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.     

Plant actin-binding protein SCAB1 is dimeric actin cross-linker with atypical pleckstrin homology domain

SCAB1 is a novel plant-specific actin-binding protein that binds, bundles, and stabilizes actin filaments and regulates stomatal movement. Here, we dissected the structure and function of SCAB1 by structural and biochemical approaches. We show that SCAB1 is composed of an actin-binding domain, two coiled-coil (CC) domains, and a fused immunoglobulin and pleckstrin homology (Ig-PH) domain. We determined crystal structures for the CC1 and Ig-PH domains at 1.9 and 1.7 Å resolution, respectively. The CC1 domain adopts an antiparallel helical hairpin that further dimerizes into a four-helix bundle. The CC2 domain also mediates dimerization. At least one of the coiled coils is required for actin binding, indicating that SCAB1 is a bivalent actin cross-linker. The key residues required for actin binding were identified. The PH domain lacks a canonical basic phosphoinositide-binding pocket but can bind weakly to inositol phosphates via a basic surface patch, implying the involvement of inositol signaling in SCAB1 regulation. Our results provide novel insights into the functional organization of SCAB1.     

Filament assembly from profilin-actin

Profilin plays a major role in the assembly of actin filament at the barbed ends. The thermodynamic and kinetic parameters for barbed end assembly from profilin-actin have been measured turbidimetrically. Filament growth from profilin-actin requires MgATP to be bound to actin. No assembly is observed from profilin-CaATP-actin. The rate constant for association of profilin-actin to barbed ends is 30% lower than that of actin, and the critical concentration for F-actin assembly from profilin-actin units is 0.3 microM under physiological ionic conditions. Barbed ends grow from profilin-actin with an ADP-Pi cap. Profilin does not cap the barbed ends and is not detectably incorporated into filaments. The EDC-cross-linked profilin-actin complex (PAcov) both copolymerizes with F-actin and undergoes spontaneous self-assembly, following a nucleation-growth process characterized by a critical concentration of 0.2 microM under physiological conditions. The PAcov polymer is a helical filament that displays the same diffraction pattern as F-actin, with layer lines at 6 and 36 nm. The PAcov filaments bound phalloidin with the same kinetics as F-actin, bound myosin subfragment-1, and supported actin-activated ATPase of myosin subfragment-1, but they did not translocate in vitro along myosin-coated glass surfaces. These results are discussed in light of the current models of actin structure.     

Red light, Phot1 and JAC1 modulate Phot2-dependent reorganization of chloroplast actin filaments and chloroplast avoidance movement

The phototropin (phot)-dependent intracellular relocation of chloroplasts is a ubiquitous phenomenon in plants. We have previously revealed the involvement of a short cp-actin (chloroplast actin) filament-based mechanism in this movement. Here, the reorganization of cp-actin filaments during the avoidance movement of chloroplasts was analyzed in higher time resolution under blue GFP (green fluorescent protein) excitation light in an actin filament-visualized line of Arabidopsis thaliana. Under standard background red light of 89 μmol m(-2) s(-1), cp-actin filaments transiently disappeared at approximately 30 s and reappeared in a biased configuration on chloroplasts approximately 70 s after blue excitation light irradiation. The timing of biased cp-actin reappearance was delayed under the background of strong red light or in the absence of red light. Consistently, chloroplast movement was delayed under these conditions. In phot1 mutants, acceleration of both the disappearance and reappearance of cp-actin filaments occurred, indicating an inhibitory action of phot1 on reorganization of cp-actin filaments. Avoidance movements began sooner in phot1 than in wild-type plants. No reorganization of cp-actin filaments was seen in phot2 or phot1phot2 mutants lacking phot2, which is responsible for avoidance movements. Surprisingly, jac1 (j-domain protein required for chloroplast accumulation response 1) mutants, lacking the accumulation response, showed no avoidance movements under the whole-cell irradiation condition for GFP observation. Cp-actin filaments in jac1 did not show a biased distribution, with a small or almost no transient decrease in the number. These results indicate a close association between the biased distribution of cp-actin filaments and chloroplast movement. Further, JAC1 is suggested to function in the biased cp-actin filament distribution by regulating their appearance and disappearance.     

