Effect of Hypothyroidism on Different Forms of Actin in Rat Cerebral Neuronal Cultures Studied by an Improved DNase I Inhibition Assay

An improved DNase I inhibition assay for the filamentous actin (F-actin) and monomeric actin (G-actin) in brain cells has been developed. Unlike other methods, the cell lysis conditions and postlysis treatments, established by us, inhibited the temporal inactivation of actin in the cell lysate and maintained a stable F-actin/G-actin ratio for at least 4-5 h after lysis. The new procedure allowed separate quantitation of the noncytoskeletal F-actin in the Triton-soluble fraction (12,000 g, 10 min supernatant) that did not readily sediment with the Triton-insoluble cytoskeletal F-actin (12,000 g, 10 min pellet). We have applied this modified assay system to study the effect of hypothyroidism on different forms of actin using primary cultures of neurons derived from cerebra of neonatal normal and hypothyroid rats. Our results showed a 20% increase in the Triton-insoluble cytoskeletal F-actin in cultures from hypothyroid brain relative to normal controls. In the Triton-soluble fraction, containing the G-actin and the noncytoskeletal F-actin, cultures from hypothyroid brain showed a 15% increase in G-actin, whereas the F-actin remained unaltered. The 10% increase in total actin observed in this fraction from hypothyroid brain could be totally accounted for by the enhancement of G-actin. The mean F-actin/G-actin ratio in this fraction was about 30% higher in the cultures from normal brain compared to that of the hypothyroid system, which indicates that hypothyroidism tends to decrease the proportion of noncytoskeletal F-actin relative to 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%.     

Regulation of endothelial nitric oxide synthase by the actin cytoskeleton

In the present study, the association ofendothelial nitric oxide synthase (eNOS) with the actin cytoskeleton inpulmonary artery endothelial cells (PAEC) was examined. We found thatthe protein contents of eNOS, actin, and caveolin-1 were significantly higher in the caveolar fraction of plasma membranes than in the noncaveolar fraction of plasma membranes in PAEC. Immunoprecipitation of eNOS from lysates of caveolar fractions of plasma membranes in PAECresulted in the coprecipitation of actin, and immunoprecipitation ofactin from lysates of caveolar fractions resulted in thecoprecipitation of eNOS. Confocal microscopy of PAEC, in which eNOS waslabeled with fluorescein, F-actin was labeled with Texasred-phalloidin, and G-actin was labeled with deoxyribonuclease Iconjugated with Texas red, also demonstrated an association betweeneNOS and F-actin or G-actin. Incubation of purified eNOS with purifiedF-actin and G-actin resulted in an increase in eNOS activity. Theincrease in eNOS activity caused by G-actin was much higher than thatcaused by F-actin. Incubation of PAEC with swinholide A, an actinfilament disruptor, resulted in an increase in eNOS activity, eNOSprotein content, and association of eNOS with G-actin and in a decrease in the association of eNOS with F-actin. The increase in eNOS activitywas higher than that in eNOS protein content in swinholide A-treatedcells. In contrast, exposure of PAEC to phalloidin, an actin filamentstabilizer, caused decreases in eNOS activity and association of eNOSwith G-actin and increases in association of eNOS with F-actin. Theseresults suggest that eNOS is associated with actin in PAEC and thatactin and its polymerization state play an important role in theregulation of eNOS activity.

     

Actin polymerization induced by pulsed electric stimulation of bone cells in vitro

Electric field pulses, capacitively applied to tissue cultures of embryonic bone cells, were shown to induce changes in the state of cellular actin. Three actin states could be defined by DNAase I inhibition. A rapidly (20-30 s) inhibiting fraction, attributed to monomeric G-actin, amounts to 55% of total actin in nonstimulated cells. An additional fraction of 8% required approx. 20 min to reach full inhibition and was tentatively defined as polymeric 'F'-actin. The remaining 37% could be detected only after treatment of the cells with 0.75 M guanidine hydrochloride, which dissociates actin from all its protein interactions. This fraction, N-actin (network actin) is believed to represent F-actin integrated into some supramolecular structure, where it is not accessible to DNAase I. Upon short electric stimulation the distribution changed to 40% G-actin, 12% F-actin and 48% N-actin. 3-Isobutyl-1-methylxanthine (IBMX; an inhibitor of cAMP phosphodiesterase), depletion of extracellular calcium, and calmodulin inhibitors abolished this field effect.     

