The actin filament severing protein actophorin promotes the formation of rigid bundles of actin filaments crosslinked with alpha-actinin

The actin filament severing protein, Acanthamoeba actophorin, decreases the viscosity of actin filaments, but increases the stiffness and viscosity of mixtures of actin filaments and the crosslinking protein alpha-actinin. The explanation of this paradox is that in the presence of both the severing protein and crosslinker the actin filaments aggregate into an interlocking meshwork of bundles large enough to be visualized by light microscopy. The size of these bundles depends on the size of the containing vessel. The actin filaments in these bundles are tightly packed in some areas while in others they are more disperse. The bundles form a continuous reticulum that fills the container, since the filaments from a particular bundle may interdigitate with filaments from other bundles at points where they intersect. The same phenomena are seen when rabbit muscle aldolase rather than alpha-actinin is used as the crosslinker. We propose that actophorin promotes bundling by shortening the actin filaments enough to allow them to rotate into positions favorable for lateral interactions with each other via alpha-actinin. The network of bundles is more rigid and less thixotropic than the corresponding network of single actin filaments linked by alpha-actinin. One explanation may be that alpha-actinin (or aldolase) normally in rapid equilibria with actin filaments may become trapped between the filaments increasing the effective concentration of the crosslinker.     

Actin-binding proteins regulate the work performed by myosin II motors on single actin filaments.

Regulation of actin/myosin II force generation by calcium [Kamm and Stull, Annu. Rev. Physiol. 51:299-313, 1989] and phosphorylation of myosin II light chains [Sellers and Adelstein, "The Enzymes," Vol. 18, Orlando, FL: Academic Pres, 1987, pp. 381-418] is well established. However, additional regulation of actin/myosin II force generation/contraction may result from actin-binding proteins [Stossel et al., Ann. Rev. Cell Biol. 1:353-402, 1985; Pollard and Cooper, Ann. Rev. Biochem. 55:987-1035, 1986] as they affect the gel state of the actin cytomatrix [reviewed in Taylor and Condeelis, Int. Rev. Cytol., 56:57-143, 1979]. Regulation of the gel state of actin may determine whether an isotonic or isometric contraction results from the interaction between myosin and actin. We have extended the single actin filament motility assay of Kron and Spudich [Proc. Natl. Acad. Sci. U.S.A. 83:6272-6276, 1986] by including filamin or alpha-actinin on the substrate with myosin II to examine how actin-crosslinking proteins regulate the movements of single actin filaments. Increasing amounts of actin-crosslinking proteins inhibit filament velocity and decrease the number of filaments moving. Reversal of crosslinking yields increased velocities and numbers of moving filaments. These results support the solation-contraction coupling hypothesis [see Taylor and Fechheimer, Phil. Trans. Soc. London B 299:185-197, 1982] which proposes that increased crosslinking of actin inhibits myosin-based contraction. This study also illustrates the potentially varied roles of different actin-crosslinking proteins and offers a novel method to examine actin-binding protein activity and their regulation of motility at the single molecule level.     

Interaction of microtubule-associated protein 2 with actin filaments

The interaction of unphosphorylated and phosphorylated microtubule-associated protein 2 (MAP-2) with actin filaments was examined by electron microscopic, electrophoretic, and dark-field light microscopic techniques. Unphosphorylated MAP-2 was observed to cross-link and bundle individual actin filaments. Chymotryptic fragments of MAP-2 protein were produced which bound to, but could not cross-link, actin polymer; these fragments encompassed the tubulin binding domain of MAP-2. The phosphorylation of intact MAP-2, by means of endogenous protein kinases, inhibited the ability of this molecule to cross-link and bundle actin filaments. Phosphorylation did not, however, inhibit the binding of MAP-2 to F-actin. The chymotryptic fragments of phosphorylated MAP-2 that retained their ability to bind to actin and promote microtubule assembly also encompassed the tubulin binding domain of this molecule. An analysis of MAP-2 fragments by nonequilibrium pH gradient electrophoresis indicated that most of the polypeptide backbone is relatively acidic with the exception of the tubulin binding domain. This region was determined to be the most basic (positively charged) region of the MAP-2 molecule. Biochemical and morphological evidence is presented to demonstrate that both unphosphorylated MAP-2 and phosphorylated MAP-2 have the capacity to use actin, in addition to microtubules, as a separate anchoring substrate. The presence of tubulin, however, strongly inhibits the interaction of MAP-2 with actin filaments.     