Light-induced structural changes of cytoskeleton in squid photoreceptor microvilli detected by rapid-freeze method

The cytoskeleton in squid photoreceptor microvilli was studied by freeze-substitution electron microscopy combined with rapid freezing using liquid helium, under dark-adapted and light-illuminated conditions. In the dark-adapted microvilli, actin filaments were regularly associated with granular structures on their surface; these granular structures were cross-linked to the rhodopsin-bearing plasma membranes through slender strands. Upon exposure to light, the granular components detached from the actin filaments, which then appeared to be fragmented and/or depolymerized. These observations have led us to conclude that light stimulation triggers the breakdown of the microvillar actin filament complex in squid photoreceptor cells. The results are discussed with special reference to the physiological role of actin filaments in photoreception.     

Structure and mobility of actin filaments as measured by quasielastic light scattering, viscometry, and electron microscopy

Actin filaments of different lengths were prepared by polymerizing actin in the presence of various concentrations of gelsolin, a protein which accelerates actin polymerization by stabilizing nuclei from which filaments grow and which binds to their fast growing ends. The lengths of the actin filaments following polymerization were measured by electron microscopy and showed that the number-average filament length agreed with the predicted length if each gelsolin molecule acted as a seed for the growth of an actin filament. The distribution of lengths was independent of the actin:gelsolin ratio and was similar to that of actin filaments polymerized in the absence of gelsolin (Lw/Ln = 1.8). The mobility of these filaments in solution was studied by quasielastic light scattering and by viscometry. The translational diffusion constant determined by quasielastic light scattering was in agreement with the infinite dilution values calculated from the dimensions and the distribution of lengths determined by electron microscopy for relatively short filament lengths. Under conditions where overlap of the rotational domains of the filaments would be expected to occur, the measured diffusion rates deviated from their predicted dilute solution values and the solution viscosity increased abruptly. The dependence of the diffusion constant and the solution viscosity on the length of the actin filaments can be explained in terms of a theory that describes the restraints on diffusion of independent rigid rods in semi-dilute solution. The results suggest that the rheology of actin filaments can be accounted for by steric restraints. The length of cytoplasmic actin filaments in some cell types is such that these steric constraints are significant and could produce large changes in physical properties with small changes in filament length.     

Actin filament nucleation by the bacterial pathogen, Listeria monocytogenes

Shortly after Listeria is phagocytosed by a macrophage, it dissolves the phagosomal membrane and enters the cytoplasm. 1 h later, actin filaments coat the Listeria and then become rearranged to form a tail with which the Listeria moves to the macrophage surface as a prelude to spreading. If infected macrophages are treated with cytochalasin D, all the actin filaments associated with the Listeria break down leaving a fine, fibrillar material that does not decorate with subfragment 1 of myosin. This material is associated with either the surface of the Listeria (the cloud stage) or one end (the tail stage). If the cytochalasin-treated infected macrophages are detergent extracted and then incubated in nuclei-free monomeric actin under polymerizing conditions, actin filaments assemble from the fine, fibrillar material, the result being that each Listeria has actin filaments radiating from its surface like the spokes of a wheel (cloud form) or possesses a long tail of actin filaments formed from the fine, fibrillar material located at one end of the Listeria. Evidence that the fine fibrillar material is involved in nucleating actin assembly comes from a Listeria mutant. Although the mutant replicates at a normal rate in macrophages, actin filaments do not form on its surface (cloud stage) or from one end (tail stage), nor does the bacterium spread. Furthermore it does not form the fine fibrillar material. Evidence that the nucleating material is a secretory product of Listeria and not the macrophage comes from experiments using chloramphenicol, which inhibits protein synthesis in Listeria but not in macrophages. If chloramphenicol is applied 1 h after infection, a time before actin filaments are found attached to the Listeria in untreated macrophages, actin filaments never assemble on the Listeria even when fixed 3 h later. Furthermore the fine fibrillar material is absent, although there is a coat of dense granular material.     

