Roles of type II myosin and a tropomyosin isoform in retrograde actin flow in budding yeast

Retrograde flow of cortical actin networks and bundles is essential for cell motility and retrograde intracellular movement, and for the formation and maintenance of microvilli, stereocilia, and filopodia. Actin cables, which are F-actin bundles that serve as tracks for anterograde and retrograde cargo movement in budding yeast, undergo retrograde flow that is driven, in part, by actin polymerization and assembly. We find that the actin cable retrograde flow rate is reduced by deletion or delocalization of the type II myosin Myo1p, and by deletion or conditional mutation of the Myo1p motor domain. Deletion of the tropomyosin isoform Tpm2p, but not the Tpm1p isoform, increases the rate of actin cable retrograde flow. Pretreatment of F-actin with Tpm2p, but not Tpm1p, inhibits Myo1p binding to F-actin and Myo1p-dependent F-actin gliding. These data support novel, opposing roles of Myo1p and Tpm2 in regulating retrograde actin flow in budding yeast and an isoform-specific function of Tpm1p in promoting actin cable function in myosin-driven anterograde cargo transport.     

Non-polymerizing long-pitch actin dimers that interact with myosin

The production of a soluble actomyosin complex would be a significant step toward elucidating molecular interactions responsible for biological movement. We took a systematic approach to producing soluble long-pitch actin dimers that are polymerization-deficient yet retain biological protein-protein interactions, including myosin binding. Actin mutant proteins and chemical crosslinking were combined with different polymerization inhibition strategies, including ADP-ribosylation, or the use of a polymerization-deficient actin mutant protein. While all of the long-pitch actin dimers retained interactions reflective of F-actin activity, each displayed different interactions with myosin. Myosin did not interact productively with long-pitch actin dimers capped with DNase-I, and led to filament formation of unmodified long-pitch actin dimers or dimers possessing a polymerization-deficient actin subunit. However, ADP-ribosylated long-pitch actin dimers interacted with myosin, giving this dimer great potential for producing a soluble actomyosin complex, which could greatly improve our understanding of the molecular basis of movement in cells, tissues, and organisms.     

Relative distribution of myosin, actin, and alpha-actinin in adherent monocytes.

The characteristic amoeboid movement of human leucocytes uses mechanical energy derived from the hydrolysis of adenosine triphosphate through a mechanochemical system of the contractile proteins myosin, actin, and the actin-associated protein alpha-actinin. We observed the relative distribution of myosin, actin, and alpha-actinin in adherent monocytes during movement by a double-fluorescence staining procedure. The results indicate that myosin and alpha-actinin are closely associated with the actin cable network, and that alpha-actinin is in close association with the plasma membrane and anchors filamentous actin (F-actin) beneath the plasma membrane; F-actin and alpha-actinin play an important role at the leading edge during the formation of lamellipodia. These findings should be helpful in clarifying the mechanism of leucocyte movement from a morphologic standpoint.     

The biochemical kinetics underlying actin movement generated by one and many skeletal muscle myosin molecules

To better understand how skeletal muscle myosin molecules move actin filaments, we determine the motion-generating biochemistry of a single myosin molecule and study how it scales with the motion-generating biochemistry of an ensemble of myosin molecules. First, by measuring the effects of various ligands (ATP, ADP, and P(i)) on event lifetimes, tau(on), in a laser trap, we determine the biochemical kinetics underlying the stepwise movement of an actin filament generated by a single myosin molecule. Next, by measuring the effects of these same ligands on actin velocities, V, in an in vitro motility assay, we determine the biochemistry underlying the continuous movement of an actin filament generated by an ensemble of myosin molecules. The observed effects of P(i) on single molecule mechanochemistry indicate that motion generation by a single myosin molecule is closely associated with actin-induced P(i) dissociation. We obtain additional evidence for this relationship by measuring changes in single molecule mechanochemistry caused by a smooth muscle HMM mutation that results in a reduced P(i)-release rate. In contrast, we observe that motion generation by an ensemble of myosin molecules is limited by ATP-induced actin dissociation (i.e., V varies as 1/tau(on)) at low [ATP], but deviates from this relationship at high [ATP]. The single-molecule data uniquely provide a direct measure of the fundamental mechanochemistry of the actomyosin ATPase reaction under a minimal load and serve as a clear basis for a model of ensemble motility in which actin-attached myosin molecules impose a load.     

