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PubMed1991

Three different actin filament assemblies occur in every hair cell: each contains a specific actin crosslinking protein.

The apex of hair cells of the chicken auditory organ contains three different kinds of assemblies of actin filaments in close spatial proximity. These are (a) paracrystals of actin filaments with identical polarity in stereocilia, (b) a dense gellike meshwork of actin filaments forming the cuticular plate, and (c) a bundle of parallel actin filaments with mixed polarities that constitute the circumferential filament belt attached to the cytoplasmic aspect of the zonula adhaerens (ZA). Each different supramolecular assembly of actin filaments contains a specific actin filament cross-linking protein which is unique to that particular assembly. Thus fimbrin appears to be responsible for paracrystallin packing of actin filaments in stereocillia; an isoform of spectrin resides in the cuticular plate where it forms the whisker-like crossbridges, and alpha actinin is the actin crosslinking protein of the circumferential ZA bundle. Tropomyosin, which stabilizes actin filaments, is present in all the actin filament assemblies except for the stereocilia. Another striking finding was that myosin appears to be absent from the ZA ring and cuticular plate of hair cells although present in the ZA ring of supporting cells. The abundance of myosin in the ZA ring of the surrounding supporting cells means that it may be important in forming a supporting tensile cellular framework in which the hair cells are inserted.

Actin Cytoskeleton↗
PubMed1994

Actin mutations that show suppression with fimbrin mutations identify a likely fimbrin-binding site on actin.

Actin interacts with a large number of different proteins that modulate its assembly and mediate its functions. One such protein is the yeast actin-binding protein Sac6p, which is homologous to vertebrate fimbrin (Adams, A. E. M., D. Botstein, and D. G. Drubin. 1991. Nature (Lond.). 354:404-408.). Sac6p was originally identified both genetically (Adams, A. E. M., and D. Botstein. 1989. Genetics. 121:675-683.) by dominant, reciprocal suppression of a temperature-sensitive yeast actin mutation (act1-1), as well as biochemically (Drubin, D. G., K. G. Miller, and D. Botstein. 1988. J. Cell Biol. 107: 2551-2561.). To identify the region on actin that interacts with Sac6p, we have analyzed eight different act1 mutations that show suppression with sac6 mutant alleles, and have asked whether (a) these mutations occur in a small defined region on the crystal structure of actin; and (b) the mutant actins are defective in their interaction with Sac6p in vitro. Sequence analysis indicates that all of these mutations change residues that cluster in the small domain of the actin crystal structure, suggesting that this region is an important part of the Sac6p-binding domain. Biochemical analysis reveals defects in the ability of several of the mutant actins to bind Sac6p, and a reduction in Sac6p-induced cross-linking of mutant actin filaments. Together, these observations identify a likely site of interaction of fimbrin on actin.

Actin Cytoskeleton↗
PubMed2002

The three mouse actin-depolymerizing factor/cofilins evolved to fulfill cell-type-specific requirements for actin dynamics.

Actin-depolymerizing factor (ADF)/cofilins are essential regulators of actin filament turnover. Several ADF/cofilin isoforms are found in multicellular organisms, but their biological differences have remained unclear. Herein, we show that three ADF/cofilins exist in mouse and most likely in all other mammalian species. Northern blot and in situ hybridization analyses demonstrate that cofilin-1 is expressed in most cell types of embryos and adult mice. Cofilin-2 is expressed in muscle cells and ADF is restricted to epithelia and endothelia. Although the three mouse ADF/cofilins do not show actin isoform specificity, they all depolymerize platelet actin filaments more efficiently than muscle actin. Furthermore, these ADF/cofilins are biochemically different. The epithelial-specific ADF is the most efficient in turning over actin filaments and promotes a stronger pH-dependent actin filament disassembly than the two other isoforms. The muscle-specific cofilin-2 has a weaker actin filament depolymerization activity and displays a 5-10-fold higher affinity for ATP-actin monomers than cofilin-1 and ADF. In steady-state assays, cofilin-2 also promotes filament assembly rather than disassembly. Taken together, these data suggest that the three biochemically distinct mammalian ADF/cofilin isoforms evolved to fulfill specific requirements for actin filament dynamics in different cell types.

Actin Depolymerizing Factors↗
PubMed2007

Expression of rotavirus NSP4 alters the actin network organization through the actin remodeling protein cofilin.

