Micro- and macrorheological properties of actin networks effectively cross-linked by depletion forces

The structure and rheology of cytoskeletal networks are regulated by actin binding proteins. Aside from these specific interactions, depletion forces can also alter the properties of cytoskeletal networks. Here we demonstrate that the addition of poly(ethylene glycol) (PEG) as a depletion agent results not only in severe structural changes, but also in alterations in mechanical properties of actin solutions. In the plateau of the elastic modulus two regimes can be distinguished by micro and macrorheological methods. In the first, the elastic modulus increases only slightly with increasing depletion agent, whereas above a critical concentration c*, a strong increase of cPEG6k3.5 is observed in a distinct second regime. Microrheological data and electron microscopy images show a homogenous network of actin filaments in the first regime, whereas at higher PEG concentrations a network of actin bundles is observed. The concentration dependence of the plateau modulus G0, the shift in entanglement time taue, and the nonlinear response indicate that below c* the network becomes effectively cross-linked, whereas above c* G0(cPEG6k) is primarily determined by the network of bundles that exhibits a linearly increasing bundle thickness.     

Role of the actin bundling protein fascin in growth cone morphogenesis: localization in filopodia and lamellipodia

Growth cones at the distal tips of growing nerve axons contain bundles of actin filaments distributed throughout the lamellipodium and that project into filopodia. The regulation of actin bundling by specific actin binding proteins is likely to play an important role in many growth cone behaviors. Although the actin binding protein, fascin, has been localized in growth cones, little information is available on its functional significance. We used the large growth cones of the snail Helisoma to determine whether fascin was involved in temporal changes in actin filaments during growth cone morphogenesis. Fascin localized to radially oriented actin bundles in lamellipodia (ribs) and filopodia. Using a fascin antibody and a GFP fascin construct, we found that fascin incorporated into actin bundles from the beginning of growth cone formation at the cut end of axons. Fascin associated with most of the actin bundle except the proximal 6--12% adjacent to the central domain, which is the region associated with actin disassembly. Later, during growth cone morphogenesis when actin ribs shortened, the proximal fascin-free zone of bundles increased, but fascin was retained in the distal, filopodial portion of bundles. Treatment with tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), which phosphorylates fascin and decreases its affinity for actin, resulted in loss of all actin bundles from growth cones. Our findings suggest that fascin may be particularly important for the linear structure and dynamics of filopodia and for lamellipodial rib dynamics by regulating filament organization in bundles.     

Binding of actin filaments to connectin

The binding of actin filaments to connectin, a muscle elastic protein, was investigated by means of turbidity and sedimentation measurements and electron microscopy. In the presence of less than 0.12 M KCl at pH 7.0, actin filaments bound to connectin. Long actin filaments formed bundles. Short actin filaments also aggregated into irregular bundles or a meshwork, and were frequently attached perpendicularly to long bundles. The binding of F-actin to connectin was saturated at an equal weight ratio (molar ratio, 50 : 1), as determined by a cosedimentation assay. Larger amounts of sonicated short actin filaments appeared to bind to connectin than intact F-actin. Myosin S1-decorated actin filaments did not bind to connectin. The addition of S1 to connectin-induced actin bundles resulted in partial disaggregation. Thus, connectin does not appear to interfere with actin-myosin interactions, since myosin S1 binds to actin more strongly than connectin.     