Influence of the C terminus of Wiskott-Aldrich syndrome protein (WASp) and the Arp2/3 complex on actin polymerization

The 70 C-terminal amino acids of Wiskott-Aldrich syndrome protein (WASp WA) activate the actin nucleation activity of the Arp2/3 complex. WASp WA binds both the Arp2/3 complex and actin monomers, but the mechanism by which it activates the Arp2/3 complex is not known. We characterized the effect of WASp WA on actin polymerization in the absence and presence of the human Arp2/3 complex. WASp WA binds actin monomers with an apparent K(d) of 0.4 microM, inhibiting spontaneous nucleation and subunit addition to pointed ends, but not addition to barbed ends. A peptide containing only the WASp homology 2 motif behaves similarly but with a 10-fold lower affinity. In contrast to previously published results, neither WASp WA nor a similar region of the protein Scar1 significantly depolymerizes actin filaments under a variety of conditions. WASp WA and the Arp2/3 complex nucleate actin filaments, and the rate of this nucleation is a function of the concentrations of both WASp WA and the Arp2/3 complex. With excess WASp WA and <10 nM Arp2/3 complex, there is a 1:1 correspondence between the Arp2/3 complex and the concentration of filaments produced, but the filament concentration plateaus at an Arp2/3 complex concentration far below the cellular concentration determined to be 9.7 microM in human neutrophils. Preformed filaments increase the rate of nucleation by WASp WA and the Arp2/3 complex but not the number of filaments that are generated. We propose that filament side binding by the Arp2/3 complex enhances its activation by WASp WA.     

Cofilin, a protein in porcine brain that binds to actin filaments and inhibits their interactions with myosin and tropomyosin

Cofilin, a 21 000 molecular weight protein of porcine brain, reacts stoichiometrically with actin in a 1:1 molar ratio. Upon binding of cofilin, the fluorescence of pyrene-labeled actin under polymerizing conditions is changed into the monomer form, irrespective of whether cofilin is added to actin before or after polymerization. Cofilin decreases the viscosity of actin filaments but increases the light-scattering intensity of the filaments. The centrifugation assay and the DNase I inhibition assay demonstrate that cofilin binds to actin filaments in a 1:1 molar ratio of cofilin to actin monomer in the filament and that cofilin increases the monomeric actin to a limited extent (up to 1.1-1.5 microM monomer) in the presence of physiological concentrations of Mg2+ and KCl. Cofilin is also able to bind to monomeric actin, as demonstrated by gel filtration. Electron microscopy showed that actin filaments are shortened and slightly thickened in the presence of cofilin. No bundle formation was observed in the presence of various concentrations of cofilin. The gel point assay using an actin cross-linking protein and the nucleation assay also suggested that cofilin shortens the actin filaments and hence increases the filament number. Cofilin blocks the binding of tropomyosin to actin filaments. Tropomyosin is dissociated from actin filaments by the binding of cofilin to actin filaments. Cofilin was found to inhibit the superprecipitation of actin-myosin mixtures as well as the actin-activated myosin ATPase. All these results suggest that cofilin is a new type of actin-associated protein.     

