Cytochemical dopamine visualization as a method for estimation of globular actin content in cytosol of living cells

The possibility to reveal globular (G-) actin in cell cytosole by means of microscopy has been studied. The applicability of this method was in particular evaluated for diagnostics of malignant cells, whose main pathocytological feature is an anomalously high content of G-actin in cytosol. The cells of a common origin but with different states of cytosolic actin were analyzed by means of cytochemical reaction for biogenic amines using Falck-Hillarp method after 40-h incubation of the cells in dopamine-containing cultivation medium. Mouse embryo cell line BALB/3T3, clone A31 with differentiated actin cytoskeleton were used as a control cell line. The same cells infected with pathogenic virus SV-40 (cell line 3T3B-SV40) exhibited a malignant phenotype; their cytosol mainly consisted of G-actin. Manifold increase in fluorescence intensity of cytosol and karyoplasms, the loci with the highest G-actin concentration, was revealed in malignant cells in comparison with their healthy prototype. Thus, it was shown that G-actin of malignant cells is a diagnostic target for dopamine, which, as it was earlier shown, penetrates into the cytosol, polymerizes G-actin, incorporating into the filaments as integral component in the 100:1 ratio, and thus fluorescently labels G-actin due to conversion into isoquinoline by reaction with formaldehyde. Besides, dopamine exhibited a strong cytotoxicity that considerably reduced the viability of malignant cells. The data suggest that the content of Gactin in cytosol of living cells can be quantitatively estimated by fluorescence intensity of cytosol following incubation of the cells in dopamine-containing medium.     

Dopamine as a possible substance for oncotherapy and for quantitative valuation of cytosolic G-Actin

Viability, histology and ultrastructure of normal cells and cells of different degrees of malignancy after interaction with dopamine as well as the ability of these cells and isolated G-actin in model experiments to stain by Falck technique were studied. It is shown that dopamine, virtually having no effect on the viability of the “normal” non-tumorigenic transformed cells, noticeably reduces cell viability of slightly tumorigenic cells, causes a significant reduction in viability of attachable cancerous cells and a very significant decrease in cell viability of cancerous cells growing in suspension. The intensity of fluorescence of the cytosol in cells treated with dopamine, has been very high and varied in different cultures, and that of isolated actin directly depended on its concentration. Common to all cell morphological feature of damage from the action of dopamine and the putative substrate of fluorescence was actodopamine filaments network strands (identified on the structure and size), which appears in the cytosol loci, where they were absent in control. The data show that dopamine can be used as an oncotherapeutic remedy and diagnostic tool interacting with G-actin as a cellular target.     

Cytochalasin D acts as an inhibitor of the actin-cofilin interaction

Cofilin, a key regulator of actin filament dynamics, binds to G- and F-actin and promotes actin filament turnover by stimulating depolymerization and severance of actin filaments. In this study, cytochalasin D (CytoD), a widely used inhibitor of actin dynamics, was found to act as an inhibitor of the G-actin-cofilin interaction by binding to G-actin. CytoD also inhibited the binding of cofilin to F-actin and decreased the rate of both actin polymerization and depolymerization in living cells. CytoD altered cellular F-actin organization but did not induce net actin polymerization or depolymerization. These results suggest that CytoD inhibits actin filament dynamics in cells via multiple mechanisms, including the well-known barbed-end capping mechanism and as shown in this study, the inhibition of G- and F-actin binding to cofilin.     