Mutual effects of alpha-actinin, calponin and filamin on actin binding

The mutual effect of three actin-binding proteins (alpha-actinin, calponin and filamin) on the binding to actin was analyzed by means of differential centrifugation and electron microscopy. In the absence of actin alpha-actinin, calponin and filamin do not interact with each other. Calponin and filamin do not interfere with each other in the binding to actin bundles. Slight interference was observed in the binding of alpha-actinin and calponin to actin bundles. Higher ability of calponin to depress alpha-actinin binding can be due to the higher stoichiometry calponin/actin in the complexes formed. The largest interference was observed in the pair filamin-alpha-actinin. These proteins interfere with each other in the binding to the bundled actin filaments; however, neither of them completely displaced another protein from its complexes with actin. The structure of actin bundles formed in the presence of any one actin-binding protein was different from that observed in the presence of binary mixtures of two actin-binding proteins. In the case of calponin or its binary mixtures with alpha-actinin or filamin the total stoichiometry actin-binding protein/actin was larger than 0.5. This means that alpha-actinin, calponin and filamin may coexist on actin filaments and more than mol of any actin-binding protein is bound per two actin monomers. This may be important for formation of different elements of cytoskeleton.     

Mutual effects of α-actinin,calponin and filamin on actin binding

The mutual effect of three actin-binding proteins (α-actinin, calponin and filamin) on the binding to actin was analyzed by means of differential centrifugation and electron microscopy. In the absence of actin α-actinin, calponin and filamin do not interact with each other. Calponin and filamin do not interfere with each other in the binding to actin bundles. Slight interference was observed in the binding of α-actinin and calponin to actin bundles. Higher ability of calponin to depress α-actinin binding can be due to the higher stoichiometry calponin/actin in the complexes formed. The largest interference was observed in the pair filamin–α-actinin. These proteins interfere with each other in the binding to the bundled actin filaments; however, neither of them completely displaced another protein from its complexes with actin. The structure of actin bundles formed in the presence of any one actin-binding protein was different from that observed in the presence of binary mixtures of two actin-binding proteins. In the case of calponin or its binary mixtures with α-actinin or filamin the total stoichiometry actin-binding protein/actin was larger than 0.5. This means that α-actinin, calponin and filamin may coexist on actin filaments and more than mol of any actin-binding protein is bound per two actin monomers. This may be important for formation of different elements of cytoskeleton.     

Modelling the dynamics of F-actin in the cell

The regulation of the interactions between the actin binding proteins and the actin filaments are known to affect the cytoskeletal structure of F-actin. We develop a model depicting the formation of actin cytoskeleton, bundles and orthogonal networks, via activation or inactivation of different types of actin binding proteins. It is found that as the actin filament density increases in the cell, a spontaneous tendency to organize into bundles or networks occurs depending on the active actin binding protein concentration. Also, a minute change in the relative binding affinity of the actin binding proteins in the cell may lead to a major change in the actin cytoskeleton. Both the linear stability analysis and the numerical results indicate that the structures formed are highly sensitive to changes in the parameters, in particular to changes in the parameter ϕ, denoting the relative binding affinity and concentration of the actin binding proteins.     

Effect of ethylene glycol on the interaction of different myosin subfragment 1.nucleotide complexes with actin

To elucidate the structure of the cross-bridge intermediates in the actomyosin ATPase cycle, several laboratories have added both ethylene glycol and AMP-PNP to muscle fibers. These studies suggested that ethylene glycol shifts the structure of myosin.AMP-PNP toward the weak-binding conformation, i.e., toward the structure of myosin.ATP. Since only the weak-binding conformation of myosin subfragment 1 (S-1) binds with no apparent cooperativity to the troponin-tropomyosin-actin complex (regulated actin), we used this as a probe to examine the conformation of various S-1.nucleotide complexes in ethylene glycol. Our results show that ethylene glycol markedly weakens the binding strength of S-1, S-1.ADP, and S-1.AMP-PNP to actin but has almost no effect on the binding strength of S-1.ATP. As in muscle fibers, at 40% ethylene glycol, the binding strength of S-1.AMP-PNP to actin becomes very similar to the binding strength of S-1.ATP. In the presence of troponin-tropomyosin, the binding of S-1.AMP-PNP to actin shows no apparent cooperativity in 40% ethylene glycol. Therefore, our results confirm that ethylene glycol shifts the structure of the myosin.AMP-PNP toward the weak-binding conformation. However, our results also suggest that ethylene glycol has a direct effect on the regulated actin complex. This is shown by the fact that ethylene glycol markedly increases the cooperative binding of S-1.ADP to regulated actin both in the presence and in the absence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regeneration of actin bundles in Chara: polarized growth and orientation by endoplasmic flow

