Analysis of the Actin–Myosin II System in Fish Epidermal Keratocytes: Mechanism of Cell Body Translocation

While the protrusive event of cell locomotion is thought to be driven by actin polymerization, the mechanism of forward translocation of the cell body is unclear. To elucidate the mechanism of cell body translocation, we analyzed the supramolecular organization of the actin–myosin II system and the dynamics of myosin II in fish epidermal keratocytes. In lamellipodia, long actin filaments formed dense networks with numerous free ends in a brushlike manner near the leading edge. Shorter actin filaments often formed T junctions with longer filaments in the brushlike area, suggesting that new filaments could be nucleated at sides of preexisting filaments or linked to them immediately after nucleation. The polarity of actin filaments was almost uniform, with barbed ends forward throughout most of the lamellipodia but mixed in arc-shaped filament bundles at the lamellipodial/cell body boundary. Myosin II formed discrete clusters of bipolar minifilaments in lamellipodia that increased in size and density towards the cell body boundary and colocalized with actin in boundary bundles. Time-lapse observation demonstrated that myosin clusters appeared in the lamellipodia and remained stationary with respect to the substratum in locomoting cells, but they exhibited retrograde flow in cells tethered in epithelioid colonies. Consequently, both in locomoting and stationary cells, myosin clusters approached the cell body boundary, where they became compressed and aligned, resulting in the formation of boundary bundles. In locomoting cells, the compression was associated with forward displacement of myosin features. These data are not consistent with either sarcomeric or polarized transport mechanisms of cell body translocation. We propose that the forward translocation of the cell body and retrograde flow in the lamellipodia are both driven by contraction of an actin–myosin network in the lamellipodial/cell body transition zone.     

The Role of Stress Fibers in the Shape Determination Mechanism of Fish Keratocytes

Crawling cells have characteristic shapes that are a function of their cell types. How their different shapes are determined is an interesting question. Fish epithelial keratocytes are an ideal material for investigating cell shape determination, because they maintain a nearly constant fan shape during their crawling locomotion. We compared the shape and related molecular mechanisms in keratocytes from different fish species to elucidate the key mechanisms that determine cell shape. Wide keratocytes from cichlids applied large traction forces at the rear due to large focal adhesions, and showed a spatially loose gradient associated with actin retrograde flow rate, whereas round keratocytes from black tetra applied low traction forces at the rear small focal adhesions and showed a spatially steep gradient of actin retrograde flow rate. Laser ablation of stress fibers (contractile fibers connected to rear focal adhesions) in wide keratocytes from cichlids increased the actin retrograde flow rate and led to slowed leading-edge extension near the ablated region. Thus, stress fibers might play an important role in the mechanism of maintaining cell shape by regulating the actin retrograde flow rate.     

High resolution detection of mechanical forces exerted by locomoting fibroblasts on the substrate

We have developed a new approach to detect mechanical forces exerted by locomoting fibroblasts on the substrate. Cells were cultured on elastic, collagen-coated polyacrylamide sheets embedded with 0. 2-micrometer fluorescent beads. Forces exerted by the cell cause deformation of the substrate and displacement of the beads. By recording the position of beads during cell locomotion and after cell removal, we discovered that most forces were radially distributed, switching direction in the anterior region. Deformations near the leading edge were strong, transient, and variable in magnitude, consistent with active local contractions, whereas those in the posterior region were weaker, more stable, and more uniform, consistent with passive resistance. Treatment of cells with cytochalasin D or myosin II inhibitors caused relaxation of the forces, suggesting that they are generated primarily via actin-myosin II interactions; treatment with nocodazole caused no immediate effect on forces. Immunofluorescence indicated that the frontal region of strong deformation contained many vinculin plaques but no apparent concentration of actin or myosin II filaments. Strong mechanical forces in the anterior region, generated by locally activated myosin II and transmitted through vinculin-rich structures, likely play a major role in cell locomotion and in mechanical signaling with the surrounding environment.     