Probing GFP-actin diffusion in living cells using fluorescence correlation spectroscopy

The cytoskeleton of eukaryotic cells is continuously remodeled by polymerization and depolymerization of actin. Consequently, the relative content of polymerized filamentous actin (F-actin) and monomeric globular actin (G-actin) is subject to temporal and spatial fluctuations. Since fluorescence correlation spectroscopy (FCS) can measure the diffusion of fluorescently labeled actin it seems likely that FCS allows us to determine the dynamics and hence indirectly the structural properties of the cytoskeleton components with high spatial resolution. To this end we investigate the FCS signal of GFP-actin in living Dictyostelium discoideum cells and explore the inherent spatial and temporal signatures of the actin cytoskeleton. Using the free green fluorescent protein (GFP) as a reference, we find that actin diffusion inside cells is dominated by G-actin and slower than diffusion in diluted cell extract. The FCS signal in the dense cortical F-actin network near the cell membrane is probed using the cytoskeleton protein LIM and is found to be slower than cytosolic G-actin diffusion. Furthermore, we show that polymerization of the cytoskeleton induced by Jasplakinolide leads to a substantial decrease of G-actin diffusion. Pronounced fluctuations in the distribution of the FCS correlation curves can be induced by latrunculin, which is known to induce actin waves. Our work suggests that the FCS signal of GFP-actin in combination with scanning or spatial correlation techniques yield valuable information about the local dynamics and concomitant cytoskeletal properties.     

Inhibition of sliding movement of F-actin by crosslinking emphasizes the role of actin structure in the mechanism of motility

The effects of crosslinking of monomeric and polymeric actin with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), disuccinimidyl suberate (DSS) and glutaraldehyde on the interaction with heavy meromyosin (HMM) in solution and on the sliding movement on glass-attached HMM were examined. The Vmax values of actin-activated HMM ATPase decreased in the following order: intact actin = EDC F-actin greater than DSS actin greater than glutaraldehyde F-actin = glutaraldehyde G-actin greater than EDC G-actin. The affinity of actin for HMM in the presence of ATP decreased in the following order: DSS actin greater than glutaraldehyde F-actin = glutaraldehyde G-actin greater than intact actin greater than EDC F-actin greater than EDC G-actin. However, sliding movement was inhibited only in the case of glutaraldehyde-crosslinked F and G-actin and EDC-crosslinked G-actin. Interestingly, after copolymerization of "non-motile" glutaraldehyde or EDC-crosslinked monomers with "motile" monomers of intact actin sliding of the copolymers was observed and its rate was independent of the type of crosslinked monomer, i.e. of the manner of their interaction with HMM. These data strongly indicate that inhibition of the sliding of actin by crosslinking cannot be explained entirely by changes in the Vmax value or affinity for myosin heads. We conclude that movement is generated by interaction of myosin with segments of F-actin containing a number of intact monomers, and the mechanism of inhibition involves an effect of the crosslinkers on the structure of F-actin itself.     

Measurements of spatiotemporal changes in G-actin concentration reveal its effect on stimulus-induced actin assembly and lamellipodium extension

To understand the intracellular role of G-actin concentration in stimulus-induced actin assembly and lamellipodium extension during cell migration, we developed a novel technique for quantifying spatiotemporal changes in G-actin concentration in live cells, consisting of sequential measurements of fluorescent decay after photoactivation (FDAP) of Dronpa-labeled actin. Cytoplasmic G-actin concentrations decreased by ～40% immediately after cell stimulation and thereafter the cell area extended. The extent of stimulus-induced G-actin loss and cell extension correlated linearly with G-actin concentration in unstimulated cells, even at concentrations much higher than the critical concentration of actin filaments, indicating that cytoplasmic G-actin concentration is a critical parameter for determining the extent of stimulus-induced G-actin assembly and cell extension. Multipoint FDAP analysis revealed that G-actin concentration in lamellipodia was comparable to that in the cell body. We also assessed the cellular concentrations of free G-actin, profilin- and thymosin-β4-bound G-actin, and free barbed and pointed ends of actin filaments by model fitting of jasplakinolide-induced temporal changes in G-actin concentration.     