Polymerization and depolymerization of actin with nucleotide states at filament ends

Polymerization induces hydrolysis of ATP bound to actin, followed by γ-phosphate release, which helps advance the disassembly of actin filaments into ADP-G-actin. Mechanical understanding of this correlation between actin assembly and ATP hydrolysis has been an object of intensive studies in biochemistry and structural biology for many decades. Although actin polymerization and depolymerization occur only at either the barbed or pointed ends and the kinetic and equilibrium properties are substantially different from each other, characterizing their properties is difficult to do by bulk assays, as these assays report the average of all actin filaments in solution and are therefore not able to discern the properties of individual actin filaments. Biochemical studies of actin polymerization and hydrolysis were hampered by these inherent properties of actin filaments. Total internal reflection fluorescence (TIRF) microscopy overcame this problem by observing single actin filaments. With TIRF, we now know not only that each end has distinct properties, but also that the rate of γ-phosphate release is much faster from the terminals than from the interior of actin filaments. The rate of γ-phosphate release from actin filament ends is even more accelerated when latrunculin A is bound. These findings highlight the importance of resolving structural differences between actin molecules in the interior of the filament and those at either filament end. This review provides a history of observing actin filaments under light microscopy, an overview of dynamic properties of ATP hydrolysis at the end of actin filament, and structural views of γ-phosphate release.     

Ca2+ regulation of gelsolin activity: binding and severing of F-actin.

Regulation of the F-actin severing activity of gelsolin by Ca2+ has been investigated under physiologic ionic conditions. Tryptophan fluorescence intensity measurements indicate that gelsolin contains at least two Ca2+ binding sites with affinities of 2.5 x 10(7) M-1 and 1.5 x 10(5) M-1. At F-actin and gelsolin concentrations in the range of those found intracellularly, gelsolin is able to bind F-actin with half-maximum binding at 0.14 microM free Ca2+ concentration. Steady-state measurements of gelsolin-induced actin depolymerization suggest that half-maximum depolymerization occurs at approximately 0.4 microM free Ca2+ concentration. Dynamic light scattering measurements of the translational diffusion coefficient for actin filaments and nucleated polymerization assays for number concentration of actin filaments both indicate that severing of F-actin occurs slowly at micromolar free Ca2+ concentrations. The data suggest that binding of Ca2+ to the gelsolin-F-actin complex is the rate-limiting step for F-actin severing by gelsolin; this Ca2+ binding event is a committed step that results in a Ca2+ ion bound at a high-affinity, EGTA-resistant site. The very high affinity of gelsolin for the barbed end of an actin filament drives the binding reaction equilibrium toward completion under conditions where the reaction rate is slow.     

Assembly of Acanthamoeba actin in the presence of Acanthamoeba profilin measured by fluorescence photobleaching recovery

Assembly of Acanthamoeba actin, of which trace quantities had been labeled with 5-(iodoacetamido)-fluorescein, was quantified using the modulation detection method of fluorescence photobleaching recovery (FPR). This technique permits explicit determination of the fraction of labeled actin incorporated into filaments and the translational diffusion coefficients of the filaments, from which filament length can be calculated. Addition of Acanthamoeba profilin in molar ratios to actin of about 1.1:1 and 2.3:1 retarded the initial kinetics of assembly (induced by addition of 2mM Mg+2) and reduced the fraction of actin incorporated into filaments. The diffusion coefficients of filaments formed were greatly changed by the presence of profilin at short times, but the differences became increasingly smaller at longer times. After 26 hr. the filaments formed in 1.1:1 profilin were about 12% shorter and in 2.3:1 profilin were about 20% shorter than filaments formed by actin alone under the same conditions.     

Distribution pattern changes of actin filaments during chloroplast movement in Adiantum capillus-veneris

Chloroplasts change their positions in a cell in response to light intensities. The photoreceptors involved in chloroplast photo-relocation movements and the behavior of chloroplasts during their migration were identified in our previous studies, but the mechanism of movement has yet to be clarified. In this study, the behavior of actin filaments under various light conditions was observed in Adiantum capillus-veneris gametophytes. In chloroplasts staying in one place under a weak light condition and not moving, circular structures composed of actin filaments were observed around the chloroplast periphery. In contrast, short actin filaments were observed at the leading edge of moving chloroplasts induced by partial cell irradiation. In the dark, the circular structures found under the weak light condition disappeared and then reappeared around the moving chloroplasts. Mutant analyses revealed that the disappearance of the circular actin structure was mediated by the blue light photoreceptor, phototropin2.     

