Tropomyosin directly modulates actomyosin mechanical performance at the level of a single actin filament

Muscle contraction is the result of myosin cross-bridges (XBs) cyclically interacting with the actin-containing thin filament. This interaction is modulated by the thin filament regulatory proteins, troponin and tropomyosin (Tm). With the use of an in vitro motility assay, the role of Tm in myosin’s ability to generate force and motion was assessed. At saturating myosin surface densities, Tm had no effect on thin filament velocity. However, below 50% myosin saturation, a significant reduction in actin–Tm filament velocity was observed, with complete inhibition of movement occurring at 12.5% of saturating surface densities. Under similar conditions, actin filaments alone demonstrated no reduction in velocity. The effect of Tm on force generation was assessed at the level of a single thin filament. In the absence of Tm, isometric force was a linear function of the density of myosin on the motility surface. At 50% myosin surface saturation, the presence of Tm resulted in a 2-fold enhancement of force relative to actin alone. However, no further potentiation of force was observed with Tm at saturating myosin surface densities. These results indicate that, in the presence of Tm, the strong binding of myosin cooperatively activates the thin filament. The inhibition of velocity at low myosin densities and the potentiation of force at higher myosin densities suggest that Tm can directly modulate the kinetics of a single myosin XB and the recruitment of a population of XBs, respectively. At saturating myosin conditions, Tm does not appear to affect the recruitment or the kinetics of myosin XBs.

Both N-terminal myosin-binding and C-terminal actin-binding sites on smooth muscle caldesmon are required for caldesmon-mediated inhibition of actin filament velocity

It has been suggested that the tethering caused by binding of the N-terminal region of smooth muscle caldesmon (CaD) to myosin and its C-terminal region to actin contributes to the inhibition of actin-filament movement over myosin heads in an in vitro motility assay. However, direct evidence for this assumption has been lacking. In this study, analysis of baculovirus-generated N-terminal and C-terminal deletion mutants of chicken-gizzard CaD revealed that the major myosin-binding site on the CaD molecule resides in a 30-amino acid stretch between residues 24 and 53, based on the very low level of binding of CaDΔ24–53 lacking the residues 24–53 to myosin compared with the level of binding of CaDΔ54–85 missing the adjacent residues 54–85 or of the full-length CaD. As expected, deletion of the region between residues 24 and 53 or between residues 54 and 85 had no effect on either actin-binding or inhibition of actomyosin ATPase activity. Deletion of residues 24–53 nearly abolished the ability of CaD to inhibit actin filament velocity in the in vitro motility experiments, whereas CaDΔ54–85 strongly inhibited actin filament velocity in a manner similar to that of full-length CaD. Moreover, CaD1–597, which lacks the major actin-binding site(s), did not inhibit actin-filament velocity despite the presence of the major myosin-binding site. These data provide direct evidence for the inhibition of actin filament velocity in the in vitro motility assay caused by the tethering of myosin to actin through binding of both the CaD N-terminal region to myosin and the C-terminal region to actin.

Particles move along actin filament bundles in nerve growth cones.

Organelle movement along actin filaments has been demonstrated in dissociated squid axoplasm [Kurznetsov, S. A., Langford, G.M. & Weiss, D. G. (1992) Nature (London) 356, 722-725 and Bearer, E.L., DeGiorgis, J.A., Bodner, R.A., Kao, A.W. & Reese, T.S. (1993) Proc. Natl. Acad. Sci. USA 90, 11252-11256] but has not been shown to occur in intact neurons. Here we demonstrate that intracellular transport occurs along actin filament bundles in intact neuronal growth cones. We used video-enhanced differential interference contrast microscopy to observe intracellular transport in superior cervical ganglion neurons cultured under conditions that enhance the visibility of actin bundles within growth cone lamellipodia. Intracellular particles, ranging in size from < 0.5-1.5 microns, moved along linear structures (termed transport bundles) at an average maximum rate of 0.48 micron/sec. After particle movement had been viewed, cultures were preserved by rapid perfusion with chemical fixative. To determine whether particle transport occurred along actin, we then used fluorescence microscopy to correlate this movement with actin and microtubule distributions in the same growth cones. The observed transport bundles colocalized with actin but not with microtubules. The rates of particle movement and the association of moving particles with actin filament bundles suggest that myosins may participate in the transport of organelles (or other materials) in intact neurons.

