongated and motile; both the endosomes and the mitochondria penetrated into the lamellipodia, while the nuclei remained in the rear and the actin remained in less organized structures. Defining the temporal and spatial dynamics and interplay of ions, contractile proteins, lipids, regulatory proteins, metabolites, and organelles should lead to an understanding of the molecular basis of cell migration, as well as other cellular functions.     

Feeling for Filaments: Quantification of the Cortical Actin Web in Live Vascular Endothelium

Contact-mode atomic force microscopy (AFM) has been shown to reveal cortical actin structures. Using live endothelial cells, we visualized cortical actin dynamics simultaneously by AFM and confocal fluorescence microscopy. We present a method that quantifies dynamic changes in the mechanical ultrastructure of the cortical actin web. We argue that the commonly used, so-called error signal imaging in AFM allows a qualitative, but not quantitative, analysis of cortical actin dynamics. The approach we used comprises fast force-curve-based topography imaging and subsequent image processing that enhances local height differences. Dynamic changes in the organization of the cytoskeleton network can be observed and quantified by surface roughness calculations and automated morphometrics. Upon treatment with low concentrations of the actin-destabilizing agent cytochalasin D, the cortical cytoskeleton network is thinned out and the average mesh size increases. In contrast, jasplakinolide, a drug that enhances actin polymerization, consolidates the cytoskeleton network and reduces the average mesh area. In conclusion, cortical actin dynamics can be quantified in live cells. To our knowledge, this opens a new pathway for conducting quantitative structure-function analyses of the endothelial actin web just beneath the apical plasma membrane.     

Gradient of rigidity in the lamellipodia of migrating cells revealed by atomic force microscopy

Changes in mechanical properties of the cytoplasm have been implicated in cell motility, but there is little information about these properties in specific regions of the cell at specific stages of the cell migration process. Fish epidermal keratocytes with their stable shape and steady motion represent an ideal system to elucidate temporal and spatial dynamics of the mechanical state of the cytoplasm. As the shape of the cell does not change during motion and actin network in the lamellipodia is nearly stationary with respect to the substrate, the spatial changes in the direction from the front to the rear of the cell reflect temporal changes in the actin network after its assembly at the leading edge. We have utilized atomic force microscopy to determine the rigidity of fish keratocyte lamellipodia as a function of time/distance from the leading edge. Although vertical thickness remained nearly constant throughout the lamellipodia, the rigidity exhibited a gradual but significant decrease from the front to the rear of the lamellipodia. The rigidity profile resembled closely the actin density profile, suggesting that the dynamics of rigidity are due to actin depolymerization. The decrease of rigidity may play a role in facilitating the contraction of the actin-myosin network at the lamellipodium/cell body transition zone.     

Actin-dependent lamellipodia formation and microtubule-dependent tail retraction control-directed cell migration

Migrating cells are polarized with a protrusive lamella at the cell front followed by the main cell body and a retractable tail at the rear of the cell. The lamella terminates in ruffling lamellipodia that face the direction of migration. Although the role of actin in the formation of lamellipodia is well established, it remains unclear to what degree microtubules contribute to this process. Herein, we have studied the contribution of microtubules to cell motility by time-lapse video microscopy on green flourescence protein-actin- and tubulin-green fluorescence protein-transfected melanoma cells. Treatment of cells with either the microtubule-disrupting agent nocodazole or with the stabilizing agent taxol showed decreased ruffling and lamellipodium formation. However, this was not due to an intrinsic inability to form ruffles and lamellipodia because both were restored by stimulation of cells with phorbol 12-myristate 13-acetate in a Rac-dependent manner, and by stem cell factor in melanoblasts expressing the receptor tyrosine kinase c-kit. Although ruffling and lamellipodia were formed without microtubules, the microtubular network was needed for advancement of the cell body and the subsequent retraction of the tail. In conclusion, we demonstrate that the formation of lamellipodia can occur via actin polymerization independently of microtubules, but that microtubules are required for cell migration, tail retraction, and modulation of cell adhesion.     