Rotavirus is a major cause of infantile gastroenteritis with a multifactorial pathogenesis. As with many other pathogens, rotavirus infection and replication leads to rearrangement of the cytoskeleton with disorganization of cytoskeletal elements such as actin and cytokeratin through a calcium-dependent process that has not been fully characterized. The rotavirus enterotoxin NSP4, shown previously to elevate intracellular calcium levels when added exogenously as well as when expressed intracellularly, is a key player in intracellular calcium regulation during rotavirus infection. Here, we investigated the role NSP4 may play in actin rearrangement. Expression of NSP4 fused to enhanced green fluorescent protein (NSP4-EGFP), but not expression of EGFP alone, caused stabilization of long cellular projections in fully confluent HEK 293 cells. Cells expressing NSP4-EGFP for 24 h were also resistant to cell rounding induced by cytochalasin D. Quantification of filamentous actin (F-actin) content by using rhodamine-conjugated phalloidin and flow cytometry showed an elevated F-actin content in NSP4-EGFP-expressing and rotavirus-infected cells in comparison with that in nonexpressing and noninfected cells. Normalization of intracellular calcium levels prevented alterations of F-actin content. Observed changes in F-actin amounts correlated with the increased activation of the actin-remodeling protein cofilin. These calcium-dependent actin rearrangements induced by intracellular NSP4 expression may contribute to rotavirus pathogenesis by interfering with cellular processes dependent on subcortical actin remodeling, including ion transport and viral release.

Actin Depolymerizing Factors↗
PubMed1999

Ischemia activates actin depolymerizing factor: role in proximal tubule microvillar actin alterations.

Apical membrane of renal proximal tubule cells is extremely sensitive to ischemia, with structural alterations occurring within 5 min. These changes are felt secondary to actin cytoskeletal disruption, yet the mechanism responsible is unknown. Actin depolymerizing factor (ADF), a 19-kDa actin-binding protein, has recently been shown to play an important role in regulation of actin filament dynamics. Because ADF is known to mediate pH-dependent F-actin binding, depolymerization, and severing, and because ADF activation occurs by dephosphorylation, we questioned whether ADF played a role in microvilli microfilament disruption during ischemia. To test our hypothesis, we induced renal ischemia in the rat with the clamp model. Initial immunofluorescence and Western blot studies on cortical tissue documented the presence of ADF in proximal tubule cells. Under physiological conditions, ADF was distributed homogeneously throughout the cytoplasm, primarily in the Triton X-100-soluble fraction, and both phosphorylated (pADF) and nonphosphorylated forms were identified. During ischemia, marked alterations occurred. Intraluminal vesicle/bleb structures contained extremely high concentrations of ADF along with G-actin, but not F-actin. Western blot showed a rapidly occurring duration-dependent dephosphorylation of ADF. At 0-30 min of ischemia, total ADF levels were unchanged, whereas pADF decreased significantly to 72% and 19% of control levels, at 5 and 15 min, respectively. Urine collected under physiological conditions did not contain ADF or actin, whereas urine collected after 30 min of ischemia contained both ADF and actin. Reperfusion was associated with normalization of cellular pADF levels, pADF intracellular distribution, and repair of apical microvilli. These data suggest that activation of ADF during ischemia via dephosphorylation is, in part, responsible for apical actin disruption resulting in microvillar destruction and formation of intraluminal vesicles.

Actin Depolymerizing Factors↗
PubMed1997

Low molecular-weight G-actin binding proteins involved in the regulation of actin assembly during myofibrillogenesis.

We previously demonstrated that small G-actin binding proteins, cofilin, ADF and profilin, are involved in the actin dynamics during myofibrillogenesis (OBINATA, T. (1993). Int. Rev. Cytol., 143: 153-189.). To better understand how they are responsible for the regulation of actin assembly, the amounts of the actin-binding proteins were quantified by means of quantitative immunoblotting and compared with that of G-actin pool. The sum of the amounts of cofilin, ADF and profilin was insufficient at early developmental stages but sufficient at later stages to account for the pool of G-actin in muscle cells. We detected expression of thymosin beta 4 at a considerable level in young embryonic but not in adult skeletal muscles. We, therefore, conclude that the G-actin pool in young embryonic skeletal muscle is mainly due to cofilin, ADF, profilin and thymosin beta 4. Switching from a non-muscle-type (NM-) cofilin to a muscle-type (M-) cofilin was observed during muscle development of mammals. In order to clarify cofilin-dependent regulation of actin assembly in muscle cells, cofilin tagged with fluorescence dyes was introduced into C2 myoblasts by a micro injection method. The exogeneous cofilin, but not ADF, caused quick disassembly of actin filaments and accumulated in furrow region of dividing cells. The analogs of the unphosphorylated form (A3-cofilin) and the phosphorylated form (D3-cofilin) were prepared by converting Ser3, a regulatory phosphorylation site, to Ala or Asp. When A3-cofilin and D3-cofilin were injected into living cells, the former was concentrated at the membrane ruffles and cleavage furrow, while the latter showed only diffuse distribution in the cytoplasm. These results suggest that the subcellular distribution of cofilin as well as its interaction with actin in vivo is regulated by its phosphorylation and dephosphorylation.