Three-dimensional structure of actin filaments and of an actin gel made with actin-binding protein

Purified muscle actin and mixtures of actin and actin-binding protein were examined in the transmission electron microscope after fixation, critical point drying, and rotary shadowing. The three-dimensional structure of the protein assemblies was analyzed by a computer-assisted graphic analysis applicable to generalized filament networks. This analysis yielded information concerning the frequency of filament intersections, the filament length between these intersections, the angle at which filaments branch at these intersections, and the concentration of filaments within a defined volume. Purified actin at a concentration of 1 mg/ml assembled into a uniform mass of long filaments which overlap at random angles between 0 degrees and 90 degrees. Actin in the presence of macrophage actin-binding protein assembled into short, straight filaments, organized in a perpendicular branching network. The distance between branch points was inversely related to the molar ratio of actin-binding protein to actin. This distance was what would be predicted if actin filaments grew at right angles off of nucleation sites on the two ends of actin-binding protein dimers, and then annealed. The results suggest that actin in combination with actin-binding protein self-assembles to form a three- dimensional network resembling the peripheral cytoskeleton of motile cells.     

Calcium-dependent interaction of actin filaments with actin binding protein in the presence of calmodulin and caldesmon

Caldesmon, calmodulin-, and actin-binding protein of chicken gizzard did not affect the process of polymerization of actin induced by 0.1 M KCl. Caldesmon binds to F-actin, thus inhibiting the gelation action of actin binding protein (ABP; filamin). Low shear viscosity and flow birefringence measurements revealed that in a system of calmodulin, caldesmon, ABP, and F-actin, gelation occurs in the presence of micromolar Ca2+ concentrations, but not in the absence of Ca2+. Electron microscopic observations showed the Ca2+-dependent formation of actin bundles in this system. These results were interpreted by the flip-flop mechanism: in the presence of Ca2+, a calmodulin-caldesmon complex is released from actin filaments on which ABP exerts its gelating action. On the other hand, in the absence of Ca2+, caldesmon remains bound to actin filaments, thus preventing the action of ABP.     

Interaction of lipid-bound myelin basic protein with actin filaments and calmodulin

Myelin basic protein (MBP) binds to negatively charged lipids on the cytosolic surface of oligodendrocytes (OLs) and is believed to be responsible for adhesion of these surfaces in the multilayered myelin sheath. MBP in solution has been shown by others to bind to both G- and F-actin, to bundle F-actin filaments, and to induce polymerization of G-actin. Here we show that MBP bound to acidic lipids can also bind to both G- and F-actin and cause their sedimentation together with MBP-lipid vesicles. Thus it can simultaneously utilize some of its basic residues to bind to the lipid bilayer and some to bind to actin. The amount of actin bound to the MBP-lipid vesicles decreased with increasing net negative surface charge of the lipid vesicles. It was also less for vesicles containing the lipid composition predicted for the cytosolic surface of myelin than for PC vesicles containing a similar amount of an acidic lipid. Calmodulin caused dissociation of actin from MBP and of the MBP-actin complex from the vesicles. However, it did not cause dissociation of bundles of actin filaments once these had formed as long as some MBP was still present. These results suggest that MBP could be a membrane actin-binding protein in OLs/myelin and its actin binding can be regulated by calmodulin and by the lipid composition of the membrane. Actin binding to MBP decreased the labeling of MBP by the hydrophobic photolabel 3-(trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine (TID), indicating that it decreased the hydrophobic interactions of MBP with the bilayer. This change in interaction of MBP with the bilayer could then create a cytosol to membrane signal caused by changes in interaction of the cytoskeleton with the membrane.     