Localised depletion of polymerised actin at the front of Walker carcinosarcoma cells increases the speed of locomotion

Spontaneously migrating Walker carcinosarcoma cells usually form lamellipodia at the front. Combined treatment with 10(-5)M colchicine and 10(-7)M latrunculin A produces large defects in the cortical F-actin layer at the leading front and suppresses lamellipodia. However, the cortical actin layer at the rear is intact and shows myosin IIA accumulation. These cells, showing no or little detectable cortical F-actin at the front and no morphologically recognisable protrusions, migrate faster than control cells with lamellipodia and an intact cortical actin layer. This documents that the cortical actin layer or actin-powered force generation at the front is redundant for locomotion. Colchicine and latrunculin A have synergistic effects in compromising the cortical layer at the front and in increasing the speed of locomotion, but antagonistic effects on the relative amount of F-actin per cell. Colchicine but not latrunculin A, can increase the proportion of polarised and locomoting cells under appropriate conditions. Locomotion and polarity of cells treated with latrunculin A and colchicine is inhibited at latrunculin A concentrations >10(-7)M, by the myosin inhibitor BDM or the ROCK inhibitor Y-27632. Colchicine and Y-27632 have antagonistic effects on polarity and the speed of locomoting cells. The data show that locomotion of metazoan cells, which normally form lamellipodia, can be driven by actomyosin contraction behind the front (cell body, uropod). They are best compatible with a cortical contraction/frontal expansion model, but they are not compatible with models implying that actin polymerisation or actomyosin contraction at the front drive locomotion of the cells studied.     

Redundancy of lamellipodia in locomoting Walker carcinosarcoma cells

Locomoting metazoan cells usually form lamellipodia at the leading front and it is widely accepted that lamellipodia are required for locomotion. In this case, suppression of lamellipodia must stop locomotion. However, the experiments show that lamellipodia are redundant for locomotion of Walker carcinosarcoma cells. Low latrunculin A concentrations (10(-7) M) transform polarised locomoting cells with lamellipodia into cells without morphologically recognisable protrusions showing an increased speed of locomotion and a reduced amount of cellular F-actin. Whereas untreated cells show a fairly linear distribution of F-actin along the plasma membrane, cells lacking morphologically recognizable protrusions at the front show modifications at the front consisting in an irregular distribution of F-actin with formation of small or large patches of F-actin alternating with small or large gaps in the F-actin layer. This is associated with a reduced resistance to deformation pressure at the front of the cell. High concentrations of latrunculin A (>10(-7) M) compromising contraction at the rear stop locomotion, suggesting that cortical contraction is important for locomotion to occur in these cells. The results are consistent with the view that actin polymerization is important for formation of lamellipodia but they are not compatible with the view that lamellipodia are essential for locomotion of Walker carcinosarcoma cells. A unifying hypothesis for the formation of different types of protrusions is proposed.     

How do in vitro reconstituted actin-based motility assays provide insight into in vivo behavior?

Recent live cell image analysis of actin dynamics in lamellipodia of motile cells has shown that regulated treadmilling, which supports actin-based propulsion of functionalized particles in biomimetic reconstituted motility assays, is also responsible for lamellipodia extension. In both cases, filaments are created by branching with Arp2/3 complex only at the membrane or particle surface, grow transiently and are capped; ADF/cofilin enhances the treadmilling but does not sever filaments in the body of the meshwork. Differences between the cellular and biomimetic systems suggest that additional regulatory mechanisms take place in lamellipodia.     

Differentially oriented populations of actin filaments generated in lamellipodia collaborate in pushing and pausing at the cell front

Eukaryotic cells advance in phases of protrusion, pause and withdrawal. Protrusion occurs in lamellipodia, which are composed of diagonal networks of actin filaments, and withdrawal terminates with the formation of actin bundles parallel to the cell edge. Using correlated live-cell imaging and electron microscopy, we have shown that actin filaments in protruding lamellipodia subtend angles from 15-90 degrees to the front, and that transitions from protrusion to pause are associated with a proportional increase in filaments oriented more parallel to the cell edge. Microspike bundles of actin filaments also showed a wide angular distribution and correspondingly variable bilateral polymerization rates along the cell front. We propose that the angular shift of filaments in lamellipodia serves in adapting to slower protrusion rates while maintaining the filament densities required for structural support; further, we suggest that single filaments and microspike bundles contribute to the construction of the lamella behind and to the formation of the cell edge when protrusion ceases. Our findings provide an explanation for the variable turnover dynamics of actin filaments in lamellipodia observed by fluorescence speckle microscopy and are inconsistent with a current model of lamellipodia structure that features actin filaments branching at 70 degrees in a dendritic array.     