Actin Depolymerizing Factors↗
PubMed1991

Inhibition of actin polymerization by a synthetic dodecapeptide patterned on the sequence around the actin-binding site of cofilin.

Cofilin is an F-actin side-binding and -depolymerizing protein with an apparent molecular mass of 21 kDa. By means of the end label fingerprinting method, the amino acid residue on cofilin sequence cross-linked to actin by zero length cross-linker, 1-ethyl-3-(3-dimethylamino propyl)carbodiimide, was identified as Lys112 and/or Lys114. A synthetic dodecapeptide patterned on the sequence around the actin-cross-linking site of cofilin (Trp104-Met115) inhibited the binding of cofilin to actin. Moreover, the dodecapeptide was found to be a potent inhibitor of actin polymerization. Thus, we conclude that the dodecapeptide sequence constitutes the region essential for the actin-binding and -depolymerizing activity of cofilin. A sequence similar to the dodecapeptide is found in other actin-depolymerizing proteins, destrin, actin-depolymerizing factor, and depactin. Therefore, the dodecapeptide sequence may be a consensus sequence essential for actin-binding and -depolymerizing activity in actin-depolymerizing proteins.

Actin Depolymerizing Factors↗
PubMed1999

New anti-actin drugs in the study of the organization and function of the actin cytoskeleton.

The high degree of structural and molecular complexity of the actin-based cytoskeleton, combined with its ability to reorganize rapidly and locally in response to stimuli, and its force-generating properties, have made it difficult to assess how the different actin structures are assembled in cells, and how they regulate cell behavior. An obvious approach to study the relationships between actin organization, dynamics, and functions is the specific perturbation of actin structures using pharmacological means. Until recently there were only a few agents available that interfered with cellular activities by binding to actin and most of our knowledge concerning the involvement of actin in basic cellular processes was based on the extensive use of the cytochalasins. In recent years we have identified an increasing number of actin-targeted marine natural products, including the latrunculins, jasplakinolides (jaspamides), swinholide A, misakinolide A, halichondramides, and pectenotoxin II, which are discussed in this article. All these marine-sponge-derived compounds are unusual macrolides and can be classified into several major families, each with its own distinct chemical structures. We describe the current state of knowledge concerning the actin-binding properties of these compounds and show that each class of drugs alters the distribution patterns of actin in a unique way, and that even within a chemical class, structurally similar compounds can have different biochemical properties and cellular effects. We also discuss the effects of these new drugs on fenestrae formation in liver endothelial cells as an example of their usefulness as powerful tools to selectively unmask actin-mediated dynamic processes.

Actins↗
PubMed1996

Viscoelastic properties of F-actin solutions in the presence of normal and mutated actin-binding proteins.

A minimal level of viscoelasticity in the cytoskeleton is an essential prerequisite of cellular motility. To determine the influence of the F-actin crosslinking proteins alpha-actinin and actin-binding protein (ABP)120 gelation factor from Dictyostelium discoideum on the properties of actin gels we used a torsion pendulum to measure directly viscoelastic changes of the filamentous networks. Using the capping proteins severin and DS151 to control actin filament length, both crosslinkers were found to increase the elasticity and the viscosity of F-actin solutions. In the case of alpha-actinin, this activity was completely blocked by micromolar concentrations of Ca2+. The inhibitory functions of the two EF hands of alpha-actinin were further investigated by introducing point mutations into either one or both of the Ca(2+)-binding regions. Mutations in the Ca(2+)-coordinating amino acid residues in the first or in both EF hands left the dynamic storage and loss moduli of the F-actin solution unaltered, independent of the Ca2+ concentration. However, alpha-actinin mutated in the second EF hand increased the viscoelasticity of actin gels like the wild-type protein in the absence of Fa2+. The ABP120 gelation factor exhibited only negligible differences to alpha-actinin in viscometry measurements, whereas its impact on the ratio G"/G' (the ratio of energy lost compared to elastically stored during a deformation) of F-actin solutions was clearly smaller than that of alpha-actinin. We conclude from these data that: (i) a torsion pendulum is an excellent tool to determine small changes of activity in normal and mutated actin-binding proteins, (ii) the first EF hand of alpha-actinin is crucial for its crosslinking function, and (iii) the viscoelastic properties of F-actin gels crosslinked by either alpha-actinin or the ABP120 gelation factor are different.