The role of actin and myosin in ascidian sperm mitochondrial translocation

Fertilization-related sperm mitochondrial movement occurs at a rate comparable to other actin-myosin-driven movements and is inhibited by cytochalasin B and N-ethyl maleimide in Ascidia ceratodes sperm. F-actin was demonstrated in the tails and mitochondria using NBD-phallacidin fluorescence. Both actin and myosin were also detected on the mitochondrion and in the tail by indirect immunofluorescence. Western blot analysis verified the presence of these proteins. Boltenia villosa and Cnemidocarpa finmarkiensis also have mitochondrion and tail localized actin and myosin. In the tails of all 3 species the fluorescence takes the form of discrete spots 0.25-0.5 micron apart. Boltenia and Cnemidocarpa sperm have additional actin at the tip of the head and additional myosin at the base of the head. The presence of actin and myosin on the mitochondrion and in the tail supports a means by which the force for mitochondrial movement is generated.     

Pak1 regulates focal adhesion strength, myosin IIA distribution, and actin dynamics to optimize cell migration

Cell motility requires the spatial and temporal coordination of forces in the actomyosin cytoskeleton with extracellular adhesion. The biochemical mechanism that coordinates filamentous actin (F-actin) assembly, myosin contractility, adhesion dynamics, and motility to maintain the balance between adhesion and contraction remains unknown. In this paper, we show that p21-activated kinases (Paks), downstream effectors of the small guanosine triphosphatases Rac and Cdc42, biochemically couple leading-edge actin dynamics to focal adhesion (FA) dynamics. Quantitative live cell microscopy assays revealed that the inhibition of Paks abolished F-actin flow in the lamella, displaced myosin IIA from the cell edge, and decreased FA turnover. We show that, by controlling the dynamics of these three systems, Paks regulate the protrusive activity and migration of epithelial cells. Furthermore, we found that expressing Pak1 was sufficient to overcome the inhibitory effects of excess adhesion strength on cell motility. These findings establish Paks as critical molecules coordinating cytoskeletal systems for efficient cell migration.     

Motor function and regulation of myosin X.

Myosin X is a member of the diverse myosin superfamily that is ubiquitously expressed in various mammalian tissues. Although its association with actin in cells has been shown, little is known about its biochemical and mechanoenzymatic function at the molecular level. We expressed bovine myosin X containing the entire head, neck, and coiled-coil domain and purified bovine myosin X in Sf9 cells. The Mg(2+)-ATPase activity of myosin X was significantly activated by actin with low K(ATP). The actin-activated ATPase activity was reduced at Ca(2+) concentrations above pCa 5 in which 1 mol of calmodulin light chain dissociates from the heavy chain. Myosin X translocates F-actin filaments with the velocity of 0.3 microm/s with the direction toward the barbed end. The actin translocating activity was inhibited at concentrations of Ca(2+) at pCa 6 in which no calmodulin dissociation takes place, suggesting that the calmodulin dissociation is not required for the inhibition of the motility. Unlike class V myosin, which shows a high affinity for F-actin in the presence of ATP, the K(actin) of the myosin X ATPase was much higher than that of myosin V. Consistently nearly all actin dissociated from myosin X in the presence of ATP. ADP did not significantly inhibit the actin-activated ATPase activity of myosin X, suggesting that the ADP release step is not rate-limiting. These results suggest that myosin X is a nonprocessive motor. Consistently myosin X failed to support the actin translocation at low density in an in vitro motility assay where myosin V, a processive motor, supports the actin filament movement.     