Carcinoembryonic antigen cell adhesion molecule 1 directly associates with cytoskeleton proteins actin and tropomyosin

CEA cell adhesion molecule 1 (CEACAM1), a type 1 transmembrane and homotypic cell adhesion protein belonging to the carcinoembryonic antigen (CEA) gene family and expressed on epithelial cells, is alternatively spliced to produce four major isoforms with three or four Ig-like ectodomains and either long (CEACAM1-L) or short (CEACAM1-S) cytoplasmic domains. When murine MC38 (methylcholanthrene-induced adenocarcinoma 38) cells were transfected with human CEACAM1-L and stimulated with sodium pervanadate, actin was found to co-localize with CEACAM1-L at cell-cell boundaries but not in untreated cells. When CEACAM1-L was immunoprecipitated from pervanadate-treated MC38/CEACAM1-L cells and the associated proteins were analyzed by two-dimensional gel analysis and mass spectrometry, actin and tropomyosin, among other proteins, were identified. Whereas a glutathione S-transferase (GST) fusion protein containing the l-isoform (GST-Cyto-L) bound poorly to F-actin in a co-sedimentation assay, the S-isoform fusion protein (GST-Cyto-S) co-sedimented with F-actin, especially when incubated with G-actin during polymerization (K(D) = 7.0 microm). Both GST-Cyto-S and GST-Cyto-L fusion proteins bind G-actin and tropomyosin by surface plasmon resonance studies with binding constants of 0.7 x 10(-8) and 1.0 x 10(-7) m for GST-Cyto-L to G-actin and tropomyosin, respectively, and 3.1 x 10(-8) and 1.3 x 10(-7) m for GST-Cyto-S to G-actin and tropomyosin, respectively. Calmodulin or EDTA inhibited binding of the GST-Cyto-L fusion protein to G-actin, whereas calmodulin and G-actin, but not EDTA, stimulated binding to tropomyosin. A biotinylated 14-amino acid peptide derived from the juxtamembrane portion of the cytoplasmic domain of CEACAM1-L associated with both G-actin and tropomyosin with K(D) values of 1.3 x 10(-5) and 1.8 x 10(-5) m, respectively. These studies demonstrate the direct interaction of CEACAM1 isoforms with G-actin and tropomyosin and the direct interaction of CEACAM1-S with F-actin.     

Membrane-associated actin from the microvillar membranes of ascites tumor cells

A membrane fraction (MF2) has been purified from isolated microvilli of the MAT-C1 subline of the 13762 rat mammary ascites adenocarcinoma under conditions which cause F-actin depolymerization. This membrane preparation contains actin as a major component, although no filamentous structures are observed by transmission electron microscopy. Membranes were extracted with a Triton X-100-containing actin-stabilizing buffer (S buffer) or actin-destabilizing buffer (D buffer). In D buffer greater than 90% of metabolically labeled protein and glycoprotein was extracted, and 80-90% of these labeled species was extracted in S buffer. When S buffer extracts of MF2 were fractionated by either gel filtration on Sepharose 6 B or rate-zonal sucrose density gradient centrifugation, most of the actin was found to be intermediate in size between G- and F-actin. In D buffer most of the MF2 actin behaved as G-actin. Extraction and gel filtration of intact microvilli in S buffer also showed the presence of the intermediate form of actin, indicating that it did not arise during membrane preparation. When [35S]methionine-labeled G-actin from ascites cells was added to S buffer extracts of MF2 and chromatographed, all of the radioactivity chromatographed as G-actin, indicating that the intermediate form of actin did not result from an association of G-actin molecules during extraction or chromatography. The results of this study suggest that the microvillar membrane fraction is enriched in an intermediate form of actin smaller than F-actin and larger than G-actin.     

Comparisons of Endothelial Cell G- and F-Actin Distribution in Situ and in Vitro

Numerous studies have described the F-actin cytoskeleton; however, little information relevant to C-actin is available. The actin pools of bovine aortic endothelial cells were examined using in situ and in vitro conditions and fluorescent probes for G-(deoxyribonuclease I.0.3 μM) or F-actin (phalloidin, 0.2 μM). Cells in situ displayed a diffuse G-actin distribution, while F-actin was concentrated in the cell periphery and in fine stress fibers that traversed some cells. Cells of subconfluent or just confluent cultures demonstrated intense fluorescence, with many F-actin stress fibers. Postconfluent cultures resembled the condition in situ; peripheral F-actin was prominent, traversing actin stress fibers were greatly reduced and fluorescent intensity was diminished. Postconfluency had little influence on G-actin. with only an enhancement in the intensity of G-actin punctate fluorescence. When post-confluent cultures were incubated with cytochalasin D (15 min; 10^--⁴ M), F-actin networks were disrupted and actin punctate and diffuse fluorescence increased. G-actin fluorescence was not altered by the incubation. Although its unstructured nature may account for the minor changes observed, the stability of the G-actin pool in the presence of notable F-actin modulations suggested that filamentous actin was the key constituent involved in these actin cytoskeletal alterations. A separate finding illustrated that the concomitant use of actin probes with image enhancement and fluorescent microscopy could reveal simultaneously the G- and F-actin pools within the same cell.     