Ku80 as a novel receptor for thymosin beta4 that mediates its intracellular activity different from G-actin sequestering

Our data demonstrate that increased intracellular expression of thymosin beta4(Tbeta4) is necessary and sufficient to induce plasminogen activator inhibitor type 1 (PAI-1) gene expression in endothelial cells. To describe the mechanism of this effect, we produced Tbeta4 mutants with impaired functional motifs and tested their intracellular location and activity. Cytoplasmic distributions of Tbeta4((AcSDKPT/4A)), Tbeta4((KLKKTET/7A)), and Tbeta4((K16A)) mutants fused with green fluorescent protein did not differ significantly from those of wild-type Tbeta4. Overexpression of Tbeta4, Tbeta4((AcSDKPT/4A)), and Tbeta4((K16A)) affected intracellular formation of actin filaments. As expected, Tbeta4((K16A)) uptake by nuclei was impaired. On the other hand, overexpression of Tbeta4((KLKKTET/7A)) resulted in developing the actin filament network typical of adhering cells, indicating that the mutant lacked the actin binding site. The mechanism by which intracellular Tbeta4 induced the PAI-1 gene did not depend upon the N-terminal tetrapeptide AcSDKP and depended only partially on its ability to bind G-actin or enter the nucleus. Both Tbeta4 and Tbeta4((AcSDKPT/4A)) induced the PAI-1 gene to the same extent, whereas mutants Tbeta4((KLKKTET/7A)) and Tbeta4((K16A)) retained about 60% of the original activity. By proteomic analysis, the Ku80 subunit of ATP-dependent DNA helicase II was found to be associated with Tbeta4. Ku80 and Tbeta4 consistently co-immunoprecipitated in a complex from endothelial cells. Co-transfection of endothelial cells with the Ku80 deletion mutants and Tbeta4 showed that the C-terminal arm domain of Ku80 is directly involved in this interaction. Furthermore, down-regulation of Ku80 by specific short interference RNA resulted in dramatic reduction in PAI-1 expression at the level of both mRNA and protein synthesis. These data suggest that Ku80 functions as a novel receptor for Tbeta4 and mediates its intracellular activity.     

Locomotion of fish epidermal keratocytes on spatially selective adhesion patterns

Cell migration results from forces generated by assembly, contraction, and adhesion of the cytoskeleton. To address how these forces integrate in space and time, novel assays are required that allow spatial separation of the different force categories. We used micro-contact printing of fibronectin on glass substrates to study the effect of adhesion patterns on fish epidermal keratocytes locomotion. Cells migrated at similar speeds on homogeneously adhesive substrates and on patterns with 5 microm-wide adhesive stripes interleaved by non-adhesive stripes with a width varied between 5 and 13 microm. The leading edge protruded on adhesive stripes and lagged behind on non-adhesive stripes. On patterns with non-adhesive stripes wider than 13 microm cells halted, although the lamellipodium did not collapse. High correlation was found between the widths of protruding and lagging edge segments and the widths of the underlying stripes. We explain our data by the force balances between actin polymerization, contraction and adhesion on fibronectin stripes; and between actin polymerization, contraction and lamellipodium-internal elastic tension on non-adhesive stripes. We tested our model further by blocking lamellipodium actin network contraction and polymerization. In both experiments we observed that cells eventually lost their ability to move. However, the two perturbations induced distinct morphological responses. The data suggested that forces powering forward motion of keratocytes are largely associated with network assembly whereas contraction maintains cell polarity. This study establishes spatially selective adhesion substrates and cell morphological readouts as a means to elucidate the mechanical balance between substrate adhesion and cytoskeleton-internal tension in cell migration.     

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.     