Lamellipodial actin mechanically links myosin activity with adhesion-site formation

Cell motility proceeds by cycles of edge protrusion, adhesion, and retraction. Whether these functions are coordinated by biochemical or biomechanical processes is unknown. We find that myosin II pulls the rear of the lamellipodial actin network, causing upward bending, edge retraction, and initiation of new adhesion sites. The network then separates from the edge and condenses over the myosin. Protrusion resumes as lamellipodial actin regenerates from the front and extends rearward until it reaches newly assembled myosin, initiating the next cycle. Upward bending, observed by evanescence and electron microscopy, results in ruffle formation when adhesion strength is low. Correlative fluorescence and electron microscopy shows that the regenerating lamellipodium forms a cohesive, separable layer of actin above the lamellum. Thus, actin polymerization periodically builds a mechanical link, the lamellipodium, connecting myosin motors with the initiation of adhesion sites, suggesting that the major functions driving motility are coordinated by a biomechanical process.     

Role of actin polymerization and actin cables in actin-patch movement in Schizosaccharomyces pombe

Factors that are involved in actin polymerization, such as the Arp2/3 complex, have been found to be packaged into discrete, motile, actin-rich foci. Here we investigate the mechanism of actin-patch motility in S. pombe using a fusion of green fluorescent protein (GFP) to a coronin homologue, Crn1p. Actin patches are associated with cables and move with rates of 0.32 microm s(-1) primarily in an undirected manner at cell tips and also in a directed manner along actin cables, often away from cell tips. Patches move more slowly or stop when actin polymerization is attenuated by Latrunculin A or in arp3 and cdc3 (profilin) mutants. In a cdc8 (tropomyosin) mutant, actin cables are absent, and patches move with similar speed but in a non-directed manner. Patches are sites of Arp3-dependent F-actin polymerization in vitro. Rapid F-actin turnover rates in vivo indicate that patches and cables are maintained continuously by actin polymerization. Our studies give rise to a model in which actin patches are centres for actin polymerization that drive their own movement on actin cables using Arp2/3-based actin polymerization.     

Apical myosin XI anticipates F‐actin during polarized growth of Physcomitrella patens cells

Tip growth is essential for land colonization by bryophytes, plant sexual reproduction and water and nutrient uptake. Because this specialized form of polarized cell growth requires both a dynamic actin cytoskeleton and active secretion, it has been proposed that the F‐actin‐associated motor myosin XI is essential for this process. Nevertheless, a spatial and temporal relationship between myosin XI and F‐actin during tip growth is not known in any plant cell. Here, we use the highly polarized cells of the moss Physcomitrella patens to show that myosin XI and F‐actin localize, in vivo, at the same apical domain and that both signals fluctuate. Surprisingly, phase analysis shows that increase in myosin XI anticipates that of F‐actin; in contrast, myosin XI levels at the tip fluctuate in identical phase with a vesicle marker. Pharmacological analysis using a low concentration of the actin polymerization inhibitor latrunculin B showed that the F‐actin at the tip can be significantly diminished while myosin XI remains elevated in this region, suggesting that a mechanism exists to cluster myosin XI‐associated structures at the cell's apex. In addition, this approach uncovered a mechanism for actin polymerization‐dependent motility in the moss cytoplasm, where myosin XI‐associated structures seem to anticipate and organize the actin polymerization machinery. From our results, we inferred a model where the interaction between myosin XI‐associated vesicular structures and F‐actin polymerization‐driven motility function at the cell's apex to maintain polarized cell growth. We hypothesize this is a general mechanism for the participation of myosin XI and F‐actin in tip growing cells.     