Actinin↗
PubMed1981

Crystalline actin tubes. III. The interaction of scandium and yttrium with skeletal muscle actin.

The effect of the trivalent cations scandium (Sc3+) and yttrium (Y3+) on the conformation of G-actin was examined using ultraviolet difference and high resolution 1H-NMR spectroscopy. A comparison was made with data obtained previously with the trivalent lanthanide cations (Ln3+). These results indicate that the first and subsequent Y ions (ionic radius 101.9 pm) behave exactly like Ln3+. Sc3+ is a smaller ion (87 pm) than any of the Ln3+. The first Sc3+ binds to a site on actin that is inaccessible to Mg2+, Y3+ and Ln3+. However, the second Sc3+ to bind behaves like an Ln3+. On replacing the native divalent cation (Mg2+), both Y3+ and Sc3+ mobilize the adenine ring of ATP bound to actin, thus exposing underlying residues to the solvent. When Y3+ and Sc3+ saturate their binding sites on actin, and when the ionic strength is raised to 0.1 M with KCl at pH 6.9, the actin aggregates. Y3+ binds to actin with a ratio of 6 : 1 and induces the aggregation of actin into crystalline actin tubes, whilst Sc3+ binds with a ratio of 8 : 1 and induces amorphous actin aggregates. These results are consistent with the suggestion that actin tubes are induced by trivalent cations, principally on the basis of their binding stoichiometry, which is determined by ionic radius.

Actins↗
PubMed1988

Comparison of the structure of myosin subfragment 1 bound to actin and free in solution. A neutron scattering study using actin made "invisible" by deuteration.

The structure of subfragment 1 (S1) bound to F-actin has been compared to the structure of free S1 using neutron scattering. The F-actin was rendered "invisible" to neutrons by selective deuteration and solvent contrast matching. Highly deuterated actin was purified from the slime mold Dictyostelium discoideum, which was fed deuterated Escherichia coli. The properties of this actin were found to be similar to those of protonated actin. The neutron-scattering pattern of S1 bound to this "invisible" actin was compared to that of free S1. At near-physiological ionic strength, a strong interference effect was observed, which arose from pairs of S1 molecules cross-linking actin filaments. However, at low ionic strength the only differences that could be observed were attributed to interference effects between neutrons scattered from S1s bound randomly to equivalent sites on an actin filament. These effects became negligible as the fraction of actin sites occupied by S1 approached zero. Thus, we conclude that the scattering by S1 attached to F-actin is identical with that of free S1, to a resolution of about 2.5 nm. The difference in apparent radii of gyration is less than 0.05 nm. Modeling calculations have been carried out to determine the sensitivity of neutron scattering to possible S1 deformations. The calculations showed that deformations of the structure of S1 that are large enough ultimately to produce a powerstroke of 5 nm or greater are only consistent with the data if they involve at most about 20% of the S1 mass. These results restrict the class of plausible models describing force generation in muscle contraction.

Actins↗
PubMed1994

Polymerization of actin induced by actin-binding fragments of caldesmon.

Our earlier studies revealed that caldesmon causes assembly of G-actin into polymers morphologically indistinguishable from those formed in the presence of salt (Gałazkiewicz, B., Belagyi, J. and Dabrowska, R. (1989) Eur. J. Biochem. 181, 607-614). In this work we have investigated the effect of actin-binding fragments of caldesmon on actin polymerization process followed by measurements of the changes in fluorescence of pyrenyl conjugated with G-actin and ATP hydrolysis. The results indicate that C-terminal 34 kDa fragment of caldesmon containing two actin-binding sites and 19 kDa containing high-affinity binding site have similar capability to polymerize actin to that of intact molecule. Binding of each of these fragments to G-actin causes bypassing of nucleation phase. The 11.5 kDa fragment comprising low affinity actin-binding site has much lower potency to polymerize actin. Conformation of actin monomers in filaments formed upon 19 kDa fragment and that formed upon 11.5 kDa fragment differs. The former fragment seems to resemble more conformation of monomers in filaments formed upon intact caldesmon than the latter one.