Thermal activation energy for bidirectional movement of actin along bipolar tracks of myosin filaments

Previous in vitro motility assays using bipolar myosin thick filaments demonstrated that actin filaments were capable of moving in both directions along the myosin filament tracks. The movements; however, were slower in the direction leading away from the central bare zone than towards it. To understand the mechanism underlying these different direction-dependent motilities, we have examined the effects of temperature on the velocities of the bidirectional movements along reconstituted myosin filaments. Activation energies of the movements were determined by Arrhenius plots at high and low concentrations of ATP. As a result, the thermal activation energy of the movement away from the central bare zone was significantly higher than that of the movement toward the zone. Given that the backward movement away from the central bare zone would cause the myosin heads to be constrained and the stiffness of the cross-bridges to increase, these results suggest that elastic energy required for the cross-bridge transition is supplied by thermal fluctuations.     

ADP-ribosylated actin caps the barbed ends of actin filaments

The mode of action on actin polymerization of skeletal muscle actin ADP-ribosylated on arginine 177 by perfringens iota toxin was investigated. ADP-ribosylated actin decreased the rate of nucleated actin polymerization at substoichiometric ratios of ADP-ribosylated actin to monomeric actin. ADP-ribosylated actin did not tend to copolymerize with actin. Actin filaments were depolymerized by the addition of ADP-ribosylated actin. The maximal monomer concentration reached by addition of ADP-ribosylated actin was similar to the critical concentration of the pointed ends of actin filaments. ADP-ribosylated actin had no effect on the rate of polymerization of gelsolin-capped actin filaments which polymerize at the pointed ends. The results suggest that ADP-ribosylated actin acts as a capping protein which binds to the barbed ends of actin filaments to inhibit polymerization. Based on an analysis of the depolymerizing effect of ADP-ribosylated actin, the equilibrium constant for binding of ADP-ribosylated actin to the barbed ends of actin filaments was determined to be about 10(8) M-1. As actin is ADP-ribosylated by perfringens iota toxin and by botulinum C2 toxin, it appears that conversion of actin into a capping protein by ADP-ribosylation is a pathophysiological reaction catalyzed by bacterial toxins which ultimately leads to inhibition of actin assembly.     

Formation and destabilization of actin filaments with tetramethylrhodamine-modified actin

Actin labeling at Cys(374) with tethramethylrhodamine derivatives (TMR-actin) has been widely used for direct observation of the in vitro filaments growth, branching, and treadmilling, as well as for the in vivo visualization of actin cytoskeleton. The advantage of TMR-actin is that it does not lock actin in filaments (as rhodamine-phalloidin does), possibly allowing for its use in investigating the dynamic assembly behavior of actin polymers. Although it is established that TMR-actin alone is polymerization incompetent, the impact of its copolymerization with unlabeled actin on filament structure and dynamics has not been tested yet. In this study, we show that TMR-actin perturbs the filaments structure when copolymerized with unlabeled actin; the resulting filaments are more fragile and shorter than the control filaments. Due to the increased severing of copolymer filaments, TMR-actin accelerates the polymerization of unlabeled actin in solution also at mole ratios lower than those used in most fluorescence microscopy experiments. The destabilizing and severing effect of TMR-actin is countered by filament stabilizing factors, phalloidin, S1, and tropomyosin. These results point to an analogy between the effects of TMR-actin and severing proteins on F-actin, and imply that TMR-actin may be inappropriate for investigations of actin filaments dynamics.     