Interaction of phalloidin with chemically modified actin

Modification of Tyr-69 with tetranitromethane impairs the polymerizability of actin in accordance with the previous report [Lehrer, S. S. and Elzinga, M. (1972) Fed. Proc. 31, 502]. Phalloidin induces this chemically modified actin to form the same characteristic helical thread-like structure as normal F-actin. The filaments bind myosin heads and activate the myosin ATPase activity as effectively as normal F-actin. When a dansyl group is introduced at the same point [Chantler, P. D. and Gratzer, W. B. (1975) Eur. J. Biochem. 60, 67-72], phalloidin still induces the polymerization. The filaments bind myosin heads and activate the myosin ATPase activity. These results indicate that Tyr-69 is not directly involved in either an actin-actin binding site or the myosin binding site on actin. Moreover, the results suggest that phalloidin binds to actin monomer in the presence of salt and its binding induces a conformational change in actin which is essential for polymerization, or that actin monomer fluctuates between in unpolymerizable and polymerizable form while phalloidin binds to actin only in the polymerizable form and its binding locks the conformation which causes the irreversible polymerization of actin. Modification of Tyr-53 with 5-diazonium-(1H)tetrazole blocks actin polymerization [Bender, N., Fasold, H., Kenmoku, A., Middelhoff, G. and Volk, K. E. (1976) Eur. J. Biochem. 64, 215-218]. Phalloidin is unable to induce the polymerization of this modified actin nor does it bind to it. Phalloidin does not induce the polymerization of the trypsin-digested actin core. These results indicate that the site at which phalloidin binds is involved in polymerization and the probable conformational change involved in polymerization may be modulated through this site.     

In vitro motility of skeletal muscle myosin and its proteolytic fragments

We have compared actin-activated myosin ATPase activity, myosin binding to actin, and the velocity of myosin-induced actin sliding in order to understand the mechanism of myosin motility. In our in vitro assay, F-actin slides at a constant velocity, regardless of length. The F-actin could slide over myosin heads at KCl concentrations below a critical value (60 mM with myosin and HMM, 100 mM with S-1), and the sliding velocities were quite similar below the critical KCl concentration. However, at KCl concentrations close to the critical value, the sliding F-actin is attached to only one or a few particular points on the surface, each of which perhaps consists of a single head of myosin. The KATPase values for actin-activated ATPase were approximately 300 microM for S-1 and approximately 200 microM with HMM below the critical KCl concentration, and approximately 5,000 microM above the critical KCl concentration. This increase in KATPase is due to a drastic reduction in the binding affinity of myosin heads to F-actin, as determined by a proteolytic digestion method and direct observation by fluorescence microscopy. We also show that the Vmax of actin-activated myosin ATPase activity decreases steadily with increasing KCl concentration, even though the velocity of F-actin sliding remains unchanged. This result provides evidence that the ATPase activity is not necessarily linked to motility. We discuss possible models that do not require a tight coupling between myosin ATPase and motility.     

19F NMR study of the myosin and tropomyosin binding sites on actin

Actin was labeled with pentafluorophenyl isothiocyanate at Lys-61. The label was sufficiently small not to affect the rate or extent of actin polymerization unlike the much larger fluorescein 5-isothiocyanate which completely inhibits actin polymerization [Burtnick, L. D. (1984) Biochim. Biophys. Acta 791, 57-62]. Furthermore, the label resonances in the 376.3-MHz 19F NMR spectrum were unaffected by actin polymerization. However, the binding of the relaxing protein tropomyosin resulted in the fluorinated Lys-61 resonances broadening out beyond detection due to a substantial increase in the effective correlation time of the label. Similarly, the binding of myosin subfragment 1 to F-actin resulted in the dramatic broadening of the labeled Lys-61 resonances. Thus, Lys-61 on actin appears to be closely associated with the binding sites for both tropomyosin and myosin, suggesting that both these proteins can compete for the same site on actin. The other region of actin known to be involved in myosin binding, Cys-10, was found to be more remote from the actin-actin interfaces than Lys-61. Labels on Cys-10 exhibited substantially greater mobility than fluorescein 5-isothiocyanate attached to Lys-61 which appeared to be held down on the surface of the actin monomer. This may sterically hinder the actin-actin interaction about 1 nm from the tropomyosin/myosin binding site.     