Thymosin-beta(4) changes the conformation and dynamics of actin monomers

Thymosin-beta(4) (Tbeta(4)) binds actin monomers stoichiometrically and maintains the bulk of the actin monomer pool in metazoan cells. Tbeta(4) binding quenches the fluorescence of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS) conjugated to Cys(374) of actin monomers. The K(d) of the actin-Tbeta(4) complex depends on the cation and nucleotide bound to actin but is not affected by the AEDANS probe. The different stabilities are determined primarily by the rates of dissociation. At 25 degrees C, the free energy of Tbeta(4) binding MgATP-actin is primarily enthalpic in origin but entropic for CaATP-actin. Binding is coupled to the dissociation of bound water molecules, which is greater for CaATP-actin than MgATP-actin monomers. Proteolysis of MgATP-actin, but not CaATP-actin, at Gly(46) on subdomain 2 is >12 times faster when Tbeta(4) is bound. The C terminus of Tbeta(4) contacts actin near this cleavage site, at His(40). By tritium exchange, Tbeta(4) slows the exchange rate of approximately eight rapidly exchanging amide protons on actin. We conclude that Tbeta(4) changes the conformation and structural dynamics ("breathing") of actin monomers. The conformational change may reflect the unique ability of Tbeta(4) to sequester actin monomers and inhibit nucleotide exchange.     

Pinocytosis and locomotion of amoebae. XIX. Immunocytochemical demonstration of actin and myosin in Amoeba proteus

The spatial distribution of cytoplasmic actin and myosin in 1. normal locomoting, 2. immobilized, and 3. pinocytosing Amoeba proteus was demonstrated by indirect immunofluorescence microscopy. In orthotactic and polytactic cells fixed during normal locomotion actin is mainly located in a cortical layer delineating the granuloplasm from the peripheral hyaloplasm. In cell areas lacking a hyaloplasmic sheet the actin layer immediately borders the plasma membrane. The amount of actin within the continuous layer seems to increase from the advancing front to the middle cell region and to decrease again toward the uroid. The distribution of myosin is largely congruent to the display of actin, with the exception that the myosin-based fluorescence of the cortical layer gradually increases from the front to the uroid. A considerable amount of actin and myosin is also distributed around the nucleus and the contractile vacuole. In immobilized cells contracted by the external application of 10(-4)M procaine hydrochloride the cortical layer distinctly increases in thickness. In contrast to normal locomoting cells actin and myosin show a uniform distribution within the cell cortex along the entire surface. In pinocytosing cells, up to three cortical layers conspicuously rich in actin are produced during the process of channel formation. One of these layers is located in close proximity to the plasma membrane of the pinocytotic channels and the vacuoles. The immunocytochemical results are discussed with respect to earlier observations on the distribution of actin and myosin in Amoeba proteus as obtained by other methods.     

Tracking retrograde flow in keratocytes: news from the front

Actin assembly at the leading edge of the cell is believed to drive protrusion, whereas membrane resistance and contractile forces result in retrograde flow of the assembled actin network away from the edge. Thus, cell motion and shape changes are expected to depend on the balance of actin assembly and retrograde flow. This idea, however, has been undermined by the reported absence of flow in one of the most spectacular models of cell locomotion, fish epidermal keratocytes. Here, we use enhanced phase contrast and fluorescent speckle microscopy and particle tracking to analyze the motion of the actin network in keratocyte lamellipodia. We have detected retrograde flow throughout the lamellipodium at velocities of 1-3 microm/min and analyzed its organization and relation to the cell motion during both unobstructed, persistent migration and events of cell collision. Freely moving cells exhibited a graded flow velocity increasing toward the sides of the lamellipodium. In colliding cells, the velocity decreased markedly at the site of collision, with striking alteration of flow in other lamellipodium regions. Our findings support the universality of the flow phenomenon and indicate that the maintenance of keratocyte shape during locomotion depends on the regulation of both retrograde flow and actin polymerization.     

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.     