Calmodulin-regulated binding of the 90-kDa heat shock protein to actin filaments

We have found that the 90-kDa heat shock protein (HSP90) prepared from a mouse lymphoma exists in homodimeric form under physiological conditions and has the ability to bind to F-actin (Koyasu, S., Nishida, E., Kadowaki, T., Matsuzaki, F., Iida, K., Harada, F., Kasuga, M., Sakai, H., and Yahara, I. (1986) Proc. Natl. Acad. Sci. U.S.A., in press). Here we show that calmodulin regulates the binding of HSP90 to F-actin in a Ca2+-dependent manner. The binding of HSP90 to F-actin occurred optimally under physiological solution conditions, i.e. in 2 mM MgCl2 + 100 mM KCl. The binding was saturable in a molar ratio of about 1 HSP90 (dimer) to 10 actins. HSP90 was dissociated from F-actin by the binding of tropomyosin to F-actin. Calmodulin was found to inhibit the binding of HSP90 to F-actin in a Ca2+-dependent manner. Moreover, the equilibrium gel filtration demonstrated that calmodulin binds to HSP90 in the presence of Ca2+, but not in the absence of Ca2+. These data indicate that HSP90 complexed with Ca2+-calmodulin is unable to bind to F-actin. Ca2+-dependent interaction of HSP90 with calmodulin as well as calmodulin-regulated binding of HSP90 to F-actin revealed here may provide new insight into the function of HSP90 and the regulation of actin structure in cells.     

Actin-binding protein G (AbpG) participates in modulating the actin cytoskeleton and cell migration in Dictyostelium discoideum

Cell migration is involved in various physiological and pathogenic events, and the complex underlying molecular mechanisms have not been fully elucidated. The simple eukaryote Dictyostelium discoideum displays chemotactic locomotion in stages of its life cycle. By characterizing a Dictyostelium mutant defective in chemotactic responses, we identified a novel actin-binding protein serving to modulate cell migration and named it actin-binding protein G (AbpG); this 971–amino acid (aa) protein contains an N-terminal type 2 calponin homology (CH2) domain followed by two large coiled-coil regions. In chemoattractant gradients, abpG⁻ cells display normal directional persistence but migrate significantly more slowly than wild-type cells; expressing Flag-AbpG in mutant cells eliminates the motility defect. AbpG is enriched in cortical/lamellipodial regions and colocalizes well with F-actin; aa 401–600 and aa 501–550 fragments of AbpG show the same distribution as full-length AbpG. The aa 501–550 region of AbpG, which is essential for AbpG to localize to lamellipodia and to rescue the phenotype of abpG⁻ cells, is sufficient for binding to F-actin and represents a novel actin-binding protein domain. Compared with wild-type cells, abpG⁻ cells have significantly higher F-actin levels. Collectively our results suggest that AbpG may participate in modulating actin dynamics to optimize cell locomotion.     

Comparison of filamin A-induced cross-linking and Arp2/3 complex-mediated branching on the mechanics of actin filaments.

We compared the effects of human filamin A (FLNa) and the activated human Arp2/3 complex on mechanical properties of actin filaments. As little as 1 FLNa to 800 polymerizing actin monomers induces a sharp concentration-dependent increase in the apparent viscosity of 24 microm actin, a parameter classically defined as a gel point. The activated Arp2/3 complex, at concentrations up to 1:25 actins had no detectable actin gelation activity, even in the presence of phalloidin, to stabilize actin filaments against debranching. Increasing the activated Arp2/3 complex to actin ratio raises the FLNa concentration required to induce actin gelation, an effect ascribable to Arp2/3-mediated actin nucleation resulting in actin filament length diminution. Time lapse video microscopy of microparticles attached to actin filaments or photoactivation of fluorescence revealed actin filament immobilization by FLNa in contrast to diffusion of Arp2/3-branched actin filaments. The experimental results support theories predicting that polymer branching absent cross-linking does not lead to polymer gelation and are consistent with the observation that cells deficient in actin filament cross-linking activity have unstable surfaces. They suggest complementary roles for actin branching and cross-linking in cellular actin mechanics in vivo.     