Expression of a nonpolymerizable actin mutant in Sf9 cells

We have succeeded in expressing actin in the baculovirus/Sf9 cell system in high yield. The wild-type (WT) actin is functionally indistinguishable from tissue-purified actin in its ability to activate ATPase activity and to support movement in an in vitro motility assay. Having achieved this feat, we used a mutational strategy to express a monomeric actin that is incapable of polymerization. Native actin requires actin binding proteins or chemical modification to maintain it in a monomeric state. The mutant actin sediments in the analytical ultracentrifuge as a homogeneous monomeric species of 3.2 S in 100 mM KCl and 2 mM MgCl(2), conditions that cause WT actin to polymerize. The two point mutations that render actin nonpolymerizable are in subdomain 4 (A204E/P243K; "AP-actin"), distant from the myosin binding site. AP-actin binds to skeletal myosin subfragment 1 (S1) and forms a homogeneous complex as demonstrated by analytical ultracentrifugation. The ATPase activity of a cross-linked AP-actin.S1 complex is higher than that of S1 alone, although less than that supported by filamentous actin (F-actin). AP-Actin is an excellent candidate for structural studies of complexes of actin with motor proteins and other actin-binding proteins.     

The bound nucleotide of actin

The extent of actin polymerization has been studied for samples in which the bound nucleotide of the actin was ATP, ADP, or an analog of ATP that was not split (AMPPNP). The equilibrium constants for the addition of a monomer to a polymer end were determined from the concentration of monomer coexisting with the polymer. An analysis of these results concludes that the bound ATP on G-actin provides little energy to promote the polymerization of the actin. AMPPNP was incorporated into F-actin and the interaction of F-actin · AMPPNP with myosin was studied. F-actin · AMPPNP activated the ATPase of myosin to the same extent as did F-actin · ADP. However, the rate of superprecipitation was slower in the case of F-actin · AMPPNP than in the control.     

Modeling cell protrusion predicts how myosin II and actin turnover affect adhesion-based signaling

Orchestration of cell migration is essential for development, tissue regeneration, and the immune response. This dynamic process integrates adhesion, signaling, and cytoskeletal subprocesses across spatial and temporal scales. In mesenchymal cells, adhesion complexes bound to extracellular matrix mediate both biochemical signal transduction and physical interaction with the F-actin cytoskeleton. Here, we present a mathematical model that offers insight into both aspects, considering spatiotemporal dynamics of nascent adhesions, active signaling molecules, mechanical clutching, actin treadmilling, and nonmuscle myosin II contractility. At the core of the model is a positive feedback loop, whereby adhesion-based signaling promotes generation of barbed ends at, and protrusion of, the cell’s leading edge, which in turn promotes formation and stabilization of nascent adhesions. The model predicts a switch-like transition and optimality of membrane protrusion, determined by the balance of actin polymerization and retrograde flow, with respect to extracellular matrix density. The model, together with new experimental measurements, explains how protrusion can be modulated by mechanical effects (nonmuscle myosin II contractility and adhesive bond stiffness) and F-actin turnover.     

G146V mutation at the hinge region of actin reveals a myosin class-specific requirement of actin conformations for motility

The G146V mutation in actin is dominant lethal in yeast. G146V actin filaments bind cofilin only minimally, presumably because cofilin binding requires the large and small actin domains to twist with respect to one another around the hinge region containing Gly-146, and the mutation inhibits that twisting motion. A number of studies have suggested that force generation by myosin also requires actin filaments to undergo conformational changes. This prompted us to examine the effects of the G146V mutation on myosin motility. When compared with wild-type actin filaments, G146V filaments showed a 78% slower gliding velocity and a 70% smaller stall force on surfaces coated with skeletal heavy meromyosin. In contrast, the G146V mutation had no effect on either gliding velocity or stall force on myosin V surfaces. Kinetic analyses of actin-myosin binding and ATPase activity indicated that the weaker affinity of actin filaments for myosin heads carrying ADP, as well as reduced actin-activated ATPase activity, are the cause of the diminished motility seen with skeletal myosin. Interestingly, the G146V mutation disrupted cooperative binding of myosin II heads to actin filaments. These data suggest that myosin-induced conformational changes in the actin filaments, presumably around the hinge region, are involved in mediating the motility of skeletal myosin but not myosin V and that the specific structural requirements for the actin subunits, and thus the mechanism of motility, differ among myosin classes.     