Actin protein inside DMPC GUVs and its mechanical response to AC electric fields

Cells are dynamic systems with complex mechanical properties, regulated by the presence of different species of proteins capable to assemble (and disassemble) into filamentous forms as required by different cells functions. Giant unilamellar vesicles (GUVs) of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) are systems frequently used as a simplified model of cells because they offer the possibility of assaying separately different stimuli, which is no possible in living cells. Here we present a study of the effect of acting protein on mechanical properties of GUVs, when the protein is inside the vesicles in either monomeric G-actin or filamentous F-actin. For this, rabbit skeletal muscle G-actin is introduced inside GUVs by the electroformation method. Protein polymerization inside the GUVs is promoted by adding to the solution MgCl₂ and the ion carrier A23187 to allow the transport of Mg⁺² ions into the GUVs. To determine how the presence of actin changes the mechanical properties of GUVs, the vesicles are deformed by the application of an AC electric field in both cases with G-actin and with polymerized F-actin. The changes in shape of the vesicles are characterized by optical microscopy and from them the bending stiffness of the membrane are determined. It is found that G-actin has no appreciable effect on the bending stiffness of DMPC GUVs, but the polymerized actin makes the vesicles more rigid and therefore more resistant to deformations. This result is supported by evidence that actin filaments tend to accumulate near the membrane.     

Visualization of F-actin and G-actin equilibrium using fluorescence resonance energy transfer (FRET) in cultured cells and neurons in slices

The plasticity of excitatory synapses has conventionally been studied from a functional perspective. Recent advances in neuronal imaging techniques have made it possible to study another aspect, the plasticity of the synaptic structure. This takes place at the dendritic spines, where most excitatory synapses are located. Actin is the most abundant cytoskeletal component in dendritic spines, and thus the most plausible site of regulation. The mechanism by which actin is regulated has not been characterized because of the lack of a specific method for detection of the polymerization status of actin in such a small subcellular structure. Here we describe an optical approach that allows us to monitor F-actin and G-actin equilibrium in living cells through the use of two-photon microscopy to observe fluorescence resonance energy transfer (FRET) between actin monomers. Our protocol provides the first direct method for looking at the dynamic equilibrium between F-actin and G-actin in intact cells.     

Hyper-mobility of water around actin filaments revealed using pulse-field gradient spin-echo H NMR and fluorescence spectroscopy

This paper reports that water molecules around F-actin, a polymerized form of actin, are more mobile than those around G-actin or in bulk water. A measurement using pulse-field gradient spin-echo ¹H NMR showed that the self-diffusion coefficient of water in aqueous F-actin solution increased with actin concentration by ∼5%, whereas that in G-actin solution was close to that of pure water. This indicates that an F-actin/water interaction is responsible for the high self-diffusion of water. The local viscosity around actin was also investigated by fluorescence measurements of Cy3, a fluorescent dye, conjugated to Cys 374 of actin. The steady-state fluorescence anisotropy of Cy3 attached to F-actin was 0.270, which was lower than that for G-actin, 0.334. Taking into account the fluorescence lifetimes of the Cy3 bound to actin, their rotational correlation times were estimated to be 3.8 and 9.1 ns for F- and G-actin, respectively. This indicates that Cy3 bound to F-actin rotates more freely than that bound to G-actin, and therefore the local water viscosity is lower around F-actin than around G-actin.     

Hyper-mobility of water around actin filaments revealed using pulse-field gradient spin-echo 1H NMR and fluorescence spectroscopy

This paper reports that water molecules around F-actin, a polymerized form of actin, are more mobile than those around G-actin or in bulk water. A measurement using pulse-field gradient spin-echo (1)H NMR showed that the self-diffusion coefficient of water in aqueous F-actin solution increased with actin concentration by ～5%, whereas that in G-actin solution was close to that of pure water. This indicates that an F-actin/water interaction is responsible for the high self-diffusion of water. The local viscosity around actin was also investigated by fluorescence measurements of Cy3, a fluorescent dye, conjugated to Cys 374 of actin. The steady-state fluorescence anisotropy of Cy3 attached to F-actin was 0.270, which was lower than that for G-actin, 0.334. Taking into account the fluorescence lifetimes of the Cy3 bound to actin, their rotational correlation times were estimated to be 3.8 and 9.1ns for F- and G-actin, respectively. This indicates that Cy3 bound to F-actin rotates more freely than that bound to G-actin, and therefore the local water viscosity is lower around F-actin than around G-actin.     

