Characterization of phalloidin-negative nuclear actin filaments in U2OS cells expressing cytoplasmic actin-EGFP.

Actin is a major structural component of the cytoskeleton in eukaryotic cells, including fungi, plants, and animals, and exists not only in the cytoplasm as cytoskeleton but also in the nucleus. Recently, we developed a novel actin probe, ß-actin-EGFP fusion protein, which exhibited similar monomeric to filamentous ratio as that of endogenous actin, in contrast to the widely used EGFP-ß-actin fusion protein that over-assembles in cells. Unexpectedly, this novel probe visualized an interconnected meshwork of slightly curved beam-like bundles of actin filaments in the nucleus of U2OS cells. These structures were not labeled with rhodamine phalloidin, Lifeact-EGFP or anti-actin antibodies. In addition, immunofluorescence staining and expression of cofilin-EGFP revealed that this nuclear actin structures contained cofilin. We named these actin filaments as phalloidin-negative intranuclear (PHANIN) actin filaments. Since PHANIN actin filaments could not be detected by general detection methods for actin filaments, we propose that PHANIN actin filaments are different from previously reported nuclear actin structures.

Muscular Arrangement and Sclerite Morphology in the Haptor of Tetraonchus monenteron (Monogenea, Dactylogyridea).

BACKGROUND : Tetraonchidae is a small and relatively monomorphic family of dactylogyroid monogeneans. Since the morphology of sclerites of the attachment organ (haptor) is one of the primary criteria in tetraonchid taxonomy, the confocal study of sclerites and associated musculature may provide valuable taxonomic information. PURPOSE : The aim of this study was to examine the haptoral sclerites and musculature in Tetraonchus monenteron (Wagener, 1857), a common parasite of pike. METHODS: Haptoral musculature of T. monenteron was studied using phalloidin staining and confocal microscopy, with sclerites visualized in the reflection confocal mode. RESULTS: Haptoral armature of T. monenteron consists of ventral and dorsal pairs of anchors, a ventral bar, eight pairs of marginal hooks and at least three pairs of accessory sclerites. Anchors are operated by 14 muscles, of which the most prominent are extrinsic muscles, the transverse muscle interconnecting the ventral anchors, three muscles connecting the ventral anchor to the ventral bar, and four muscles of the dorsal and ventral anchors inserting on the haptoral wall. The extrinsic muscles are attached to the braceshaped sclerites, which in turn are connected to the dorsal anchors via muscle bundles. CONCLUSION: The gaffing action of the dorsal anchors is likely to be achieved by the extrinsic muscles and the transverse muscle that clamps the extrinsic muscles against the body wall. The ventral anchors are probably held in attached position by the transverse muscle and four muscles inserting on the ventral bar and haptoral wall. The haptoral musculature may have potential utility for tetraonchid taxonomy.

Dense small molecule labeling enables activator-dependent STORM by proximity mapping.

Stochastic optical reconstruction microscopy (STORM) enables high-resolution imaging, but multi-channel 3D imaging is problematic because of chromatic aberrations and alignment errors. The use of activator-dependent STORM in which spectrally distinct activators can be coupled with a single reporter can circumvent such issues. However, the standard approach of linking activators and reporters to a single antibody molecule is hampered by low labeling density and the large size of the antibody. We proposed that small molecule labels might enable activator-dependent STORM if the reporter or activator were linked to separate small molecules that bound within 3.5 nm of each other. This would greatly increase the labeling density and therefore improve resolution. We tested various mixtures of phalloidin- or mCling-conjugated fluorophore to demonstrate this feasibility. The specific activation was dependent on the choice of activator, its density, a matching activating laser and its power. In addition to providing an effective means of multi-channel 3D STORM imaging, this method also provides information about the local proximity between labels, potentially enabling super-resolved mapping of the conformation of the labeled structures.

Amatoxin and phallotoxin concentration in Amanita phalloides spores and tissues.