Strong irradiation of localized areas of the alga Chara produces chloroplast damage and extensive loss of the actin bundles responsible for cytoplasmic streaming. Immunofluorescence using a monoclonal antibody binding to the actin bundles has been used to follow their regrowth. Bundle regeneration is polarized so that new bundles develop from the ends of the actin bundles delivering endoplasm to the damaged area and not from bundles removing endoplasm. According to the previously established polarity of the actin filaments this growth is occurring from the "barbed" but not the "pointed" ends of the component filaments. The frequently irregular orientation of the regenerated bundles contrasts with the straight, parallel arrangement of the bundles before destruction. The arrangement of the regenerated bundles is suggested to depend on orientation by passive endoplasmic flow rather than a cortical template. As a result, bundles follow sweeping curves and can form a U-turn connecting oppositely polarized bundles normally separated by the neutral line. In addition to development in continuity with the free ends of pre-existing bundles, visualization of small, discrete fluorescent structures suggests that bundles can begin to form in isolation within the damaged areas. The results are discussed in terms of the polarized actin polymerisation seen in vitro, additional controls which may operate on bundle growth in vivo, and the ability of flow to orient F-actin. The relevance of the findings to normal cell ontogeny is assessed.     

The actin gelling activity of chicken gizzard alpha-actinin at physiological temperature is triggered by water sequestration

At 37 degrees C, in the presence of 6% (w/v) polyethylene glycol 6000, 30 nM alpha-actinin from chicken gizzard induces the gelation of 12 microM actin. Static measurement shows that the addition of 30 nM alpha-actinin increases the rigidity of the system from 23.5 to 54 dynes/cm2. According to the theory of osmoelastic coupling, also large additives, such as the proteins of the cell sap, are able to cause an osmotic stress equivalent to that caused by polyethylene glycol. We thus conclude that, in vivo, alpha-actinin acts as an actin gelling protein.     

The two actin-binding regions on the myosin heads of cardiac muscle

In the presence of myosin S1 or myosin heads, actin filaments tend to form bundles. The biological meaning of the bundling of actin filaments has been unclear. In this study, we found that the cardiac myosin heads can form the bundles of actin filaments more rapidly than can skeletal S1, as monitored by light scattering and electron microscopy. Moreover, the actin bundles formed by cardiac S1 were found to be more stable against mechanical agitation. The distance between actin filaments in the bundles was approximately 20 nm, which is comparable to the length of a myosin head and two actin molecules. This suggests the direct binding of S1 tails to the adjacent actin filament. The "essential" light chain of cardiac myosin could be cross-linked to the actin molecule in the bundle. When monomeric actin molecules were added to the bundle, the bundles could be dispersed into individual filaments. The three-dimensional structure of the dispersed actin filaments was reconstructed from electron cryo-microscopic images of the single actin filaments dispersed by monomer actin. We were able to demonstrate that cardiac myosin heads bind to two actin molecules: one actin molecule at the conventional actin-binding region and the other at the essential light-chain-binding region. This capability of cardiac myosin heads to bind two actin molecules is discussed in view of lower ATPase activity and slower shortening velocity than those of skeletal ones.     