Microtubule-associated protein 4 binds to actin filaments and modulates their properties

We previously reported that an isoform of microtubule-associated protein 4 (MAP4) is localized to the distal area of developing neurites, where microtubules are relatively scarce, raising the possibility that MAP4 interacts with another major cytoskeletal component, actin filaments. In the present study, we examined the in vitro interaction between MAP4 and actin filaments, using bacterially expressed MAP4 and its truncated fragments. Sedimentation assays revealed that MAP4 and its microtubule-binding domain fragments bind to actin filaments under physiological conditions. The apparent dissociation constant and the binding stoichiometry of the fragments to actin were about 0.1 μm and 1 : 3 (MAP4/actin), respectively. Molecular dissection studies revealed that the actin-binding site on MAP4 is situated at the C-terminal part of the proline-rich region, where the microtubule-binding site is also located. Electron microscopy revealed that the MAP4-bound actin filaments become straighter and longer and that the number of actin bundles increases with greater concentrations of added MAP4 fragment, indicating that MAP4 binding alters the properties of the actin filaments. A multiple sequence alignment of the proline-rich regions of MAP4 and tau revealed two putative actin-binding consensus sequences.     

Equilibrium constant for binding of an actin filament capping protein to the barbed end of actin filaments

Depolymerization of treadmilling actin filaments by a capping protein isolated from bovine brain was used for determination of the equilibrium constant for binding of the capping protein to the barbed ends of actin filaments. When the capping protein blocks monomer consumption at the lengthening barbed ends, monomers continue to be produced at the shortening pointed ends until a new steady state is reached in which monomer production at the pointed ends is balanced by monomer consumption at the uncapped barbed ends. In this way the ratio of capped to uncapped filaments could be determined as a function of the capping protein concentration. Under the experimental conditions (100 mM KCl and 2 mM MgCl2, pH 7.5, 37 degrees C) the binding constant was found to be about 2 X 10(9) M-1. Capping proteins effect the actin monomer concentration only at capping protein concentrations far above the reciprocal of their binding constant. Half-maximal increase of the monomer concentration requires capping of about 99% of the actin filaments. A low proportion of uncapped filaments has a great weight in determining the monomer concentration because association and dissociation reactions occur at the dynamic barbed ends with higher frequencies than at the pointed ends.     

The plant-specific actin binding protein SCAB1 stabilizes actin filaments and regulates stomatal movement in Arabidopsis

Microfilament dynamics play a critical role in regulating stomatal movement; however, the molecular mechanism underlying this process is not well understood. We report here the identification and characterization of STOMATAL CLOSURE-RELATED ACTIN BINDING PROTEIN1 (SCAB1), an Arabidopsis thaliana actin binding protein. Plants lacking SCAB1 were hypersensitive to drought stress and exhibited reduced abscisic acid-, H(2)O(2)-, and CaCl(2)-regulated stomatal movement. In vitro and in vivo analyses revealed that SCAB1 binds, stabilizes, and bundles actin filaments. SCAB1 shares sequence similarity only with plant proteins and contains a previously undiscovered actin binding domain. During stomatal closure, actin filaments switched from a radial orientation in open stomata to a longitudinal orientation in closed stomata. This switch took longer in scab1 plants than in wild-type plants and was correlated with the delay in stomatal closure seen in scab1 mutants in response to drought stress. Our results suggest that SCAB1 is required for the precise regulation of actin filament reorganization during stomatal closure.     

Formin-induced nucleation of actin filaments

Formins are proteins best defined by the presence of the unique, highly conserved formin homology domain 2 (FH2). FH2 is necessary and sufficient to nucleate an actin filament in vitro. The FH2 domain also binds to the filament's barbed end, modulating its elongation and protecting it from capping proteins. FH2 itself appears to be a processive cap that walks with the barbed end as it elongates.     