Structural basis of actin sequestration by thymosin-beta4: implications for WH2 proteins

The WH2 (Wiscott-Aldridge syndrome protein homology domain 2) repeat is an actin interacting motif found in monomer sequestering and filament assembly proteins. We have stabilized the prototypical WH2 family member, thymosin-beta4 (Tbeta4), with respect to actin, by creating a hybrid between gelsolin domain 1 and the C-terminal half of Tbeta4 (G1-Tbeta4). This hybrid protein sequesters actin monomers, severs actin filaments and acts as a leaky barbed end cap. Here, we present the structure of the G1-Tbeta4:actin complex at 2 A resolution. The structure reveals that Tbeta4 sequesters by capping both ends of the actin monomer, and that exchange of actin between Tbeta4 and profilin is mediated by a minor overlap in binding sites. The structure implies that multiple WH2 motif-containing proteins will associate longitudinally with actin filaments. Finally, we discuss the role of the WH2 motif in arp2/3 activation.     

Separation of propulsive and adhesive traction stresses in locomoting keratocytes

Strong, actomyosin-dependent, pinching tractions in steadily locomoting (gliding) fish keratocytes revealed by traction imaging present a paradox, since only forces perpendicular to the direction of locomotion are apparent, leaving the actual propulsive forces unresolved. When keratocytes become transiently "stuck" by their trailing edge and adopt a fibroblast-like morphology, the tractions opposing locomotion are concentrated into the tail, leaving the active pinching and propulsive tractions clearly visible under the cell body. Stuck keratocytes can develop approximately 1 mdyn (10,000 pN) total propulsive thrust, originating in the wings of the cell. The leading lamella develops no detectable propulsive traction, even when the cell pulls on its transient tail anchorage. The separation of propulsive and adhesive tractions in the stuck phenotype leads to a mechanically consistent hypothesis that resolves the traction paradox for gliding keratocytes: the propulsive tractions driving locomotion are normally canceled by adhesive tractions resisting locomotion, leaving only the pinching tractions as a resultant. The resolution of the traction pattern into its components specifies conditions to be met for models of cytoskeletal force production, such as the dynamic network contraction model (Svitkina, T.M., A.B. Verkhovsky, K.M. McQuade, and G.G. Borisy. 1997. J. Cell Biol. 139:397-415). The traction pattern associated with cells undergoing sharp turns differs markedly from the normal pinching traction pattern, and can be accounted for by postulating an asymmetry in contractile activity of the opposed lateral wings of the cell.     

Location of actin, myosin, and microtubular structures during directed locomotion of Dictyostelium amebae

During their life cycle, amebae of the cellular slime mould Dictyostelium discoideum aggregate to form multicellular structures in which differentiation takes place. Aggregation depends upon the release of chemotactic signals of 3',5'-cAMP from aggregation centers. In response to the signals, aggregating amebae elongate, actively more toward the attractive source, and may be easily identified from the other cells because of their polarized appearance. To examine the role of cytoskeletal components during ameboid locomotion, immunofluorescence microscopy with antibodies to actin, myosin, and to a microtubule-associated component was used. In addition, rhodamine-labeled phallotoxin was employed. Actin and myosin display a rather uniform distribution in rounded unstretched cells. In polarized locomoting cells, actin fluorescence (due to both labeled phallotoxin and specific antibody) is prevalently concentrated in the anterior pseudopod while myosin fluorescence appears to be excluded from the pseudopod. Similarly, microtubules in locomoting cells are excluded from the leading pseudopod. The cell nucleus is attached to the microtubule network by way of a nucleus-associated organelle serving as a microtubule-organizing center and seems to be maintained in a rather fixed position by the microtubules. These findings, together with available morphological and biochemical evidences, are consistent with a mechanism in which polymerized actin is moved into the pseudopod through its interaction with myosin at the base of the pseudopod. Microtubules, apparently, do not actively participate in movement but seem to behave as anchorage structures for the nucleus and possibly other cytoplasmic organelles.     