On the elastic properties of tetramethylrhodamine F-actin

(Iodoacetamido)tetramethylrhodamine disrupts F-actin. At the 1:1 fluorophore to actin (as monomer) ratio approximately 80% of the protein becomes non-sedimentable. The fluorescent, non-sedimentable actin copolymerizes with G-actin to yield fluorescent filaments. The tensile strength of these filaments changes with the ratio of the fluorescent non-sedimentable actin to the G-actin, being 1.6 pN, 2.9 pN and 3.6 pN at the 1/4, 2/3 and 1/1 ratios, respectively. These tensile strengths are approximately two orders of magnitude lower than those obtained by decoration of F-actin with phalloidin.     

Dissociative mechanism of F-actin thermal denaturation

We have applied differential scanning calorimetry to investigate thermal unfolding of F-actin. It has been shown that the thermal stability of F-actin strongly depends on ADP concentration. The transition temperature, T(m), increases with increasing ADP concentration up to 1 mM. The T(m) value also depends on the concentration of F-actin: it increases by almost 3 degrees C as the F-actin concentration is increased from 0.5 to 2.0 mg/ml. Similar dependence of the T(m) value on protein concentration was demonstrated for F-actin stabilized by phalloidin, whereas it was much less pronounced in the presence of AlF4(-). However, T(m) was independent of protein concentration in the case of monomeric G-actin. The results suggest that at least two reversible stages precede irreversible thermal denaturation of F-actin; one of them is dissociation of ADP from actin subunits, and another is dissociation of subunits from the ends of actin filaments. The model explains why unfolding of F-actin depends on both ADP and protein concentration.     

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.     

State of actin in gastric parietal cells

Remodeling of theapical membrane-cytoskeleton has been suggested to occur when gastricparietal cells are stimulated to secrete HCl. The present experimentsassayed the relative amounts of F-actin and G-actin in gastric glandsand parietal cells, as well as the changes in the state of actin onstimulation. Glands and cells were treated with a Nonidet P-40extraction buffer for separation into detergent-soluble (supernatant)and detergent-insoluble (pellet) pools. Two actin assays were used toquantitate actin: the deoxyribonuclease I binding assay to measureG-actin and F-actin content in the two pools and a simple Western blotassay to quantitate the relative amounts of actin in the pools.Functional secretory responsiveness was assayed by aminopyrineaccumulation. About 5% of the total parietal cell protein is actin,with about 90% of the actin present as F-actin. Stimulation of acidsecretion resulted in no measurable change in the relative amounts ofG-actin and cytoskeletal F-actin. Treatment of gastric glands withcytochalasin D inhibited acid secretion and resulted in a decrease inF-actin and an increase in G-actin. No inhibition of parietal cellsecretion was observed when phalloidin was used to stabilize actinfilaments. These data are consistent with the hypothesis thatmicrofilamentous actin is essential for membrane recruitment underlyingparietal cell secretion. Although the experiments do not eliminate theimportance of rapid exchange between G- and F-actin for the secretoryprocess, the parietal cell maintains actin in a highly polymerizedstate, and no measurable changes in the steady-state ratio of G-actin to F-actin are associated with stimulation to secrete acid.

     

Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.     

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.     

Imaging remodeling of the actin cytoskeleton in vascular smooth muscle cells after mechanosensitive arteriolar constriction

Experiments were performed to determine whether remodeling of the actin cytoskeleton contributes to arteriolar constriction. Mouse tail arterioles were mounted on cannulae in a myograph and superfused with buffer solution. The alpha1-adrenergic agonist phenylephrine (0.1-1 micromol/l) caused constriction that was unaffected by cytochalasin D (300 nmol/l) or latrunculin A (100 nmol/l), inhibitors of actin polymerization. In contrast, each compound abolished the mechanosensitive constriction (myogenic response) evoked by elevation in transmural pressure (PTM; 10-60 or 90 mmHg). Arterioles were fixed, permeabilized, and stained with Alexa-568 phalloidin and Alexa-488 DNAse I to visualize F-actin and G-actin, respectively, using a Zeiss 510 laser scanning microscope. Elevation in PTM, but not phenylephrine (1 micromol/l), significantly increased the intensity of F-actin and significantly decreased the intensity of G-actin staining in arteriolar vascular smooth muscle cells (VSMCs). The increase in F-actin staining caused by an elevation in PTM was inhibited by cytochalasin D. In VSMCs at 10 mmHg, prominent F-actin staining was restricted to the cell periphery, whereas after elevation in PTM, transcytoplasmic F-actin fibers were localized through the cell interior, running parallel to the long axis of the cells. Phenylephrine (1 micromol/l) did not alter the architecture of the actin cytoskeleton. In contrast to VSMCs, the actin cytoskeleton of endothelial or adventitial cells was not altered by an elevation in PTM. Therefore, the actin cytoskeleton of VSMCs undergoes dramatic alteration after elevation in PTM of arterioles and plays a selective and essential role in mechanosensitive myogenic constriction.     