Overexpression of a NAC-domain protein promotes shoot branching in rice

For a better understanding of shoot branching in rice (Oryza sativa), a rice activation-tagging library was screened for mutations in tiller development. Here, an activation-tagging mutant Ostil1 (Oryza sativa tillering1) was characterized, which showed increased tillers, enlarged tiller angle and semidwarf phenotype. Flanking sequence was obtained by plasmid rescue. RNA-interfering and overexpression transgenic rice plants were produced using Agrobacterium-mediated transformation. The mutant phenotype was cosegregated with the reallocation of Ds element, and the flanking region of the reallocated Ds element was identified as part of the OsNAC2 gene. Northern analysis showed that expression of OsNAC2 was greatly induced in the mutant plants. Transgenic rice overexpressing the OsNAC2 resulted in recapture of the mutant phenotype, while downregulation of OsNAC2 in the Ostil1 mutant through RNA interfering (RNAi) complemented the mutant phenotype, confirming that the Ostil1 was caused by overexpression of OsNAC2. Overexpression of OsNAC2 regulates shoot branching in rice. Overexpression of OsNAC2 contributes tiller bud outgrowth, but does not affect tiller bud initiation. This suggests that OsNAC2 has potential utility for improving plant structure for higher light-use efficiency and higher yield potential in rice.     

Kinetics of the cooperative association of actin to actin filaments.

The cooperative formation of actin filaments from monomers was followed by light scattering and electron microscopy. The results are well described by a simple model mechanism in which the growth and destruction of filaments occurs by stepwise addition or dissociation of protomers. All steps except the dimerisation step are assumed to have identical rate constants. These were found to be 5 X 10(3) M-1 - sec-1 and 3 X 10(-2) sec-1 for the association and dissociation, respectively (at pH 7.5 and in the presence of 10(-3) M calcium chloride). The equilibrium constant of elongation as obtained from the critical concentration is 1.7 X 10(5) M-1. The corresponding equilibrium constant of dimerisation is about 10 million times smaller (cooperativity parameter sigma = 2 X 10(-7)). This makes the nucleation extremely difficult and cooperativity very high. A best fit of the model to the experimental data is achieved when the destruction of a dimer is much faster than the addition of a third protomer (fast monomer- dimer pre-equilibrium). The size of the nucleus from which propagation becomes faster than dissociation is 3.     

Regulation of the interaction of actin filaments with microtubule-associated protein 2 by calmodulin-dependent protein kinase II

Phosphorylation of microtubule-associated protein 2 (MAP 2) by Ca²⁺-, calmodulin-dependent protein kinase II (protein kinase II) inhibited the actin filament cross-linking activity of MAP 2. This inhibition required the presence of ATP, Mg²⁺, Ca²⁺ and calmodulin. The minimal concentration of MAP 2 required for gel formation of actin filaments was increased with increasing amounts of phosphate incorporated into MAP 2, and the phosphorylated MAP 2, into which 10.3 mol of phosphate/mol of protein had been incorporated, did not cause actin filaments to gel under the experimental conditions used. The phosphorylation of MAP 2 by Ca²⁺-, phospholipid-dependent protein kinase (protein kinase C) and cAMP-dependent protein kinase also inhibited the actin filament cross-linking activity of MAP 2. The extent and rate of phosphorylation of MAP 2 by protein kinase II were higher than those of the phosphorylation by protein kinase C and cAMP-dependent protein kinase. The interaction of actin filaments with MAP 2 was inhibited more by the actions of protein kinase II and protein kinase C than by cAMP-dependent protein kinase. The actin filament cross-linking activity of MAP 2 phosphorylated either by protein kinase II, cAMP-dependent protein kinase or protein kinase C was retrieved when phosphorylated MAP 2 was treated by protein phosphatase. These results indicate that the interaction of actin filaments with MAP 2 is regulated by the phosphorylation-dephosphorylation of MAP 2.