Contribution of the LIM Domain and Nebulin-Repeats to the Interaction of Lasp-2 with Actin Filaments and Focal Adhesions

Lasp-2 binds to actin filaments and concentrates in the actin bundles of filopodia and lamellipodia in neural cells and focal adhesions in fibroblastic cells. Lasp-2 has three structural regions: a LIM domain, a nebulin-repeat region, and an SH3 domain; however, the region(s) responsible for its interactions with actin filaments and focal adhesions are still unclear. In this study, we revealed that the N-terminal fragment from the LIM domain to the first nebulin-repeat module (LIM-n1) retained actin-binding activity and showed a similar subcellular localization to full-length lasp-2 in neural cells. The LIM domain fragment did not interact with actin filaments or localize to actin filament bundles. In contrast, LIM-n1 showed a clear subcellular localization to filopodial actin bundles. Although truncation of the LIM domain caused the loss of F-actin binding activity and the accumulation of filopodial actin bundles, these truncated fragments localized to focal adhesions. These results suggest that lasp-2 interactions with actin filaments are mediated through the cooperation of the LIM domain and the first nebulin-repeat module in vitro and in vivo. Actin filament binding activity may be a major contributor to the subcellular localization of lasp-2 to filopodia but is not crucial for lasp-2 recruitment to focal adhesions.     

Molecular and biochemical characterization of a novel actin bundling protein in Acanthamoeba

Actin binding proteins play key roles in cell structure and movement particularly as regulators of the assembly, stability and localization of actin filaments in the cytoplasm. In the present study, a cDNA clone encoding an actin bundling protein named as AhABP was isolated from Acanthamoeba healyi, a causative agent of granulomatous amebic encephalitis. This clone exhibited high similarity with genes of Physarum polycephalum and Dictyostelium discoideum, which encode actin bundling proteins. Domain search analysis revealed the presence of essential conserved regions, i.e., an active actin binding site and 2 putative calcium binding EF-hands. Transfected amoeba cells demonstrated that AhABP is primarily localized in phagocytic cups, peripheral edges, pseudopods, and in cortical cytoplasm where actins are most abundant. Moreover, AhABP after the deletion of essential regions formed ellipsoidal inclusions within transfected cells. High-speed co-sedimentation assays revealed that AhABP directly interacted with actin in the presence of up to 10 microM of calcium. Under the electron microscope, thick parallel bundles were formed by full length AhABP, in contrast to the thin actin bundles formed by constructs with deletion sites. In the light of these results, we conclude that AhABP is a novel actin bundling protein that is importantly associated with actin filaments in the cytoplasm.     

Intranuclear actin bundles induced by dimethyl sulfoxide in interphase nucleus of Dictyostelium

Electron microscopic evidence demonstrated that dimethyl sulfoxide (DMSO) induces formation of giant intranuclear microfilament bundles in the interphase nucleus of a cellular slime mold, Dictyostelium. These giant bundles are approximately giant bundles are approximately 3 micrometer long, 0.85 micrometer wide, and composed of microfilaments 6 nm in diameter. Studies in which glycerinated cells were used showed that these microfilaments bind rabbit skeletal muscle heavy meromyosin, forming typical decorated "arrowhead" structures, and that this binding can be reverted by Mg-adenosine triphosphate. These data verify that the intranuclear microfilaments are the contractile protein actin, and that DMSO affects intranuclear actin, inducing the formation of such giant bundles. The intranuclear actin bundles appear at any developmental stage in two different species of cellular slime molds after treatment with DMSO. The native form of the intranuclear actin molecules and their possible functions are discussed, and it is proposed that the contractile protein has essential functions in the cell nucleus.     

Interactions in affinity partition studied using fluorescence spectroscopy.

Fluorescence titration has been used to determine the binding constant and number of binding sites for the textile triazine dye Procion Yellow HE-3G to lactate dehydrogenase from rabbit muscle (E.C. 1.1.1.27). Triazine dye was either free in solution or attached to one of the polymer carriers, polyethylene glycol or dextran. Titrations were performed in solutions of buffer, dextran, and polyethylene glycol. Aqueous two-phase systems composed of polyethylene glycol and dextran were prepared and the binding constant and number of binding sites for ligand polyethylene glycol-Procion Yellow to lactate dehydrogenase were determined in both upper and lower phases of these systems. Affinity partition of lactate dehydrogenase in a PEG-dextran system was also performed using PEG-Procion Yellow as ligand, and partition coefficients of lactate dehydrogenase showed good agreement with theoretical partition coefficients calculated from the binding constant and number of binding sites obtained from fluorescence titration. The advantage of using fluorescence titration to determine affinity of a polymer ligand for a protein is that measurement of binding strength can be made in the actual environment encountered by protein-ligand complex during the purification process.     