Multiple actin binding domains of Ena/VASP proteins determine actin network stiffening

Vasodilator-stimulated phosphoprotein (Ena/VASP) is an actin binding protein, important for actin dynamics in motile cells and developing organisms. Though VASP’s main activity is the promotion of barbed end growth, it has an F-actin binding site and can form tetramers, and so could additionally play a role in actin crosslinking and bundling in the cell. To test this activity, we performed rheology of reconstituted actin networks in the presence of wild-type VASP or mutants lacking the ability to tetramerize or to bind G-actin and/or F-actin. We show that increasing amounts of wild-type VASP increase network stiffness up to a certain point, beyond which stiffness actually decreases with increasing VASP concentration. The maximum stiffness is 10-fold higher than for pure actin networks. Confocal microscopy shows that VASP forms clustered actin filament bundles, explaining the reduction in network elasticity at high VASP concentration. Removal of the tetramerization site results in significantly reduced bundling and bundle clustering, indicating that VASP’s flexible tetrameric structure causes clustering. Removing either the F-actin or the G-actin binding site diminishes VASP’s effect on elasticity, but does not eliminate it. Mutating the F-actin and G-actin binding site together, or mutating the F-actin binding site and saturating the G-actin binding site with monomeric actin, eliminates VASP’s ability to increase network stiffness. We propose that, in the cell, VASP crosslinking confers only moderate increases in linear network elasticity, and unlike other crosslinkers, VASP’s network stiffening activity may be tuned by the local concentration of monomeric actin.     

Tolrestat对高糖所致肾小球系膜细胞肌动蛋白去组装的影响

研究了醛糖还原酶抑制剂Tolrestat对高浓度葡萄糖(HG)所致肾小球系膜细胞(MC)肌动蛋白(actin)组装的影响。结果证明,与正常浓度葡萄糖(NG)相比,在HG培养的MC,F-actin失去束状外观呈不规则网状,显示F-actin部分去组装;F-actin荧光强度降低,G-actin荧光强度升高和F-/G-actin荧光强度比值下降。Tolrestat加入培养后,明显防止HG引起的F-actin去组装及F-和G-actin荧光强度的变化。提示多元醇通路激活在HG引起的MCactin去组装改变中起一定作用。     

Comparison of actin changes and calcium metabolism in plastic- and fibronectin-adherent human neutrophils.

Human neutrophils adherent to a polystyrene plastic surface are vigorously activated, whereas those adherent to fibronectin manifest only a priming response. The basis of these metabolic differences was further characterized; polystyrene-adherent cells, which were shown to spread quickly upon adhesion, exhibited an increase of cytoskeleton-associated actin (F-actin) (measured by a nitrobenzoxadiazole-phallacidin fluorescent staining assay) and a decrease of monomeric G-actin concentration (measured by a DNase inhibition assay); in contrast, fibronectin-adherent cells exhibited little spreading and decreased their F-actin, after 1.5 min of adhesion, to 33.49 +/- 6.9% (mean +/- SD, n = 5) of initial levels found in suspended cells before plating. Actin depolymerization in fibronectin-adherent cells was confirmed by measuring G-actin, which sharply increased during the first minute of adhesion, rising from 0.065 +/- 0.007 to 0.20 +/- 0.035 microgram/microgram of protein (mean +/- SEM, p less than 0.05), and then remained elevated during 5 min of observation. In contrast, soluble fibronectin induced a decrease of G-actin in suspended cells. Cells pretreated with 1 microM cytochalasin D and allowed to adhere to a plastic surface did not spread, failed to generate O2-, and exhibited elevated concentrations of G-actin (0.1 to 0.2 microgram/microgram of protein) during the 5 min of observation. Actin changes, as well as respiratory burst, in adherent cells were shown to proceed through a pertussis toxin-insensitive pathway. Fluo-3 measurements of intracellular Ca2+ concentrations ([Ca2+]i) showed a fourfold and twofold [Ca2+]i increase in polystyrene- and fibronectin-adherent cells, respectively, after 2 min. The small rise in [Ca2+]i in fibronectin-adherent cells corresponds to a primed response of these cells to subsequent activation with FMLP. Ionomycin (1 microM) added to neutrophils just before adhesion on fibronectin induced full activation, i.e., O2- production and actin polymerization. The metabolic events controlling metabolic priming and actin depolymerization are as yet uncharacterized, but fibronectin receptor-linked responses beyond the mediation of cell adhesion have now been identified, suggesting complex metabolic functions of integrin receptors.     