Most of the fatal cases of mushroom poisoning are caused by Amanita phalloides. The amount of toxin in mushroom varies according to climate and environmental conditions. The aim of this study is to measure α-, ß-, and γ-amanitin with phalloidin and phallacidin toxin concentrations. Six pieces of A. phalloides mushrooms were gathered from a wooded area of Düzce, Turkey, on November 23, 2011. The mushrooms were broken into pieces as spores, mycelium, pileus, gills, stipe, and volva. α-, ß-, and γ-Amanitin with phalloidin and phallacidin were analyzed using reversed-phase high-performance liquid chromatography. As a mobile phase, 50 mM ammonium acetate + acetonitrile (90 + 10, v/v) was used with a flow rate of 1 mL/min. C18 reverse phase column (150 × 4.6 mm; 5 µm particle) was used. The least amount of γ-amanitin toxins was found at the mycelium. The other toxins found to be in the least amount turned out to be the ones at the spores. The maximum amounts of amatoxins and phallotoxin were found at gills and pileus, respectively. In this study, the amount of toxin in the spores of A. phalloides was published for the first time, and this study is pioneering to deal with the amount of toxin in mushrooms grown in Turkey.

Preparation of developing Xenopus muscle for sarcomeric protein localization by high-resolution imaging.

Mutations in several sarcomeric proteins have been linked to various human myopathies. Therefore, having an in vivo developmental model available that develops quickly and efficiently is key for investigators to elucidate the critical steps, components and signaling pathways involved in building a myofibril; this is the pivotal foundation for deciphering disease mechanisms as well as the development of myopathy-related therapeutics. Although striated muscle cell culture studies have been extremely informative in providing clues to both the distribution and functions of sarcomeric proteins, myocytes in vivo develop in an irreproducible 3D environment. Xenopus laevis (frog) embryos are cost effective, compliant to protein level manipulations and develop relatively quickly (⩽ a week) in a petri dish, thus providing a powerful system for de novo myofibrillogenesis studies. Although fluorophore-conjugated phalloidin labeling is the gold standard approach for investigating actin-thin filament architecture, it is well documented that phalloidin-labeling can be challenging and inconsistent within Xenopus embryos. Therefore we highlight several techniques that can be utilized to preserve both antibody and fluorophore-conjugated phalloidin labeling within Xenopus embryos for high-resolution fluorescence microscopy.

Involvement of the sieve element cytoskeleton in electrical responses to cold shocks.

This study dealt with the visualization of the sieve element (SE) cytoskeleton and its involvement in electrical responses to local cold shocks, exemplifying the role of the cytoskeleton in Ca(2+)-triggered signal cascades in SEs. High-affinity fluorescent phalloidin as well as immunocytochemistry using anti-actin antibodies demonstrated a fully developed parietal actin meshwork in SEs. The involvement of the cytoskeleton in electrical responses and forisome conformation changes as indicators of Ca(2+) influx was investigated by the application of cold shocks in the presence of diverse actin disruptors (latrunculin A and cytochalasin D). Under control conditions, cold shocks elicited a graded initial voltage transient, ΔV1, reduced by external La(3+) in keeping with the involvement of Ca(2+) channels, and a second voltage transient, ΔV2. Cytochalasin D had no effect on ΔV1, while ΔV1 was significantly reduced with 500 nm latrunculin A. Forisome dispersion was triggered by cold shocks of 4°C or greater, which was indicative of an all-or-none behavior. Forisome dispersion was suppressed by incubation with latrunculin A. In conclusion, the cytoskeleton controls cold shock-induced Ca(2+) influx into SEs, leading to forisome dispersion and sieve plate occlusion in fava bean (Vicia faba).

The Qdot-labeled actin super-resolution motility assay measures low-duty cycle muscle myosin step size.

Myosin powers contraction in heart and skeletal muscle and is a leading target for mutations implicated in inheritable muscle diseases. During contraction, myosin transduces ATP free energy into the work of muscle shortening against resisting force. Muscle shortening involves relative sliding of myosin and actin filaments. Skeletal actin filaments were fluorescently labeled with a streptavidin conjugate quantum dot (Qdot) binding biotin-phalloidin on actin. Single Qdots were imaged in time with total internal reflection fluorescence microscopy and then spatially localized to 1-3 nm using a super-resolution algorithm as they translated with actin over a surface coated with skeletal heavy meromyosin (sHMM) or full-length ß-cardiac myosin (MYH7). The average Qdot-actin velocity matches measurements with rhodamine-phalloidin-labeled actin. The sHMM Qdot-actin velocity histogram contains low-velocity events corresponding to actin translation in quantized steps of ~5 nm. The MYH7 velocity histogram has quantized steps at 3 and 8 nm in addition to 5 nm and larger compliance compared to that of sHMM depending on the MYH7 surface concentration. Low-duty cycle skeletal and cardiac myosin present challenges for a single-molecule assay because actomyosin dissociates quickly and the freely moving element diffuses away. The in vitro motility assay has modestly more actomyosin interactions, and methylcellulose inhibited diffusion to sustain the complex while preserving a subset of encounters that do not overlap in time on a single actin filament. A single myosin step is isolated in time and space and then characterized using super-resolution. The approach provides a quick, quantitative, and inexpensive step size measurement for low-duty cycle muscle myosin.