Disruption of the cytoskeleton during Semaphorin 3A induced growth cone collapse correlates with differences in actin organization and associated binding proteins

Repulsive guidance cues induce growth cone collapse or collapse and retraction. Collapse results from disruption and loss of the actin cytoskeleton. Actin‐rich regions of growth cones contain binding proteins that influence filament organization, such as Arp2/3, cortactin, and fascin, but little is known about the role that these proteins play in collapse. Here, we show that Semaphorin 3A (Sema 3A), which is repulsive to mouse dorsal root ganglion neurons, has unequal effects on actin binding proteins and their associated filaments. The immunofluorescence staining intensity of Arp‐2 and cortactin decreases relative to total protein; whereas in unextracted growth cones fascin increases. Fascin and myosin IIB staining redistribute and show increased overlap. The degree of actin filament loss during collapse correlates with filament superstructures detected by rotary shadow electron microscopy. Collapse results in the loss of branched f‐actin meshworks, while actin bundles are partially retained to varying degrees. Taken together with the known affects of Sema 3A on actin, this suggests a model for collapse that follows a sequence; depolymerization of actin meshworks followed by partial depolymerization of fascin associated actin bundles and their movement to the neurite to complete collapse. The relocated fascin associated actin bundles may provide the substrate for actomyosin contractions that produce retraction. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Simultaneous interaction of actin with alpha-actinin and calponin

Interaction of calponin and alpha-actinin with actin was analyzed by means of cosedimentation and electron microscopy. G-actin was polymerized in the presence of calponin, alpha-actinin, or both of these actin-binding proteins (ABPs). The single and bundled actin filaments were separated, and the stoichiometry of ABPs and actin in both types of filaments was determined. Binding of calponin to the single or bundled actin filaments was not dependent on the presence of alpha-actinin and did not displace alpha-actinin from actin. In the presence of calponin, however, less alpha-actinin was bound to the bundled actin filaments, and the binding of alpha-actinin was accompanied by a partial decrease in the calponin/actin stoichiometry in the bundles of actin filaments. Calponin had no influence on the binding of alpha-actinin to the single actin filaments. The structure of actin bundles formed in the presence of the two ABPs differed from that formed in the presence of either one singly. We conclude that calponin and alpha-actinin can coexist on actin and that nearly each actin monomer can bind one of these ABPs.     

A Highlights from MBoC Selection: αE-catenin actin-binding domain alters actin filament conformation and regulates binding of nucleation and disassembly factors

The actin-binding protein αE-catenin may contribute to transitions between cell migration and cell–cell adhesion that depend on remodeling the actin cytoskeleton, but the underlying mechanisms are unknown. We show that the αE-catenin actin-binding domain (ABD) binds cooperatively to individual actin filaments and that binding is accompanied by a conformational change in the actin protomer that affects filament structure. αE-catenin ABD binding limits barbed-end growth, especially in actin filament bundles. αE-catenin ABD inhibits actin filament branching by the Arp2/3 complex and severing by cofilin, both of which contact regions of the actin protomer that are structurally altered by αE-catenin ABD binding. In epithelial cells, there is little correlation between the distribution of αE-catenin and the Arp2/3 complex at developing cell–cell contacts. Our results indicate that αE-catenin binding to filamentous actin favors assembly of unbranched filament bundles that are protected from severing over more dynamic, branched filament arrays.     