Thymosin beta4 induces a conformational change in actin monomers

Using fluorescence resonance energy transfer spectroscopy we demonstrate that thymosin beta(4) (tbeta(4)) binding induces spatial rearrangements within the small domain (subdomains 1 and 2) of actin monomers in solution. Tbeta(4) binding increases the distance between probes attached to Gln-41 and Cys-374 of actin by 2 A and decreases the distance between the purine base of bound ATP (epsilonATP) and Lys-61 by 1.9 A, whereas the distance between Cys-374 and Lys-61 is minimally affected. Distance determinations are consistent with tbeta(4) binding being coupled to a rotation of subdomain 2. By differential scanning calorimetry, tbeta(4) binding increases the cooperativity of ATP-actin monomer denaturation, consistent with conformational rearrangements in the tbeta(4)-actin complex. Changes in fluorescence resonance energy transfer are accompanied by marked reduction in solvent accessibility of the probe at Gln-41, suggesting it forms part of the binding interface. Tbeta(4) and cofilin compete for actin binding. Tbeta(4) concentrations that dissociate cofilin from actin do not dissociate the cofilin-DNase I-actin ternary complex, consistent with the DNase binding loop contributing to high-affinity tbeta(4)-binding. Our results favor a model where thymosin binding changes the average orientation of actin subdomain 2. The tbeta(4)-induced conformational change presumably accounts for the reduced rate of amide hydrogen exchange from actin monomers and may contribute to nucleotide-dependent, high affinity binding.     

Actin-myosin network reorganization breaks symmetry at the cell rear to spontaneously initiate polarized cell motility

We have analyzed the spontaneous symmetry breaking and initiation of actin-based motility in keratocytes (fish epithelial cells). In stationary keratocytes, the actin network flow was inwards and radially symmetric. Immediately before motility initiation, the actin network flow increased at the prospective cell rear and reoriented in the perinuclear region, aligning with the prospective axis of movement. Changes in actin network flow at the cell front were detectable only after cell polarization. Inhibition of myosin II or Rho kinase disrupted actin network organization and flow in the perinuclear region and decreased the motility initiation frequency, whereas increasing myosin II activity with calyculin A increased the motility initiation frequency. Local stimulation of myosin activity in stationary cells by the local application of calyculin A induced directed motility initiation away from the site of stimulation. Together, these results indicate that large-scale actin-myosin network reorganization and contractility at the cell rear initiate spontaneous symmetry breaking and polarized motility of keratocytes.     

Effect of thymosin beta15 on the branching of developing neurons

The thymosin betas (Tbetas) are polypeptide regulators of actin dynamics that are critical for the growth and branching of neurites in developing neurons. We found that mRNAs for Tbeta4, Tbeta10, and Tbeta15 were highly expressed in the developing rat brain during neuritogenesis, supporting a role for the Tbetas in this process. Overexpression of the Tbetas increased the number of neurite branches per neuron in cultured hippocampal and cerebral cortex neurons, and Tbeta15 had the greatest effect. Actin binding activity appears to be essential for the branch-promoting activity of Tbetas because two mutants of Tbeta15 lacking monomeric actin binding activity failed to stimulate branch formation. We also found that transfection of siRNA against Tbeta15 reduced branching. Taken together, these data suggest that the three Tbetas, and especially Tbeta15, stimulate neurite branching during brain development.     

Hyaluronan Synthesis Mediates the Fibrotic Response of Keratocytes to Transforming Growth Factor β

TGFβ induces fibrosis in healing corneal wounds, and in vitro corneal keratocytes up-regulate expression of several fibrosis-related genes in response to TGFβ. Hyaluronan (HA) accumulates in healing corneas, and HA synthesis is induced by TGFβ by up-regulation of HA synthase 2. This study tested the hypothesis that HA acts as an extracellular messenger, enhancing specific fibrotic responses of keratocytes to TGFβ. HA synthesis inhibitor 4-methylumbelliferone (4MU) blocked TGFβ induction of HA synthesis in a concentration-dependent manner. 4MU also inhibited TGFβ-induced up-regulation of α-smooth muscle actin, collagen type III, and extra domain A-fibronectin. Chemical analogs of 4MU also inhibited fibrogenic responses in proportion to their inhibition of HA synthesis. 4MU, however, showed no effect on TGFβ induction of luciferase by the 3TP-Lux reporter plasmid. Inhibition of HA using siRNA to HA synthase 2 reduced TGFβ up-regulation of smooth muscle actin, fibronectin, and cell division. Similarly, brief treatment of keratocytes with hyaluronidase reduced TGFβ responses. These results suggest that newly synthesized cell-associated HA acts as an extracellular enhancer of wound healing and fibrosis in keratocytes by augmenting a limited subset of the cellular responses to TGFβ.     