A concept study on identification and attribution profiling of chemical threat agents using liquid chromatography-mass spectrometry applied to Amanita toxins in food.

Accidental or deliberate poisoning of food is of great national and international concern. Detecting and identifying potentially toxic agents in food is challenging due to their large chemical diversity and the complexity range of food matrices. A methodology is presented whereby toxic agents are identified and further characterized using a two-step approach. First, generic screening is performed by LC/MS/MS to detect toxins based on a list of selected potential chemical threat agents (CTAs). After identifying the CTAs, a second LC/MS analysis is performed applying accurate mass determination and the generation of an attribution profile. To demonstrate the potential of the methodology, toxins from the mushrooms Amanita phalloides and Amanita virosa were analyzed. These mushrooms are known to produce cyclic peptide toxins, which can be grouped into amatoxins, phallotoxins and virotoxins, where α-amanitin and ß-amanitin are regarded as the most potent. To represent a typical complex food sample, mushroom stews containing either A. phalloides or A. virosa were prepared. By combining the screening method with accurate mass analysis, the attribution profile for the identified toxins and related components in each stew was established and used to identify the mushroom species in question. In addition, the analytical data was consistent with the fact that the A. virosa specimens used in this study were of European origin. This adds an important piece of information that enables geographic attribution and strengthens the attribution profile.

[Determination of main peptide toxins from Amanita pallidorosea with HPLC and their antifungal action on Blastomyces albicans].

OBJECTIVE: To detect peptide toxins in Amanita pallidorasea and to study the antifungal activities of peptide toxins against Blastomyces albicans. METHODS: We separated and identified peptide toxins and determined its contents in the fruiting body, pileus and the mixture of stipe and volva from A. pallidorasea by HPLC and ESI-MS methods. Meanwhile, we detected antifungal activities of the crude toxin and the separated peptide toxins against Blastomyces albicans JLC31680 and JLC31681 by the paper disk method. RESULTS: We totally got three peptide toxins: alpha-amanitin (alpha-AMA), beta-amanitin (beta-AMA) and phalloidin (PHD). The contents of alpha-AMA, beta-AMA and PHD were 30.3 mg/g, 6.99 mg/g and 9.95 mg/g in fruiting body, and 45.0 mg/g, 11.1 mg/g and 11.3 mg/g in pileus. The contents of alpha-AMA and PHD were 11.7 mg/g and 7.98 mg/g in the mixture of stipe and volva , but the beta-AMA was not detected in this part. The inhibition ratio of the crude toxin and alpha-AMA, beta-AMA and PHD to B. albicans JLC31680 were 11.96%, 32.52%, 23.29% (P<0.01) and 15.46% (P<0.05). The inhibition ratio of the crude toxin and beta-AMA to B. albicans JLC31681 was 10.16% and 11.10% (P < 0.01), while that of alpha-AMA's was 6.89% (P < 0.05). CONCLUSIONS: A. pallidorasea is a new resource of peptide toxins with antifungal activity.

[Fibrillar actin in the nuclear apparatus of the ciliate Paramecium caudatum].

Due to their nuclear dualism, ciliates provide a good model for studying the role of actin in spatial organization and transcription activity of the nucleus. The actin in the nuclear apparatus of the ciliate Paramecium caudatum was studied using fluorescently labeled phalloiodin and indirect immunocytochemistry. Fibrillar actin was demonstrated in both of the nuclei. Actin was revealed in the chromatin areas, and was often associated with the periphery of the amplified nucleoli in the macronucleus. Redistribution of actin was observed depending on different physiological state of the cells. Stable infection of the macronulear with the intranuclear endobionts Holospora obtuse led to the loss of nuclear actin accompanied by significant nuclear fragility and redistribution of the phosphorylated form of the actin-binding protein cofilin. Spherical bodies resembling karyosphere were found in the macronuclear anlagen.