The Myosin IXb motor activity targets the myosin IXb RhoGAP domain as cargo to sites of actin polymerization

Myosin IXb (Myo9b) is a single-headed processive myosin that exhibits Rho GTPase-activating protein (RhoGAP) activity in its tail region. Using live cell imaging, we determined that Myo9b is recruited to extending lamellipodia, ruffles, and filopodia, the regions of active actin polymerization. A functional motor domain was both necessary and sufficient for targeting Myo9b to these regions. The head domains of class IX myosins comprise a large insertion in loop2. Deletion of the large Myo9b head loop 2 insertion abrogated the enrichment in extending lamellipodia and ruffles, but enhanced significantly the enrichment at the tips of filopodia and retraction fibers. The enrichment in the tips of filopodia and retraction fibers depended on four lysine residues C-terminal to the loop 2 insertion and the tail region. Fluorescence recovery after photobleaching and photoactivation experiments in lamellipodia revealed that the dynamics of Myo9b was comparable to that of actin. The exchange rates depended on the Myo9b motor region and motor activity, and they were also dependent on the turnover of F-actin. These results demonstrate that Myo9b functions as a motorized RhoGAP molecule in regions of actin polymerization and identify Myo9b head sequences important for in vivo motor properties.     

An Open Model of Actin Dendritic Nucleation

The availability of quantitative experimental data on the kinetics of actin assembly has enabled the construction of many mathematical models focused on explaining specific behaviors of this complex system. However these ad hoc models are generally not reusable or accessible by the large community of actin biologists. In this work, we present a comprehensive model that integrates and unifies much of the in vitro data on the components of the dendritic nucleation mechanism for actin dynamics. More than 300 simulations have been run based on compartmental and three-dimensional spatial versions of this model. Several key findings are highlighted, including an explanation for the sharp boundary between actin assembly and disassembly in the lamellipodia of migrating cells. Because this model, with the simulation results, is “open source”, in the sense that it is publicly available and editable through the Virtual Cell database (http://vcell.org), it can be accessed, analyzed, modified, and extended.     

Control of actin polymerization in live and permeabilized fibroblasts

We have investigated the spatial control of actin polymerization in fibroblasts using rhodamine-labeled muscle actin in; (a) microinjection experiments to follow actin dynamics in intact cells, and (b) incubation with permeabilized cells to study incorporation sites. Rhodamine-actin was microinjected into NIH-3T3 cells which were then fixed and stained with fluorescein-phalloidin to visualize total actin filaments. The incorporation of newly polymerized actin was assayed using rhodamine/fluorescein ratio-imaging. The results indicated initial incorporation of the injected actin near the tip and subsequent transport towards the base of lamellipodia at rates greater than 4.5 microns/min. Furthermore, both fluorescein- and rhodamine-intensity profiles across lamellipodia revealed a decreasing density of actin filaments from tip to base. From this observation and the presence of centripetal flux of polymerized actin we infer that the actin cytoskeleton partially disassembles before it reaches the base of the lamellipodium. In permeabilized cells we found that, in agreement with the injection studies, rhodamine-actin incorporated predominantly in a narrow strip of less than 1-microns wide, located at the tip of lamellipodia. The critical concentration for the rhodamine-actin incorporation (0.15 microM) and its inhibition by CapZ, a barbed-end capping protein, indicated that the nucleation sites for actin polymerization most likely consist of free barbed ends of actin filaments. Because any potential monomer-sequestering system is bypassed by addition of exogenous rhodamine-actin to the permeabilized cells, these observations indicate that the localization of actin incorporation in intact cells is determined, at least in part, by the presence of specific elongation and/or nucleation sites at the tips of lamellipodia and not solely by localized desequestration of subunits. We propose that the availability of the incorporation sites at the tips of lamellipodia is because of capping activities which preferentially inhibit barbed-end incorporation elsewhere in the cell, but leave barbed ends at the tips of lamellipodia free to add subunits.     