Actomyosin transports microtubules and microtubules control actomyosin recruitment during Xenopus oocyte wound healing

BACKGROUND: Interactions between microtubules and actin filaments (F-actin) are critical for cellular motility processes ranging from directed cell locomotion to cytokinesis. However, the cellular bases of these interactions remain poorly understood. We have analyzed the role of microtubules in generation of a contractile array comprised of F-actin and myosin-2 that forms around wounds made in Xenopus oocytes. RESULTS: After wounding, microtubules are transported to the wound edge in association with F-actin that is itself recruited to wound borders via actomyosin-powered cortical flow. This transport generates sufficient force to buckle and break microtubules at the wound edge. Transport is complemented by local microtubule assembly around wound borders. The region of microtubule breakage and assembly coincides with a zone of actin assembly, and perturbation of the microtubule cytoskeleton disrupts this zone as well as local recruitment of the Arp2/3 complex and myosin-2. CONCLUSIONS: The results reveal transport of microtubules in association with F-actin that is pulled to wound borders via actomyosin-based contraction. Microtubules, in turn, focus zones of actin assembly and myosin-2 recruitment at the wound border. Thus, wounding triggers the formation of a spatially coordinated feedback loop in which transport and assembly of microtubules maintains actin and myosin-2 in close proximity to the closing contractile array. These results are surprisingly reminiscent of recent findings in locomoting cells, suggesting that similar feedback interactions may be generally employed in a variety of fundamental cell motility processes.     

Identification of Novel Graded Polarity Actin Filament Bundles in Locomoting Heart Fibroblasts: Implications for the Generation of Motile Force

We have determined the structural organization and dynamic behavior of actin filaments in entire primary locomoting heart fibroblasts by S1 decoration, serial section EM, and photoactivation of fluorescence. As expected, actin filaments in the lamellipodium of these cells have uniform polarity with barbed ends facing forward. In the lamella, cell body, and tail there are two observable types of actin filament organization. A less abundant type is located on the inner surface of the plasma membrane and is composed of short, overlapping actin bundles (0.25–2.5 μm) that repeatedly alternate in polarity from uniform barbed ends forward to uniform pointed ends forward. This type of organization is similar to the organization we show for actin filament bundles (stress fibers) in nonlocomoting cells (PtK2 cells) and to the known organization of muscle sarcomeres. The more abundant type of actin filament organization in locomoting heart fibroblasts is mostly ventrally located and is composed of long, overlapping bundles (average 13 μm, but can reach up to about 30 μm) which span the length of the cell. This more abundant type has a novel graded polarity organization. In each actin bundle, polarity gradually changes along the length of the bundle. Actual actin filament polarity at any given point in the bundle is determined by position in the cell; the closer to the front of the cell the more barbed ends of actin filaments face forward.

By photoactivation marking in locomoting heart fibroblasts, as expected in the lamellipodium, actin filaments flow rearward with respect to substrate. In the lamella, all marked and observed actin filaments remain stationary with respect to substrate as the fibroblast locomotes. In the cell body of locomoting fibroblasts there are two dynamic populations of actin filaments: one remains stationary and the other moves forward with respect to substrate at the rate of the cell body.

This is the first time that the structural organization and dynamics of actin filaments have been determined in an entire locomoting cell. The organization, dynamics, and relative abundance of graded polarity actin filament bundles have important implications for the generation of motile force during primary heart fibroblast locomotion.