[The characteristic of the coelomic fluid and coelomic epithelium cell populations of starfish Asterias rubens L., capable to attach and spread on various substrates].

Cultivation is one of the methods modeling processes occurring in vivo. The success of cultivation, in particular, is defined by a substratum choice. We studied the ability of coelomocytes and coelomic epithelial cells to attach and spread to fibronectin, laminin, polylysine, and glass. Qualitative composition of heterogeneous populations of coelomocytes and epithelial cells was determined after staining the cells with rhodamine-phalloidin and DAPI, and changes in the composition of populations evaluated in response to injury. Seven relative classes of coelomocytes has been identified, three of which has been shown to participate in the formation of clot during primary repair of wounds. There was a change in the proportion of these cells, attached to specific ligands in response to the injury. In coelomic epithelium 8 relative classes of cells has been identified, two of which are likely to be candidates for the role of progenitor cells for coelomocytes--coelomocyte-like and small epithelial cells with high nuclear-cytoplasmic ratio. The enrichment with the small cells in population of attached coelomic epithelium cells has been revealed when seeding on laminin. Continued viability of epithelial cells has been shown when cultured on laminin during 2 months.

Fluorescent visualization of macromolecules in Drosophila whole mounts.

The ability to determine the expression dynamics of individual genes "in situ" by visualizing the precise spatial and temporal distribution of their products in whole mounts by histochemical and immunocytochemical reactions has revolutionized our understanding of cellular processes. Drosophila developmental genetics was one of the fields that benefited most from these technologies, and a variety of fluorescent methods were specifically designed for investigating the localization of developmentally important proteins and cell markers during embryonic and post embryonic stages of this model organism. In this chapter we present detailed protocols for fluorescence immunocytochemistry of whole mount embryos, imaginal discs, pupal retinas, and salivary glands of Drosophila melanogaster, as well as methods for fluorescent visualization of specific subcellular structures in these tissues.

Both actin and polyproline interactions of profilin-1 are required for migration, invasion and capillary morphogenesis of vascular endothelial cells.

The objective of the present study was to evaluate how different ligand interactions of profilin-1 (Pfn1), an actin-binding protein that is upregulated during capillary morphogenesis of vascular endothelial cells (VEC), contribute to migration and capillary forming ability of VEC. We adopted a knockdown-knockin experimental system to stably express either fully functional form or mutants of Pfn1 that are impaired in binding to two of its major ligands, actin (H119E mutant) and proteins containing polyproline domains (H133S mutant), in a human dermal microvascular cell line (HmVEC) against near-null endogenous Pfn1 background. We found that silencing endogenous Pfn1 expression in HmVEC leads to slower random migration, reduced velocity of membrane protrusion and a significant impairment in matrigel-induced cord formation. Only re-expression of fully functional but not any of the two ligand-binding deficient mutants of Pfn1 rescues the above defects. We further show that loss of Pfn1 expression in VEC inhibits three-dimensional capillary morphogenesis, MMP2 secretion and ECM invasion. VEC invasion through ECM is also inhibited when actin and polyproline interactions of Pfn1 are disrupted. Together, these experimental data demonstrate that Pfn1 regulates VEC migration, invasion and capillary morphogenesis through its interaction with both actin and proline-rich ligands.

Identification of toxic oligopeptides in Amanita fungi employing capillary electrophoresis-electrospray ionization-mass spectrometry with positive and negative ion detection.

The identification of toxic oligopeptides employing CE-ESI-MS is presented. The analytes studied ama- and phallotoxins are of significant forensic interest because over 90% of the lethal cases of fungus poisoning in man are caused by species of Amanita which contain these toxins. A CE method was developed to separate the toxins alpha-, beta- and gamma-amanitin, phalloidin and phallacidin. Their fragmentation patterns in MS(n) experiments were investigated in the positive and in the negative ion mode, also the influence of the sheath liquid mixture of the used interface on the S/N. Method validation included the determination of the LOD and the repeatability of the migration time and peak area for both detection modes. With the optimized method LODs of 13-79 ng/mL (17-87 nmol/L) were reached. The CE-MS procedure was successfully applied to the identification of ama- and phallotoxins in extracts of air-dried mushroom samples.