Actin dynamics in Phytophthora infestans; rapidly reorganizing cables and immobile,long‐lived plaques

The actin cytoskeleton is a dynamic but well‐organized intracellular framework that is essential for proper functioning of eukaryotic cells. Here, we use the actin binding peptide Lifeact to investigate the in vivo actin cytoskeleton dynamics in the oomycete plant pathogen Phytophthora infestans. Lifeact–eGFP labelled thick and thin actin bundles and actin filament plaques allowing visualization of actin dynamics. All actin structures in the hyphae were cortically localized. In growing hyphae actin filament cables were axially oriented in the sub‐apical region whereas in the extreme apex in growing hyphae, waves of fine F‐actin polymerization were observed. Upon growth termination, actin filament plaques appeared in the hyphal tip. The distance between a hyphal tip and the first actin filament plaque correlated strongly with hyphal growth velocity. The actin filament plaques were nearly immobile with average lifetimes exceeding 1 h, relatively long when compared to the lifetime of actin patches known in other eukaryotes. Plaque assembly required ～30 s while disassembly was accomplished in ～10 s. Remarkably, plaque disassembly was not accompanied with internalization and the formation of endocytic vesicles. These findings suggest that the functions of actin plaques in oomycetes differ from those of actin patches present in other organisms.     

alpha-Crystallin localizes to the leading edges of migrating lens epithelial cells

alpha-crystallin (alphaA and alphaB) is a major lens protein, which belongs to the small heat-shock family of proteins and binds to various cytoskeletal proteins including actin, vimentin and desmin. In this study, we investigated the cellular localization of alphaA and alphaB-crystallins in migrating epithelial cells isolated from porcine lens. Immunofluorescence localization and confocal imaging of alphaB-crystallin in confluent and in migrating subconfluent cell cultures revealed a distinct pattern of subcellular distribution. While alphaB-crystallin localization was predominantly cytoplasmic in confluent cultures, it was strongly localized to the leading edges of cell membrane or the lamellipodia in migrating cells. In accordance with this pattern, we found abundant levels of alphaB-crystallin in membrane fractions compared to cytosolic and nuclear fractions in migrating lens epithelial cells. alphaA-crystallin, which has 60% sequence identity to alphaB-crystallin, also exhibited a distribution profile localizing to the leading edge of the cell membrane in migrating lens epithelial cells. Localization of alphaB-crystallin to the lamellipodia appears to be dependent on phosphorylation of residue serine-59. An inhibitor of p38 MAP kinase (SB202190), but not the ERK kinase inhibitor PD98059, was found to diminish localization of alphaB-crystallin to the lamellipodia, and this effect was found to be associated with reduced levels of Serine-59 phosphorylated alphaB-crystallin in SB202190-treated migrating lens epithelial cells. alphaB-crystallin localization to the lamellipodia was also altered by the treatment with RGD (Arg-Ala-Asp) peptide, dominant negative N17 Rac1 GTPase, cytochalasin D and Src kinase inhibitor (PP2), but not by the Rho kinase inhibitor Y-27632 or the myosin II inhibitor, blebbistatin. Additionally, in migrating lens epithelial cells, alphaB-crystallin exhibited a clear co-localization with the actin meshwork, beta-catenin, WAVE-1, a promoter of actin nucleation, Abi-2, a component of WAVE-1 protein complex and Arp3, a protein of the actin nucleation complex, suggesting potential interactions between alphaB-crystallin and regulatory proteins involved in actin dynamics and cell adhesion. This is the first report demonstrating specific localization of alphaA and alphaB-crystallins to the lamellipodia in migrating lens epithelial cells and our findings indicate a potential role for alpha-crystallin in actin dynamics during cell migration.     