Torsional rigidity of single actin filaments and actin–actin bond breaking force under torsion measured directly by in vitro micromanipulation

Knowledge of the elastic properties of actin filaments is crucial for considering its role in muscle contraction, cellular motile events, and formation of cell shape. The stiffness of actin filaments in the directions of stretching and bending has been determined. In this study, we have directly determined the torsional rigidity and breaking force of single actin filaments by measuring the rotational Brownian motion and tensile strength using optical tweezers and microneedles, respectively. Rotational angular fluctuations of filaments supplied the torsional rigidity as (8.0 ± 1.2) × 10−26 Nm2. This value is similar to that deduced from the longitudinal rigidity, assuming the actin filament to be a homogeneous rod. The breaking force of the actin–actin bond was measured while twisting a filament through various angles using microneedles. The breaking force decreased greatly under twist, e.g., from 600–320 pN when filaments were turned through 90°, independent of the rotational direction. Our results indicate that an actin filament exhibits comparable flexibility in the rotational and longitudinal directions, but breaks more easily under torsional load.

Vaccinia locomotion in host cells: Evidence for the universal involvement of actin-based motility sequences ABM-1 and ABM-2

Vaccinia uses actin-based motility for virion movement in host cells, but the specific protein components have yet to be defined. A cardinal feature of Listeria and Shigella actin-based motility is the involvement of vasodilator-stimulated phosphoprotein (VASP). This essential adapter recognizes and binds to actin-based motility 1 (ABM-1) consensus sequences [(D/E)FPPPPX(D/E), X = P or T] contained in Listeria ActA and in the p90 host-cell vinculin fragment generated by Shigella infection. VASP, in turn, provides the ABM-2 sequences [XPPPPP, X = G, P, L, S, A] for binding profilin, an actin-regulatory protein that stimulates actin filament assembly. Immunolocalization using rabbit anti-VASP antibody revealed that VASP concentrates behind motile virions in HeLa cells. Profilin was also present in these actin-rich rocket tails, and microinjection of 10 μM (intracellular) ABM-2 peptide (GPPPPP)3 blocked vaccinia actin-based motility. Vinculin did not colocalize with VASP on motile virions and remained in focal adhesion contacts; however, another ABM-1-containing host protein, zyxin, was concentrated at the rear of motile virions. We also examined time-dependent changes in the location of these cytoskeletal proteins during vaccinia infection. VASP and zyxin were redistributed dramatically several hours before the formation of actin rocket tails, concentrating in the viral factories of the perinuclear cytoplasm. Our findings underscore the universal involvement of ABM-1 and ABM-2 docking sites in actin-based motility of Listeria, Shigella, and now vaccinia.

Arp2/3 Complex from Acanthamoeba Binds Profilin and Cross-links Actin Filaments

The Arp2/3 complex was first purified from Acanthamoeba castellanii by profilin affinity chromatography. The mechanism of interaction with profilin was unknown but was hypothesized to be mediated by either Arp2 or Arp3. Here we show that the Arp2 subunit of the complex can be chemically cross-linked to the actin-binding site of profilin. By analytical ultracentrifugation, rhodamine-labeled profilin binds Arp2/3 complex with a Kd of 7 μM, an affinity intermediate between the low affinity of profilin for barbed ends of actin filaments and its high affinity for actin monomers. These data suggest the barbed end of Arp2 is exposed, but Arp2 and Arp3 are not packed together in the complex exactly like two actin monomers in a filament. Arp2/3 complex also cross-links actin filaments into small bundles and isotropic networks, which are mechanically stiffer than solutions of actin filaments alone. Arp2/3 complex is concentrated at the leading edge of motile Acanthamoeba, and its localization is distinct from that of α-actinin, another filament cross-linking protein. Based on localization and actin filament nucleation and cross-linking activities, we propose a role for Arp2/3 in determining the structure of the actin filament network at the leading edge of motile cells.

The interaction of Arp2/3 complex with actin: Nucleation, high affinity pointed end capping, and formation of branching networks of filaments

The Arp2/3 complex is a stable assembly of seven protein subunits including two actin-related proteins (Arp2 and Arp3) and five novel proteins. Previous work showed that this complex binds to the sides of actin filaments and is concentrated at the leading edges of motile cells. Here, we show that Arp2/3 complex purified from Acanthamoeba caps the pointed ends of actin filaments with high affinity. Arp2/3 complex inhibits both monomer addition and dissociation at the pointed ends of actin filaments with apparent nanomolar affinity and increases the critical concentration for polymerization at the pointed end from 0.6 to 1.0 μM. The high affinity of Arp2/3 complex for pointed ends and its abundance in amoebae suggest that in vivo all actin filament pointed ends are capped by Arp2/3 complex. Arp2/3 complex also nucleates formation of actin filaments that elongate only from their barbed ends. From kinetic analysis, the nucleation mechanism appears to involve stabilization of polymerization intermediates (probably actin dimers). In electron micrographs of quick-frozen, deep-etched samples, we see Arp2/3 bound to sides and pointed ends of actin filaments and examples of Arp2/3 complex attaching pointed ends of filaments to sides of other filaments. In these cases, the angle of attachment is a remarkably constant 70 ± 7°. From these in vitro biochemical properties, we propose a model for how Arp2/3 complex controls the assembly of a branching network of actin filaments at the leading edge of motile cells.