Muscular system of a peculiar parasitic cnidarian Polypodium hydriforme: a phalloidin fluorescence study.

Musculature of the free-living stages of Polypodium hydriforme has been studied using phalloidin fluorescence method and confocal microscopy. P. hydriforme is a unique cnidarian possessing only smooth muscle cells situated within the mesoglea, not epithelial muscle cells, like the rest of cnidarians. Phalloidin fluorescence on whole mount preparations demonstrates an extensively developed subepidermal muscle system mostly consisting of long parallel fibers running along the tentacles. For the first time along with contracted muscle fibers we could clearly demonstrate relaxed fibers looking as long spirals. System of thin parallel circular F-actin positive fibers has been discovered outside of longitudinal muscles. The body of the animal and the mouth cone contain weakly developed parallel muscles. No special attachment of the muscle fibers to the tips of the tentacles or to the rim of the mouth has been observed. The results are discussed in connection with the "triploblastic" organization of P. hydriforme and its phylogenetic position.

Quantifying retinal nerve fiber layer thickness in whole-mounted retina.

In order to relate optical measurement of the retinal nerve fiber layer (RNFL) to the underlying structure, one must have accurate values for RNFL thickness at the locations measured optically. The purpose of this study was to develop a method for measuring RNFL thickness at any location on retinal tissue previously studied by other optical imaging. The method developed used confocal laser scanning microscopy (cLSM) to provide both en face and cross-sectional images of a whole-mounted retina. Isolated rat retina was fixed with 3% glutaraldehyde. Nerve fiber bundles were identified by using phalloidin to label F-actin and ganglion cell bodies were identified by DAPI fluorescent counterstain of nuclei. The flat-mounted retina was examined by cLSM. 2-D images were collected through the retina to a depth at least covering the ganglion cell layer. The images were stacked to reconstruct cross-sectional images of the measured retina. Thickness of nerve fiber bundles was measured on these synthesized cross sections and compared with the measurement from conventional histologic sections. The en face image displayed individual nerve fiber bundles and ganglion cells between bundles as different colors. Blood vessels, which also bound phalloidin, were easily distinguished from nerve fiber bundles. The en face image displayed the same pattern of nerve fiber bundles as seen in imaging measurements and simplified the identification of corresponding areas in the two modalities. The cross-sectional images provided thickness measurements of the RNFL over the entire field-of-view, not just at the points represented by the conventional histologic section, resulting in a large increase in available data.

CLSM analysis of the phalloidin-stained muscle system in Nerilla antennata, Nerillidium sp. and Trochonerilla mobilis (Polychaeta; Nerillidae).

The entire muscle system of Nerilla antennata, Nerillidium sp. and Trochonerilla mobilis was three-dimensionally reconstructed from whole mounts. In juvenile and adult specimens the F-actin musculature subset was stained with FITC-conjugated phalloidin and visualized with a confocal laser scanning microscope (cLSM). The muscle system shows the following major organization: 1) circular muscles are totally absent in the body wall; 2) the longitudinal muscles are confined in two ventral and two dorsal thick bundles; 3) additional longitudinal muscles are located in the ventro- and dorsomedian axis; 4) three segmental pairs of ventral oblique muscles elongate into the periphery: the main dorsoventral muscles that run along the body side posterior and dorsally and the anterior and posterior oblique parapodial muscles, which contribute to the ventral chaetal sacs; 5) one segmental pair of dorsal oblique parapodial muscles, contributing to the dorsal chaetal sacs; 6) five to seven small dorsoventral muscles per segment; and 7) complex head and pharyngeal musculature. These results support the belief that absence of circular muscles in the polychaete body wall is much more widely distributed than is currently presumed.

Hydroxyapatite promotes superior keratocyte adhesion and proliferation in comparison with current keratoprosthesis skirt materials.