Cell migration: mechanisms of rear detachment and the formation of migration tracks

Cell migration is central to many biological and pathological processes, including embryogenesis, tissue repair and regeneration as well as cancer and the inflammatory response. In general, cell migration can be usefully conceptualized as a cyclic process. The initial response of a cell to a migration-promoting agent is to polarize and extend protrusions in the direction of migration. These protrusions can be large, broad lamellipodia or spike-like filopodia, are usually driven by actin polymerization, and are stabilized by adhering to the extracellular matrix (ECM) via transmembrane receptors of the integrin family linked to the actin cytoskeleton. These adhesions serve as traction sites for migration as the cell moves forward over them, and they must be disassembled at the cell rear, allowing it to detach. The mechanisms of rear detachment and the regulatory processes involved are not well understood. The disassembly of adhesions that is required for detachment depends on a coordinated interaction of actin and actin-binding proteins, signaling molecules and effector enzymes including proteases, kinases and phosphatases. Originally, the biochemically regulated processes leading to rear detachment of migrating cells were thought not to be necessarily accompanied by any loss of cell material. However, it has been shown that during rear detachment long tubular extensions, the retracting fibers, are formed and that "membrane ripping" occurs at the cell rear. By this process, a major fraction of integrin-containing cellular material is left behind forming characteristic migration tracks that exactly mark the way a cell has taken.     

Cortactin promotes cell motility by enhancing lamellipodial persistence

BACKGROUND: Lamellipodial protrusion, which is the first step in cell movement, is driven by actin assembly and requires activity of the Arp2/3 actin-nucleating complex. However, it is unclear how actin assembly is dynamically regulated to support effective cell migration. RESULTS: Cells deficient in cortactin have impaired cell migration and invasion. Kymography analyses of live-cell imaging studies demonstrate that cortactin-knockdown cells have a selective defect in the persistence of lamellipodial protrusions. The motility and protrusion defects are fully rescued by cortactin molecules, provided both the Arp2/3 complex and F-actin binding sites are intact. Consistent with this requirement for simultaneous contacts with Arp2/3 and F-actin, cortactin is recruited by Arp2/3 complex to lamellipodia and binds with a higher affinity to ATP/ADP-Pi-F-actin than to ADP-F-actin. In situ labeling of lamellipodia revealed that the relative levels of free barbed ends of actin filaments are reduced by over 30% in the cortactin-knockdown cells; however, there is no change in Arp2/3-complex localization to lamellipodia. Cortactin-knockdown cells also have a selective defect in the assembly of new adhesions in protrusions, as assessed by analysis of GFP-paxillin dynamics in living cells. CONCLUSIONS: Cortactin enhances lamellipodial persistence, at least in part through regulation of Arp2/3 complex. The presence of cortactin also enhances the rate of new adhesion formation in lamellipodia. In vivo, these functions may be important during directed cell motility.     

Dynamic actin patterns and Arp2/3 assembly at the substrate-attached surface of motile cells

BACKGROUND: In the cortical region of motile cells, the actin network rapidly reorganizes as required for movement in various directions and for cell-to-substrate adhesion. The analysis of actin network dynamics requires the combination of high-resolution imaging with a specific fluorescent probe that highlights the filamentous actin structures in live cells. RESULTS: Combining total internal reflection fluorescence (TIRF) microscopy with a method for labeling actin filaments, we analyze the dynamics of actin patterns in the highly motile cells of Dictyostelium. A rapidly restructured network of single or bundled actin filaments provides a scaffold for the assembly of differentiated actin complexes. Recruitment of the Arp2/3 complex characterizes stationary foci with a lifetime of 7-10 s and traveling waves. These structures are also formed in the absence of myosin-II. Arp2/3-actin assemblies similar to those driving the protrusion of a leading edge form freely at the inner face of the plasma membrane. CONCLUSIONS: The actin system of highly motile cells runs far from equilibrium and generates a multitude of patterns within a dynamic filamentous network. Traveling waves are the most complicated patterns based on recruitment of the Arp2/3 complex. They are governed by the propagated induction of actin polymerization. We hypothesize that the actin system autonomously generates primordia of specialized structures such as phagocytic cups or lamellipodia. These primordia would represent an activated state of the actin system and enable cells to respond within seconds to local stimuli by chemotaxis or phagocytic-cup formation.