In vitro movement of actin filaments on gizzard smooth muscle myosin: requirement of phosphorylation of myosin light chain and effects of tropomyosin and caldesmon

ATP-dependent movement of actin filaments on smooth muscle myosin was investigated by using the in vitro motility assay method in which myosin was fixed on the surface of a coverslip in a phosphorylated or an unphosphorylated state. Actin filaments slid on gizzard myosin phosphorylated with myosin light chain kinase (MLCK) at a rate of 0.35 micron/s, but did not slide at all on unphosphorylated myosin. The movement of actin filaments on phosphorylated myosin was stopped by perfusion of phosphatase. Subsequent perfusion with a solution containing MLCK, calmodulin, and Ca2+ enabled actin filaments to move again. The sliding velocities on monophosphorylated and diphosphorylated myosin by MLCK were not different. Actin filaments did not move on myosin phosphorylated with protein kinase C (PKC). The sliding velocity on myosin phosphorylated with both MLCK and PKC was identical to that on myosin phosphorylated only with MLCK. Gizzard tropomyosin enhanced the sliding velocity to 0.76 micron/s. Gizzard caldesmon decreased the sliding velocity with increase in its concentration. At a 5-fold molar ratio of caldesmon to actin, the movement stopped completely. This inhibitory effect of caldesmon was relieved upon addition of excess calmodulin and Ca2+.     

Control of actin turnover by a salmonella invasion protein

Salmonella force their way into nonphagocytic host intestinal cells to initiate infection. Uptake is triggered by delivery into the target cell of bacterial effector proteins that stimulate cytoskeletal rearrangements and membrane ruffling. The Salmonella invasion protein A (SipA) effector is an actin binding protein that enhances uptake efficiency by promoting actin polymerization. SipA-bound actin filaments (F-actin) are also resistant to artificial disassembly in vitro. Using biochemical assays of actin dynamics and actin-based motility models, we demonstrate that SipA directly arrests cellular mechanisms of actin turnover. SipA inhibits ADF/cofilin-directed depolymerization both by preventing binding of ADF and cofilin and by displacing them from F-actin. SipA also protects F-actin from gelsolin-directed severing and reanneals gelsolin-severed F-actin fragments. These data suggest that SipA focuses host cytoskeletal reorganization by locally inhibiting both ADF/cofilin- and gelsolin-directed actin disassembly, while simultaneously stimulating pathogen-induced actin polymerization.     

Inhibition of the relative movement of actin and myosin by caldesmon and calponin.

Contractile activity of myosin II in smooth muscle and non-muscle cells requires phosphorylation of myosin by myosin light chain kinase. In addition, these cells have the potential for regulation at the thin filament level by caldesmon and calponin, both of which bind calmodulin. We have investigated this regulation using in vitro motility assays. Caldesmon completely inhibited the movement of actin filaments by either phosphorylated smooth muscle myosin or rabbit skeletal muscle heavy meromyosin. The amount of caldesmon required for inhibition was decreased when tropomyosin is present. Similarly, calponin binding to actin resulted in inhibition of actin filament movement by both smooth muscle myosin and skeletal muscle heavy meromyosin. Tropomyosin had no effect on the amount of calponin needed for inhibition. High concentrations of calmodulin (10 microM) in the presence of calcium completely reversed the inhibition. The nature of the inhibition by the two proteins was markedly different. Increasing caldesmon concentrations resulted in graded inhibition of the movement of actin filaments until complete inhibition of movement was obtained. Calponin inhibited actin sliding in a more "all or none" fashion. As the calponin concentration was increased the number of actin filaments moving was markedly decreased, but the velocity of movement remained near control values.