AIM: Published clinical series suggest the osteoodontokeratoprosthesis (OOKP) may have a lower extrusion rate than current synthetic keratoprostheses. The OOKP is anchored in the eye wall by autologous tooth. The authors' aim was to compare adhesion, proliferation, and morphology for telomerase transformed keratocytes seeded on calcium hydroxyapatite (the principal mineral constituent of tooth) and materials used in the anchoring elements of commercially available synthetic keratoprostheses. METHODS: Test materials were hydroxyapatite, polytetrafluoroethylene (PTFE), polyhydroxyethyl methacrylate (HEMA), and glass (control). Cell adhesion and viability were quantified at 4 hours, 24 hours, and 1 week using a calcein-AM/EthD-1 viability/cytotoxicity assay. Focal contact expression and cytoskeletal organisation were studied at 24 hours by confocal microscopy with immunoflourescent labelling. Further studies of cell morphology were performed using light and scanning electron microscopy. RESULTS: Live cell counts were significantly greater on hydroxyapatite surfaces at each time point (p<0.04). Dead cell counts were significantly higher for PTFE at 7 days (p<0.002). ss(1) integrin expression was highest on hydroxyapatite. Adhesion structures were well expressed in flat, spread out keratocytes on both HA and glass. Keratocytes tended to be thinner and spindle shaped on PTFE. The relatively few keratocytes visible on HEMA test surfaces were rounded and poorly adherent. CONCLUSIONS: Keratocyte adhesion, spreading, and viability on hydroxyapatite test surfaces is superior to that seen on PTFE and HEMA. Improving the initial cell adhesion environment in the skirt element of keratoprostheses may enhance tissue integration and reduce device failure rates.

Detection of small differences in actomyosin function using actin labeled with different phalloidin conjugates.

This study shows that there is only a negligible difference in actomyosin function in the in vitro motility assay among actin filaments labeled with Rhodamine phalloidin (RhPh), Alexa-488 phalloidin (APh), and biotin-XX phalloidin (BPh). Similar results were obtained at varying ionic strengths (0.02-0.13 M), in the presence of imidazole or 3-[N-morpholino]propanesulfonic acid (MOPS) buffer, and at varying MgATP concentrations (0.1-3 mM). If RhPh- and APh-labeled filaments were studied in a given flow cell, there was minimal variability in sliding velocity between the fluorophores (standard deviation of 3% of the absolute sliding velocity). The variability was considerably smaller than that between flow cells, allowing us to use dual labeling of different actin types and then apply analysis of variance to detect minor functional differences between them. Using this method, we could statistically verify a 4% difference (P<0.001) in sliding velocity (3mM Mg ATP) between cardiac and skeletal muscle actin. Suggested improvements of the method would readily allow the detection of even smaller differences. We discuss implications of the results for nanotechnological applications, understanding actomyosin function, and reducing experimental costs and the use of laboratory animals.

Theta stimulation polymerizes actin in dendritic spines of hippocampus.

It has been proposed that the endurance of long-term potentiation (LTP) depends on structural changes entailing reorganization of the spine actin cytoskeleton. The present study used a new technique involving intracellular and extracellular application of rhodamine-phalloidin to conventional hippocampal slices to test whether induction of LTP by naturalistic patterns of afferent activity selectively increases actin polymerization in juvenile to young adult spines. Rhodamine-phalloidin, which selectively binds to polymerized actin, was detected in perikarya and proximal dendrites of CA1 pyramidal cells that received low-frequency afferent activity but was essentially absent in spines and fine dendritic processes. Theta pattern stimulation induced LTP and caused a large (threefold), reliable increase in labeled spines and spine-like puncta in the proximal dendritic zone containing potentiated synapses. The spines frequently occurred in the absence of labeling to other structures but were also found in association with fluorescent dendritic processes. These effects were replicated (>10-fold increase in labeled spines) using extracellular applications of rhodamine-phalloidin. Increases in labeling appeared within 2 min, were completely blocked by treatments that prevent LTP induction, and occurred in slices prepared from young adult rats. These results indicate that near-threshold conditions for inducing stable potentiation cause the rapid polymerization of actin in mature spines and suggest that the effect is both sufficiently discrete to satisfy the synapse-specificity rule of LTP as well as rapid enough to participate in the initial stages of LTP consolidation.