Some properties of Physarum actinin. A regulatory protein of actin polymerization.

A factor termed Physarum actinin was isolated and partially purified from plasmodia of a myxomycete, Physarum polycephalum. When Physarum actinin was mixed with purified Physarum or rabbit striated muscle G-actin in a weight ratio of about 1 actinin to 9 actin and then the polymerization of G-actin induced, G-actin polymerized to the ordinary F-actin on addition of 0.1 M KCl. However, it polymerized to Mg-polymer on addition of 2 mM MgCl2. The reduced viscosity (etasp/C) of the Mg-polymer was 1.2 dl/g, about one-seventh of that of the F-actin (7.4 dl/g). The sedimentation coefficient of the Mg-polymer was 22.8 S, almost the same as that of the F-actin (29.4 S). The Mg-polymer showed the specific ATPase activity of the order of 1 . 10(-3) mumol ATP/mg actin per min. It was shown that Physarum actinin copolymerized with G-actin to form Mg-polymer on addition of 2 mM MgCl2. The molecular weights of Physarum actinin were about 90 000 in salt-free or slat solutions and 43 000 in a dodecyl sulfate solution. The range of salting out with ammonium sulfate was 50--65% saturation, which was different from that of Physarum actin (15--35% saturation). Physarum actinin did not interact with Physarum myosin or muscle heavy meromyosin. When the weight ratio of actinin to actin increased, the flow birefringence of the formed Mg-polymer decreased, and it became almost zero at the weight ratio of 1 actinin to 5 actin. ATPase activity reached the maximum level (2.2 . 10(-3) mumol ATP/mg actin per min) at the same ratio. On the addition of Physarum actinin to purified Physarum F-actin which had been polymerized on addition of 2 mM MgCl2 the viscosity decreased rapidly, suggesting that the F-actin filaments were broken in the smaller fragments or that they transformed to Mg-polymers. A factor with properties similar to Physarum actinin was isolated from acetone powder of sea urchin eggs.     

Bacterial lipopolysaccharide induces actin reorganization,intercellular gap formation,and endothelial barrier dysfunction in pulmonary vascular endothelial cells: Concurrent F-actin depolymerization and new actin synthesis

Bacterial lipopolysaccharide (LPS) influences pulmonary vascular endothelial barrier function in vitro. We studied whether LPS regulates endothelial barrier function through actin reorganization. Postconfluent bovine pulmonary artery endothelial cell monolayers were exposed to Escherichia coli 0111:B4 LPS 10 ng/ml or media for up to 6 h and evaluated for: (1) transendothelial ¹⁴C-albumin flux, (2) F-actin organization with fluorescence microscopy, (3) F-actin quantitation by spectrofluorometry, and (4) monomeric G-actin levels by the DNAse 1 inhibition assay. LPS induced increments in ¹⁴C-albumin flux (P < 0.001) and intercellular gap formation at ≥ 2–6 h. During this same time period the endothelial F-actin pool was not significantly changed compared to simultaneous media controls. Mean (±SE) G-actin (μg/mg total protein) was significantly (P < 0.002) increased compared to simultaneous media controls at 2, 4, and 6 h but not at 0.5 or 1 h. Prior F-actin stabilization with phallicidin protected against the LPS-induced increments in G-actin (P = 0.040) as well as changes in barrier function (P < 0.0001). Prior protein synthesis inhibition unmasked an LPS-induced decrement in F-actin (P = 0.0044), blunted the G-actin increment (P = 0.010), and increased LPS-induced changes in endothelial barrier function (P < 0.0001). Therefore, LPS induces pulmonary vascular endothelial F-actin depolymerization, intercellular gap formation, and barrier dysfunction. Over the same time period, LPS increased total actin (P < 0.0001) and new actin synthesis (P = 0.0063) which may be a compensatory endothelial cell response to LPS-induced F-actin depolymerization. © 1993 Wiley-Liss, Inc.