The quantitation of G- and F-actin in cultured cells

An improved method to quantitate the amounts of filamentous (F-actin) and monomeric (globular) actin (G-actin) in cultured cells was developed. Cells are lysed into a myosin-containing buffer and F-actin is removed by centrifugation. The pelleted F-actin is then depolymerized to G-actin in a 1 mM ATP-containing buffer for 1 h before measuring the levels of G-actin using the DNase I inhibition assay. Partitioning of G-actin in the supernatant (greater than 95%) and recovery of actin in both fractions (greater than 85%) were measured by adding [3H]actin to cultured cells. Actin in the separated fractions is stable for at least 72 h at 0 degree C. Asynchronous monolayer cultures of Chinese hamster ovary (CHO) cells contain 2.5 +/- 0.2% of the total protein as actin with 72.4 +/- 5.7% as F-actin. About 10% of this F-actin is not associated with the readily sedimented Triton-cytoskeleton. CHO cells grown in suspension contain 55.8% of the actin as F-actin; following plating about 90 min is required for these cells to flatten and for the F-actin level to reach the monolayer value of about 70%.     

Localization and dynamics of nonfilamentous actin in cultured cells [published erratum appears in J Cell Biol 1993 Nov;123(3):following 767]

Although the distribution of filamentous actin is well characterized in many cell types, the distribution of nonfilamentous actin remains poorly understood. To determine the relative distribution of filamentous and nonfilamentous actin in cultured NRK cells, we have used a number of labeling agents that differ with respect to their specificities toward the filamentous or nonfilamentous form, including monoclonal and polyclonal anti-actin antibodies, vitamin D-binding protein (DBP), and fluorescent phalloidin. Numerous punctate structures were identified that bind poorly to phalloidin but stain positively with several anti-actin antibodies. These bead structures also stain with DBP, suggesting that they are enriched in nonfilamentous actin. Similar punctate structures were observed after the microinjection of fluorescently labeled actin into living cells, allowing us to examine their dynamics in living cells. The actin-containing punctate structures were observed predominantly in the region behind lamellipodia, particularly in spreading cells induced by wounding confluent monolayers. Time-lapse recording of cells injected with fluorescent actin indicated that they form continuously near the leading edge and move centripetally toward the nucleus. Our results suggest that at least part of the unpolymerized actin molecules are localized at discrete sites, possibly as complexes with monomer sequestering proteins. These structures may represent transient storage sites of G-actin within the cell which can be transformed rapidly into actin filaments upon stimulation by specific signals.     

Interactions between G-actin and myosin subfragment 1: immunochemical probing of the NH2-terminal segment on actin

The role of the N-terminal segment of actin in myosin-induced polymerization of G-actin was studied by using peptide antibodies directed against the first seven N-terminal residues of alpha-skeletal actin. Light scattering, fluorescence, and analytical ultracentrifugation experiments showed that the Fab fragments of these antibodies inhibited the polymerization of G-actin by myosin subfragment 1 (S-1) by inhibiting the binding of these proteins to each other. Fluorescence measurements using actin labeled with pyrenyliodoacetamide revealed that Fab inhibited the initial step in the binding of S-1 to G-actin. It is deduced from these results and from other literature data that the initial contact between G-actin and S-1 involves residues 1-7 on actin and residues 633-642 on the S-1 heavy chain. This interaction appears to be of major importance for the binding of S-1 and G-actin. The presence of additional myosin contact sites on G-actin was indicated by concentration-dependent recovery of S-1 binding to G-actin without displacement of Fab. The reduced Fab inhibition of S-1 binding to polymerizing and polymerized actin is consistent with the tightening of acto-S-1 binding at these sites or the creation of new sites upon formation of F-actin.     