The effect of polyethylene glycol on the mechanics and ATPase activity of active muscle fibers

We have used polyethylene glycol (PEG) to perturb the actomyosin interaction in active skinned muscle fibers. PEG is known to potentiate protein-protein interactions, including the binding of myosin to actin. The addition of 5% w/v PEG (MW 300 or 4000) to active fibers increased fiber tension and decreased shortening velocity and ATPase activity, all by 25-40%. Variation in [ADP] or [ATP] showed that the addition of PEG had little effect on the dissociation of the cross-bridge at the end of the power stroke. Myosin complexed with ADP and the phosphate analog V(i) or AlF(4) binds weakly to actin and is an analog of a pre-power-stroke state. PEG substantially enhances binding of these states both in active fibers and in solution. Titration of force with increasing [P(i)] showed that PEG increased the free energy available to drive the power stroke by about the same amount as it increased the free energy available from the formation of the actomyosin bond. Thus PEG potentiates the binding of myosin to actin in active fibers, and it provides a method for enhancing populations of some states for structural or mechanical studies, particularly those of the normally weakly bound transient states that precede the power stroke.     

Capping protein and the Arp2/3 complex regulate nonbundle actin filament assembly to indirectly control actin bundle positioning during Drosophila melanogaster bristle development

Drosophila melanogaster bristle development is dependent on actin assembly, and prominent actin bundles form against the elongating cell membrane, giving the adult bristle its characteristic grooved pattern. Previous work has demonstrated that several actin-regulating proteins are required to generate normal actin bundles. Here we have addressed how two actin regulators, capping protein, a barbed end binding protein, and the Arp2/3 complex, a potent actin assembly nucleator, function to generate properly organized bundles. As predicted from studies in motile cells, we find that capping protein and the Arp2/3 complex act antagonistically to one another during bristle development. However, these proteins do not primarily act directly on bundles, but rather on a dynamic population of actin filaments that are not part of the bundles. These nonbundle filaments, termed snarls, play an important role in determining the number and spacing of the actin bundles. Reduction of capping protein leads to an increase in snarls, which prevents actin bundles from properly attaching to the membrane. Conversely, loss of an Arp2/3 complex component leads to a loss of snarls and accumulation of excess membrane-attached bundles. These results indicate that in nonmotile cells dynamic actin filaments can function to regulate the positioning of stable actin structures. In addition, our results suggest that the Arpc1 subunit may have an additional function, independent of the rest of the Arp2/3 complex.     

Tobacco WLIM1 is a novel F-actin binding protein involved in actin cytoskeleton remodeling

We used confocal microscopy and in vitro analyses to show that Nicotiana tabacum WLIM1, a LIM domain protein related to animal Cys-rich proteins, is a novel actin binding protein in plants. Green fluorescent protein (GFP)-tagged WLIM1 protein accumulated in the nucleus and cytoplasm of tobacco BY2 cells. It associated predominantly with actin cytoskeleton, as demonstrated by colabeling and treatment with actin-depolymerizing latrunculin B. High-speed cosedimentation assays revealed the ability of WLIM1 to bind directly to actin filaments with high affinity. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching showed a highly dynamic in vivo interaction of WLIM1-GFP with actin filaments. Expression of WLIM1-GFP in BY2 cells significantly delayed depolymerization of the actin cytoskeleton induced by latrunculin B treatment. WLIM1 also stabilized actin filaments in vitro. Importantly, expression of WLIM1-GFP in Nicotiana benthamiana leaves induces significant changes in actin cytoskeleton organization, specifically, fewer and thicker actin bundles than in control cells, suggesting that WLIM1 functions as an actin bundling protein. This hypothesis was confirmed by low-speed cosedimentation assays and direct observation of F-actin bundles that formed in vitro in the presence of WLIM1. Taken together, these data identify WLIM1 as a novel actin binding protein that increases actin cytoskeleton stability by promoting bundling of actin filaments.