Actin and myosin: control of filament assembly

Actin filaments, assembled from highly purified actin from either skeletal muscle or Dictyostelium amoebae, are very stable under physiological ionic conditions. A small and limited amount of exchange of actin filament subunits for unpolymerized actin or subunits in other filaments has been measured by three techniques: fluorescence energy transfer, incorporation of 35S-labelled actin monomers into unlabelled actin filaments, and exchange of [14C]ATP with filament-bound ADP. A 40 kDa protein purified from amoebae destabilizes these otherwise stable filaments in a Ca2+-dependent manner. Myosin purified from Dictyostelium amoebae is phosphorylated both in the tail region of the heavy chain and in one of the light chains. Phosphorylation appears to regulate myosin thick-filament formation.     

Structure and activity of JAC1 J-domain implicate the involvement of the cochaperone activity with HSC70 in chloroplast photorelocation movement

Chloroplast photorelocation movement towards weak light and away from strong light is essential for plants to adapt to the fluctuation of ambient light conditions. In the previous study, we showed that blue light receptor phototropins mediated blue light-induced chloroplast movement in Arabidopsis by regulating short actin filaments localized at the chloroplast periphery (cp-actin filaments) rather than actin cables in the cytoplasm. However, the signaling pathway for the chloroplast photorelocation movement is still unclear. We also identified JAC1 (J-domain protein required for chloroplast accumulation response 1) as an essential component for the accumulation response and dark positioning in Arabidopsis. We recently determined the crystal structure of the J-domain of JAC1. The JAC1 J-domain has a positively charged surface, which forms a putative interface with the Hsc70 chaperone by analogy to that of bovine auxilin. Furthermore, the mutation of the highly conserved HPD motif in the JAC1 J-domain impaired the in vivo activity of JAC1. These data suggest that JAC1 cochaperone activity with HSC70 is essential for chloroplast photorelocation movement.Key words: Arabidopsis, auxilin, blue light, clathrin, endocytosis, J-domain, organelle movement, phototropin     

A calcium- and pH-regulated protein from Dictyostelium discoideum that cross-links actin filaments

We have purified an actin binding protein from amebas of Dictyostelium discoideum which we call 95,000-dalton protein (95K). This protein is rod shaped, approximately 40 nm long in the electron microscope, contains two subunits measuring 95,000 daltons each, and cross-links actin filaments. Cross-linking activity was demonstrated by using falling-ball viscometry, Ostwald viscometry, and electron microscopy. Cross-linking activity is optimal at 0.1 microM Ca++ and pH 6.8, but is progressively inhibited at higher Ca++ and pH levels over a physiological range. Half-maximal inhibition occurs at 1.6 microM free Ca++ and pH 7.3, respectively. Sedimentation experiments demonstrate that elevated Ca++ and pH inhibit the binding of 95K to F-actin which explains the loss of cross-linking activity. Electron microscopy demonstrates that under optimal conditions for cross-linking, 95K protein bundles actin filaments and that this bundling is inhibited by microM Ca++. Severing of actin filaments by 95K was not observed in any of the various assays under any of the solution conditions used. Hence, 95K protein is a rod-shaped, dimeric, Ca++- and pH-regulated actin binding protein that cross-links but does not sever actin filaments.     