Ecdysterone induction of actin synthesis and polymerization in a Drosophila melanogaster cultured cell line

Actin pools have been evaluated in Drosophila melanogaster Kc 0% cells, through an actin assay based on differential inhibition of DNase I by globular (G) and filamentous (F) actin. Total actin represents about 4 % of total proteins and 54 % is G-actin. In ecdysterone treated cells (0.1 μM), the total actin content increases up to 9 % of total proteins after 3 days of treatment. Ecdysterone induces increase of G-actin as well as F-actin. Increase of both actins, detectable after only 24 hrs of treatment, is roughly parallel during the first two days of treatment. For longer hormonal treatment, actin polymerization is more important than accumulation of G-actin. Indirect immunofluorescence microscopy with antibodies to exogeneous DNase I suggests that actin is widely distributed in the whole cytoplasm before and after ecdysterone treatment. These results suggest that ecdysterone induces actin synthesis and polymerization in Drosophila melanogaster cells.     

Decreased G-actin content in cells at injury

The content of G-actin was measured in aqueous extracts of rat macrophages and human lymphocytes at hyperthermia and hypoxia. The G-actin content in altered cells decreases the greater the stronger the injury. This decrease correlates well with the data on visual estimation of cell light diffusing. A possible contribution of actin polymerization and its interaction with proteins in the colloidal reactions of altered protoplasm is discussed.     

Ca2+-calmodulin-dependent polymerization of actin by myelin basic protein

The interaction between myelin basic protein (MBP) and G-actin was studied under nonpolymerizing conditions, i.e.,2mM HEPES, pH 7.5, 0.1 mM CaCl2 and 0.2 mM ATP. Fluorescence studies using pyrenyl-actin and the measurements of ATP hydrolysis rate show that MBP induces changes in the structure of the actin monomer similar to those occurring during polymerization by salt. Electron microscope observations of the MBP-G-actin complex reveal the presence of filamentous structures which appear as separate filaments or as bundles of filaments in lateral association. These filaments are polar as visualized by attachment of heavy meromyosin. The biochemical data together with electron microscope observations suggest that the binding of MBP to G-actin under non-polymerizing conditions induces an interaction between actin monomers leading to the formation of filamentous structures which may be similar to F-actin filaments. The effects of MBP on G-actin can be reversed by calmodulin in the presence of Ca2+.     

Arabidopsis Profilin Isoforms, PRF1 and PRF2Show Distinctive Binding Activities and Subcellular Distributions

Profilin is an actin-binding protein that shows complex effects on the dynamics of the actin cytoskeleton. There are five profilin isoforms in Arabidopsis thaliana L. However, it is still an open question whether these isoforms are functionally different. In the present study, two profilin isoforms from Arabidopsis, PRF1 and PRF2 were fused with green fluorescent protein (GFP) tag and expressed in Escherichia coli and A. thaliana in order to compare their biochemical properties in vitro and their cellular distributions in vivo. Biochemical analysis revealed that fusion proteins of GFP-PRF1 and GFP-PRF2 can bind to poly-L-proline and G-actin showing remarkable differences. GFP-PRF1 has much higher affinities for both poly-L-proline and G-actin compared with GFP-PRF2. Observations of living cells in stable transgsnic A. thaliana lines revealed that 35S::GFP-PRF1 formed a filamentous network, while 35S::GFP-PRF2 formed polygonal meshes. Results from the treatment with latrunculin A and a subsequent recovery experiment indicated that filamentous alignment of GFP-PRF1 was likely associated with actin filaments. However, GFP-PRF2 localized to polygonal meshes resembling the endoplasmic reticulum. Our results provide evidence that Arabidopsis profllin isoforms PRF1 and PRF2 have different biochemical affinities for poly-L-proline and G-actin, and show distinctive Iocalizations in living cells. These data suggest that PRF1 and PRF2 are functionally different isoforms.     