Actins↗
PubMed2000

Multiple-particle tracking measurements of heterogeneities in solutions of actin filaments and actin bundles.

One of the central functions of actin cytoskeleton is to provide the mechanical support required for the establishment and maintenance of cell morphology. The mechanical properties of actin filament assemblies are a consequence of both the available polymer concentration and the actin regulatory proteins that direct the formation of higher order structures. By monitoring the displacement of well-dispersed microspheres via fluorescence microscopy, we probe the degree of spatial heterogeneity of F-actin gels and networks in vitro. We compare the distribution of the time-dependent mean-square displacement (MSD) of polystyrene microspheres imbedded in low- and high-concentration F-actin solutions, in the presence and absence of the F-actin-bundling protein fascin. The MSD distribution of a 2. 6-microM F-actin solution is symmetric and its standard deviation is similar to that of a homogeneous solution of glycerol of similar zero-shear viscosity. However, increasing actin concentration renders the MSD distribution wide and asymmetric, an effect enhanced by fascin. Quantitative changes in the shape of the MSD distribution correlate qualitatively with the presence of large heterogeneities in F-actin solutions produced by increased filament concentration and the presence of actin bundles, as detected by confocal microscopy. Multiple-particle tracking offers a new, quantitative method to characterize the organization of biopolymers in solution.

Actins↗
PubMed1994

Formation of two-dimensional complexes of F-actin and crosslinking proteins on lipid monolayers: demonstration of unipolar alpha-actinin-F-actin crosslinking.

A method is described for forming two-dimensional (2-D) paracrystalline complexes of F-actin and bundling/gelation proteins on positively charged lipid monolayers. These arrays facilitate detailed structural studies of protein interactions with F-actin by eliminating superposition effects present in 3-D bundles. Bundles of F-actin have been produced using the glycolytic enzymes aldolase and glyceraldehyde-3-phosphate dehydrogenase, the cytoskeletal protein erythrocyte adducin as well as smooth muscle alpha-actinin from chicken gizzard. All of the 2-D bundles formed contain F-actin with a 13/6 helical structure. F-actin-aldolase bundles have an interfilament spacing of 12.6 nm and a superlattice arrangement of actin filaments that can be explained by expression of a local twofold axis in the neighborhood of the aldolase. Well ordered F-actin-alpha-actinin 2-D bundles have an interfilament spacing of 36 nm and contain crosslinks 33 nm in length angled approximately 25-35 degrees to the filament axis. Images and optical diffraction patterns of these bundles suggest that they consist of parallel, unipolar arrays of actin filaments. This observation is consistent with an actin crosslinking function at adhesion plaques where actin filaments are bound to the cell membrane with uniform polarity.

Actinin↗
PubMed1998

Use of a fusion protein between GFP and an actin-binding domain to visualize transient filamentous-actin structures.

Many important processes in eukaryotic cells involve changes in the quantity, location and the organization of actin filaments [1] [2] [3]. We have been able to visualize these changes in live cells using a fusion protein (GFP-ABD) comprising the green fluorescent protein (GFP) of Aequorea victoria and the 25 kDa highly conserved actin-binding domain (ABD) from the amino terminus of the actin cross-linking protein ABP-120 [4]. In live cells of the soil amoeba Dictyostelium that were expressing GFP-ABD, the three-dimensional architecture of the actin cortex was clearly visualized. The pattern of GFP-ABD fluorescence in these cells coincided with that of rhodamine-phalloidin, indicating that GFP-ABD specifically binds filamentous (F) actin. On the ventral surface of non-polarized vegetative cells, a broad ring of F actin periodically assembled and contracted, whereas in polarized cells there were transient punctate F-actin structures; cells cycled between the polarized and non-polarized morphologies. During the formation of pseudopods, an increase in fluorescence intensity coincided with the initial outward deformation of the membrane. This is consistent with the models of pseudopod extension that predict an increase in the local density of actin filaments. In conclusion, GFP-ABD specifically binds F actin and allows the visualization of F-actin dynamics and cellular behavior simultaneously.

Actins↗
PubMed2001

C-terminal actin-binding sites of smooth muscle caldesmon switch actin between conformational states.