Effect of filamin and controlled linear shear on the microheterogeneity of F-actin/gelsolin gels

We have previously established [Cortese and Frieden, J. Cell Biol. 107:1477-1487, 1988] that actin gels formed under shear are microheterogeneous. In this study, the effect of cross-linking (by chicken gizzard filamin), severing (by plasma gelsolin), and shear on actin microheterogeneity are investigated using fluorescence photobleaching recovery and video microscopy. We find that filamin and shear form microheterogeneous F-actin:gelsolin gels by different mechanisms. Bundling of actin:gelsolin filaments by filamin can be explained by an increase in the apparent length of the filaments due to interfilament binding, resulting in a decrease of the polymer number concentration at which filaments organize into anisotropic phases. Some intrafilament binding of filamin to actin filaments may also be present, and those filaments coated with filamin immobilize more slowly than actin under the same polymerization conditions. The length of F-actin/gelsolin filaments seems to be a major factor in controlling the extent of bundling relative to network formation. In contrast, the effect of shear on the microheterogeneity of actin:gelsolin filaments is consistent with our previous proposal that shear aligns actin filaments, allowing filament-filament interactions and phase formation to occur. Short filaments are unable to organize into branched actin networks, but they can create large aggregates under low shear. Longer actin filaments will exist as networks with variable levels of branching and are less sensitive to shear. The effect of the intensity of a shear field on the spatial distribution of actin may involve a progressively more random orientation of actin molecules and bundles. A regular pattern develops across the sample at low shear rates (0.04-1.39 s-1), and becomes very irregular at higher shear rates (greater than 10 s-1). We suggest here that actin-binding proteins and shear can control the transition between isotropic networks and anisotropic phases by their effect on apparent length and local filament concentration, and also that this transition can have substantial effects on the resistance of cells to mechanical stress.     

A novel regulatory effect of myosin light chain kinase from smooth muscle on the ATP-dependent interaction between actin and myosin.

The actin-binding activity of myosin light chain kinase (MLCK) from smooth muscle was studied with special reference to the ATP-dependent interaction between actin and myosin. MLCK in the presence of calmodulin endowed sensitivity to Ca2+ on the movement of actin filaments on phosphorylated myosin from smooth muscle that was fixed on a coverslip. This regulatory effect was not attributable to the kinase activity of MLCK but could be explained by its actin-binding activity. The importance of the actin-binding activity was further substantiated by results of an experiment with Nitellopsis actin-cables in which MLCK regulated the interaction under conditions where MLCK was exclusively associated with the actin-cables.     

Rate of binding of tropomyosin to actin filaments

The decrease of the rate of actin polymerization by tropomyosin molecules which bind near the ends of actin filaments was analyzed in terms of the rate of binding of tropomyosin to actin filaments. Monomeric actin was polymerized onto actin filaments in the presence of various concentrations of tropomyosin. At high concentrations of monomeric actin (c1) and low tropomyosin concentrations (ct) (c1/ct greater than 10), actin polymerization was not retarded by tropomyosin because actin polymerization was faster than binding of tropomyosin to actin filaments. At low actin concentrations and high tropomyosin concentrations (c1/ct less than 5), the rate of elongation of actin filaments was decreased because actin polymerization was slower than binding of tropomyosin at the ends of actin filaments. The results were quantitatively analyzed by a model in which it was assumed that actin-bound tropomyosin molecules which extend beyond the ends of actin filaments retard association of actin monomers with filament ends. Under the experimental conditions (100 mM KCl, 1 mM MgCl2, pH 7.5, 25 degrees C), the rate constant for binding of tropomyosin to actin filaments turned out to be about 2.5 X 10(6) to 4 X 10(6) M-1 S-1.     

Cytoskeletal functions of cytoplasmic contractile proteins

This is a review of the evidence that the cytoplasmic contractile proteins function as a cytoskeletal system inthe cytoplasmic matrix. Biochemical experiments show that cycoplasmic actin filaments can form a solid gel under conditions likely to exist in living cells. The actin filaments are associated with other proteins which may stabilize the gel and which are involved with motile force generation like myosin. Ultrastructural studies show that actin filaments are difficult to preserve, but that under stabilizing conditions networks of actin filaments are found throughout the cytoplasmic matrix.