Yeast actin: polymerization kinetic studies of wild type and a poorly polymerizing mutant.

Wild-type actin and a mutant actin were isolated from yeast (Saccharomyces cerevisiae) and the polymerization properties were examined at pH 8.0 and 20 degrees C. The polymerization reaction was followed either by an increase in pyrene-labeled actin fluorescence or by a decrease in intrinsic fluorescence in the absence of pyrene-labeled actin. While similar to the properties of skeletal muscle actin, there are several important differences between the wild-type yeast and muscle actins. First, yeast actin polymerizes more rapidly than muscle actin under the same experimental conditions. The difference in rates may result from a difference in the steps involving formation of the nucleating species. Second, as measured with pyrene-labeled yeast actin, but not with intrinsic fluorescence, there is an overshoot in the fluorescence that has not been observed with skeletal muscle actin under the same conditions. Third, in order to simulate the polymerization process of wild-type yeast actin it is necessary to assume some fragmentation of the filaments. Finally, gelsolin inhibits polymerization of yeast actin but is known to accelerate the polymerization of muscle actin. A mutant actin (R177A/D179A) has also been isolated and studied. The mutations are at a region of contact between monomers across the long axis of the actin filament. This mutant polymerizes more slowly than wild type and filaments do not appear to fragment during polymerization. Elongation rates of the wild type and the mutant differ by only about 3-fold, and the slower polymerization of the mutant appears to result primarily from poorer nucleation.

Rho protein regulates tight junctions and perijunctional actin organization in polarized epithelia.

The rho family of GTP-binding proteins regulates actin filament organization. In unpolarized mammalian cells, rho proteins regulate the assembly of actin-containing stress fibers at the cell-matrix interface. Polarized epithelial cells, in contrast, are tall and cylindrical with well developed intercellular tight junctions that permit them to behave as biologic barriers. We report that rho regulates filamentous actin organization preferentially in the apical pole of polarized intestinal epithelial cells and, in so doing, influences the organization and permeability of the associated apical tight junctions. Thus, barrier function, which is an essential characteristic of columnar epithelia, is regulated by rho.

Shigella flexneri surface protein IcsA is sufficient to direct actin-based motility.

Shigella flexneri is a Gram-negative bacterial pathogen that can grow directly in the cytoplasm of infected host cells and uses a form of actin-based motility for intra- and intercellular spread. Moving intracellular bacteria are associated with a polarized "comet tail" composed of actin filaments. IcsA, a 120-kDa outer membrane protein necessary for actin-based motility, is located at a single pole on the surface of the organism, at the junction with the actin tail. Here, we demonstrate that stable expression of IcsA on the surface of Escherichia coli is sufficient to allow actin-dependent movement of E. coli in cytoplasmic extracts, at rates comparable to the movement of S. flexneri in infected cells. Thus, IcsA is the sole Shigella-specific factor required for actin-based motility. Continuous protein synthesis and polarized distribution of the protein are not necessary for actin tail formation or movement. Listeria monocytogenes is an unrelated bacterial pathogen that exhibits similar actin-based intracytoplasmic motility. Actin filament dynamics in the comet tails associated with the two different organisms are essentially identical, which indicates that they have independently evolved mechanisms to interact with the same components of the host cytoskeleton.

mRNA binding protein mrnp 41 localizes to both nucleus and cytoplasm

We have identified and molecularly characterized a human protein with a Mr of 40,880 Da. After UV irradiation of HeLa cells, this protein was cross-linked to poly(A)-containing mRNA and was therefore designated mrnp 41 (for mRNA binding protein of 41 kDa). Cell fractionation and immunoblotting showed mrnp 41 in both the cytoplasm and the nucleus and particularly in the nuclear envelope. Immunofluorescence microscopy localized mrnp 41 to distinct foci in the nucleoplasm, to the nuclear rim, and to meshwork-like structures throughout the cytoplasm. The cytoplasmic meshwork staining was disrupted by prior treatment of cells with the actin filament- or microtubule-disrupting drugs cytochalasin or nocodazole, respectively, suggesting association of mrnp 41 with the cytoskeleton. Double immunofluorescence with antibodies against mrnp 41 and the cytoplasmic poly(A) binding protein showed colocalization to the cytoplasmic meshwork. Immunogold electronmicroscopy confirmed mrnp 41’s cytoplasmic and nucleoplasmic localization and revealed a striking labeling of nuclear pore complexes. Together these data suggest that mrnp 41 may function in nuclear export of mRNPs and/or in cytoplasmic transport on, or attachment to, the cytoskeleton. Consistent with a role of mrnp 41 in nuclear export are previous reports that mutations in homologs of mrnp 41 in Schizosaccharomyces pombe, designated Rae1p, or in Saccharomyces cerevisiae, designated Gle2p, result in mRNA accumulation in the nucleus although it is presently not known whether these homologs are mRNA binding proteins as well.