Caldesmon is a component of the thin filaments of smooth muscles where it is believed to play an essential role in regulating the thin filaments' interaction with myosin and hence contractility. We studied the effects of caldesmon and two recombinant fragments CaDH1 (residues 506-793) and CaDH2 (residues 683-767) on the structure of actin-tropomyosin by making measurements of the fluorescence polarisation of probes specifically attached to actin. CaDH1, like the parent molecule caldesmon, is an inhibitor of actin-tropomyosin interaction with myosin whilst CaDH2 is an activator. The F-actin in permeabilised and myosin free rabbit skeletal muscle 'ghost' fibres was labelled by tetramethyl rhodamine-isothiocyanate (TRITC)-phalloidin or fluorescein-5'-isothiocyanate (FITC) at lysine 61. Fluorescence polarisation measurements were made and the parameters Phi(A), Phi(E), Theta(1/2) and Nu were calculated. Phi(A) and Phi(E) are angles between the fiber axis and the absorption and emission dipoles, respectively; Theta(1/2) is the angle between the F-actin filament axis and the fiber axis; Nu is the relative number of randomly oriented fluorophores. Actin-tropomyosin interaction with myosin subfragment-1 induced changes in the parameters of the polarised fluorescence that are typical of strong binding of myosin to actin and of the 'on' conformational state of actin. Caldesmon and CaDH1 (as well as troponin in the absence of Ca(2+)) diminished the effect of S-1, whereas CaDH2 (as well as troponin in the presence of Ca(2+)) enhanced the effect of S1. Thus the structural evidence correlates with biochemical evidence that C-terminal actin-binding sites of caldesmon can modulate the structural transition of actin monomers between 'off' (caldesmon and CaDH1) and 'on' (S-1 and CaDH2) states in a manner analogous to troponin.

Actins↗
PubMed1991

Nucleotide-induced changes in the interaction of myosin subfragment 1 with actin: detection by antibodies against the N-terminal segment of actin.

The binding of myosin subfragment 1 (S-1) to actin in the presence and absence of nucleotides was determined under conditions of partial saturation of actin, up to 80%, by Fab(1-7), the antibodies against the first seven N-terminal residues on actin. In the absence of nucleotides, the binding constant of S-1 to actin (2 x 10(7) M-1) was decreased by 1 order of magnitude by Fab(1-7). The binding of S-1 to actin caused only limited displacement of Fab, and between 30 and 50% of actin appeared to bind both proteins. In the presence of MgAMP.PNP, MgADP, and MgPPi and at low S-1 concentrations, the same antibodies caused a large decrease in the binding of S-1 to actin. However, the binding of S-1.nucleotide to actin in the presence of Fab(1-7) increased cooperatively with the increase in S-1 concentration. Also, in contrast to rigor conditions, there was no indication for the binding of Fab(1-7) and S-1.nucleotide to the same actin molecules. These results show a nucleotide-induced transition in the actomyosin interface, most likely related to the different roles of the N-terminal segment of actin in the binding of S-1 and S-1.nucleotide. The possible implications of these findings to the regulation of actomyosin interactions are discussed.

Actins↗
PubMed1991

Cysteine-374 of actin resides at the gelsolin contact site in the EGTA-resistant actin-gelsolin complex.

The interaction of pig plasma gelsolin (G) and actin (A) was examined by using photoreactive 4-maleimidobenzophenone-actin (BPM-actin) in which BPM was previously conjugated to Cys-374 of actin through the maleimide moiety. In the presence of micromolar [Ca2+], the major cross-linked product observed after irradiation of the mixture of gelsolin (82 kDa) and actin (42 kDa) had an apparent molecular mass of 130 kDa although gelsolin predominantly existed in the form of an A2G complex (170 kDa). No cross-linked product was detected in the absence of Ca2+. BPM-actin itself did not give any cross-linked product. By use of fluorescent-labeled gelsolin, the cross-linked 130 kDa was shown to be an AG complex. The cross-linked complex was also formed from the A2G complex after removal of Ca2+ by [ethylenebis-(oxyethylenenitrilo)]tetraacetic acid (EGTA) followed by irradiation, indicating that it was the EGTA-resistant AG complex that was cross-linked. The results show that Cys-374 at the C-terminal segment of actin in the EGTA-resistant AG complex is 9-10 A apart from gelsolin. Furthermore, it was shown that the EGTA-resistant actin molecule once incorporated in the A2G complex did not exchange with free actin in the presence of Ca2+. This was also supported by the effect of phosphatidylinositol 4,5-bisphosphate, which did not dissociate the EGTA-resistant actin molecule from the A2G complex in the presence of Ca2+, but did after removal of Ca2+.

Actins↗