Control of actin dynamics by proteins made of beta-thymosin repeats: the actobindin family

Actobindin is an actin-binding protein from amoeba, which consists of two beta-thymosin repeats and has been shown to inhibit actin polymerization by sequestering G-actin and by stabilizing actin dimers. Here we show that actobindin has the same biochemical properties as the Drosophila or Caenorhabditis elegans homologous protein that consists of three beta-thymosin repeats. These proteins define a new family of actin-binding proteins. They bind G-actin in a 1:1 complex with thermodynamic and kinetic parameters similar to beta-thymosins. Like beta-thymosins, they slow down nucleotide exchange on G-actin and make a ternary complex with G-actin and Latrunculin A. On the other hand, they behave as functional homologs of profilin because their complex with MgATP-G-actin, unlike beta-thymosin-actin, participates in filament barbed end growth, like profilin-actin complex. Therefore these proteins play an active role in actin-based motility processes. In addition, proteins of the actobindin family interact with the pointed end of actin filaments and inhibit pointed end growth, maybe via the interaction of the beta-thymosin repeats with two terminal subunits.     

Interaction of the small heat shock protein with molecular mass 25 kDa (hsp25) with actin.

The interaction of heat shock protein with molecular mass 25 kDa (HSP25) and its point mutants S77D + S81D (2D mutant) and S15D + S77D + S81D (3D mutant) with intact and thermally denatured actin was analyzed by means of fluorescence spectroscopy and ultracentrifugation. Wild type HSP25 did not affect the polymerization of intact actin. The HSP25 3D mutant decreased the initial rate without affecting the maximal extent of intact actin polymerization. G-actin heated at 40-45 degrees C was partially denatured, but retained its ability to polymerize. The wild type HSP25 did not affect polymerization of this partially denatured actin. The 3D mutant of HSP25 increased the initial rate of polymerization of partially denatured actin. Heating at more than 55 degrees C induced complete denaturation of G-actin. Completely denatured G-actin cannot polymerize, but it aggregates at increased ionic strength. HSP25 and especially its 2D and 3D mutants effectively prevent salt-induced aggregation of completely denatured actin. It is concluded that the interaction of HSP25 with actin depends on the state of both actin and HSP25. HSP25 predominantly acts as a chaperone and preferentially interacts with thermally unfolded actin, preventing the formation of insoluble aggregates.     

Thymosin beta 4 sequesters the majority of G-actin in resting human polymorphonuclear leukocytes

Thymosin beta 4 (T beta 4), a 5-kD peptide which binds G-actin and inhibits its polymerization (Safer, D., M. Elzinga, and V. T. Nachmias. 1991. J. Biol. Chem. 266:4029-4032), appears to be the major G-actin sequestering protein in human PMNs. In support of a previous study by Hannappel, E., and M. Van Kampen (1987. J. Chromatography. 397:279-285), we find that T beta 4 is an abundant peptide in these cells. By reverse phase HPLC of perchloric acid supernatants, human PMNs contain approximately 169 fg/cell +/- 90 fg/cell (SD), corresponding to a cytoplasmic concentration of approximately 149 +/- 80.5 microM. On non-denaturing polyacrylamide gels, a large fraction of G-actin in supernatants prepared from resting PMNs has a mobility similar to the G-actin/T beta 4 complex. Chemoattractant stimulation of PMNs results in a decrease in this G-actin/T beta 4 complex. To determine whether chemoattractant induced actin polymerization results from an inactivation of T beta 4, the G-actin sequestering activity of supernatants prepared from resting and chemoattractant stimulated cells was measured by comparing the rates of pyrenyl-actin polymerization from filament pointed ends. Pyrenyl actin polymerization was inhibited to a greater extent in supernatants from stimulated cells and these results are qualitatively consistent with T beta 4 being released as G-actin polymerizes, with no chemoattractant-induced change in its affinity for G-actin. The kinetics of bovine spleen T beta 4 binding to muscle pyrenyl G-actin are sufficiently rapid to accommodate the rapid changes in actin polymerization and depolymerization observed in vivo in response to chemoattractant addition and removal.