Probes bound to myosin Cys-707 rotate during length transients in contraction

It is widely conjectured that muscle shortens because portions of myosin molecules (the “cross-bridges”) impel the actin filament to which they transiently attach and that the impulses result from rotation of the cross-bridges. Crystallography indicates that a cross-bridge is articulated–consisting of a globular catalytic/actin-binding domain and a long lever arm that may rotate. Conveniently, a rhodamine probe with detectable attitude can be attached between the globular domain and the lever arm, enabling the observer to tell whether the anchoring region rotates. Well-established signature effects observed in shortening are tension changes resulting from the sudden release or quick stretch of active muscle fibers. In this investigation we found that closely correlated with such tension changes are changes in the attitude of the rhodamine probes. This correlation strongly supports the conjecture about how shortening is achieved.

Redistribution of phosphatidylethanolamine at the cleavage furrow of dividing cells during cytokinesis

Ro09-0198 is a tetracyclic polypeptide of 19 amino acids that recognizes strictly the structure of phosphatidylethanolamine (PE) and forms a tight equimolar complex with PE on biological membranes. Using the cyclic peptide coupled with fluorescence-labeled streptavidin, we have analyzed the cell surface localization of PE in dividing Chinese hamster ovary cells. We found that PE was exposed on the cell surface specifically at the cleavage furrow during the late telophase of cytokinesis. PE was exposed on the cell surface only during the late telophase and no alteration in the distribution of the plasma membrane-bound cyclic peptide was observed during the cytokinesis, suggesting that the surface exposure of PE reflects the enhanced scrambling of PE at the cleavage furrow. Furthermore, cell surface immobilization of PE induced by adding the cyclic peptide coupled with streptavidin to prometaphase cells effectively blocked the cytokinesis at late telophase. The peptide-streptavidin complex treatment had no effect on furrowing, rearrangement of microtubules, and nuclear reconstitution, but specifically inhibited both actin filament disassembly at the cleavage furrow and subsequent membrane fusion. These results suggest that the redistribution of the plasma membrane phospholipids is a crucial step for cytokinesis and the cell surface PE may play a pivotal role in mediating a coordinate movement between the contractile ring and plasma membrane to achieve successful cell division.

Opposite Effects of Insulin on Focal Adhesion Proteins in 3T3-L1 Adipocytes and in Cells Overexpressing the Insulin Receptor

Insulin can regulate the abundance and organization of filamentous actin within cells in culture. Early studies using cell lines that overexpress the insulin receptor demonstrated that insulin caused a rapid reversible disassembly of actin filaments that coincided with the rapid tyrosine dephosphorylation of focal adhesion kinase. We have extended these studies by demonstrating that paxillin, another focal adhesion protein, and Src undergo tyrosine dephosphorylation in response to insulin in Chinese hamster ovary (CHO) and rat hepatoma (HTC) cells that overexpress the insulin receptor. This contrasted with the effect of insulin in parental CHO and HTC cells in which focal adhesion proteins were not dephosphorylated in response to the hormone. In addition, insulin caused a dispersion of focal adhesion proteins and disruption of actin filament bundles only in cells that overexpressed the insulin receptor. Moreover, in 3T3-L1 adipocytes, which are considered prototypic insulin-responsive cells, actin filament assembly was stimulated, and focal adhesion protein tyrosine phosphorylation was not altered. 3T3-L1 cells have more insulin receptors than either parental CHO or HTC cells but have fivefold less insulin receptors than the overexpressing cell lines. We hypothesize that a threshold may exist in which the overexpression of insulin receptors determines how insulin signaling pathways regulate the actin cytoskeleton.

The small GTPase RalA targets filamin to induce filopodia

The Ras-related small GTPases Rac, Rho, Cdc42, and RalA bind filamin, an actin filament-crosslinking protein that also links membrane and other intracellular proteins to actin. Of these GTPases only RalA binds filamin in a GTP-specific manner, and GTP-RalA elicits actin-rich filopods on surfaces of Swiss 3T3 cells and recruits filamin into the filopodial cytoskeleton. Either a dominant negative RalA construct or the RalA-binding domain of filamin 1 specifically block Cdc42-induced filopod formation, but a Cdc42 inhibitor does not impair RalA’s effects, which, unlike Cdc42, are Rac independent. RalA does not generate filopodia in filamin-deficient human melanoma cells, whereas transfection of filamin 1 restores the functional response. RalA therefore is a downstream intermediate in Cdc42-mediated filopod production and uses filamin in this pathway.