F-actin Patterns Research Articles

Development of a spotted sea bass (Lateolabrax maculatus) bulbus arteriosus cell line and its application to fish virology and immunology

The bulbus arteriosus tissue of teleosts, which is located at the forefront of the heart, is used to reduce the pulse pressure. In this study, we constructed a permanent cell line (LmAB) for the first time using bulbus arteriosus tissue from spotted sea bass (Lateolabrax maculatus). This cell line has been passaged more than 80 times. Currently, it can be subcultured in L-15 medium with 8 % fetal bovine serum added. The optimal fetal bovine serum concentration and culture temperature for LmAB cells at 62 passages are 20 % and 28 °C, respectively. This cell line consists predominantly of epithelial-like cells. We used 18S rRNA gene sequencing to confirm that LmAB cells originated from spotted sea bass. Karyotype analysis revealed that 43 % of LmAB cells in passage 63 had 48 chromosomes. Exogenous plasmid transfection revealed that LmAB cells can express the green fluorescent protein gene with a transfection efficiency of up to 40 %, indicating that these cells can be used for in vitro genetic research. LmAB cells showed susceptibility to nervous necrosis virus, largemouth bass ulcer syndrome virus, and infectious spleen and kidney necrosis virus, which results in severe cytopathic effects. PCR analysis verified that these viruses can replicate in LmAB cells, and analysis of cytoskeletal F-actin patterns verified that infected cells exhibit serious changes in their actin cytoskeleton. LmAB cells infected with these three viruses showed increased expressions of interferon signaling pathway genes (IFNd, IFNγ-rel, and ISG15), indicating that the host interferon signaling pathway participates in the antiviral immune response. These findings indicate that our newly developed LmAB cell line is a valuable resource for future research in genetics, virology, and immunology.

Distinct platelet F-actin patterns and traction forces on von Willebrand factor versus fibrinogen

Upon vascular injury, platelets form a hemostatic plug by binding to the subendothelium and to each other. Platelet-to-matrix binding is initially mediated by von Willebrand factor (VWF) and platelet-to-platelet binding is mediated mainly by fibrinogen and VWF. After binding, the actin cytoskeleton of a platelet drives its contraction, generating traction forces that are important to the cessation of bleeding. Our understanding of the relationship between adhesive environment, F-actin morphology, and traction forces is limited. Here, we examined F-actin morphology of platelets attached to surfaces coated with fibrinogen and VWF. We identified distinct F-actin patterns induced by these protein coatings and found that these patterns were identifiable into three classifications via machine learning: solid, nodular, and hollow. We observed that traction forces for platelets were significantly higher on VWF than on fibrinogen coatings and these forces varied by F-actin pattern. In addition, we analyzed the F-actin orientation in platelets and noted that their filaments were more circumferential when on fibrinogen coatings and having a hollow F-actin pattern, while they were more radial on VWF and having a solid F-actin pattern. Finally, we noted that subcellular localization of traction forces corresponded to protein coating and F-actin pattern: VWF-bound, solid platelets had higher forces at their central region while fibrinogen-bound, hollow platelets had higher forces at their periphery. These distinct F-actin patterns on fibrinogen and VWF and their differences in F-actin orientation, force magnitude, and force localization could have implications in hemostasis, thrombus architecture, and venous versus arterial thrombosis.

Young and Senescent Cells: Distinct Nuclear F-actin Patterns Upon Latrunculin B Induction

Both cellular senescence and cytoskeleton are involved in the formation of many diseases and cell signaling pathways. Although recent studies have shown that F-actin is involved in DNA damage repair, chromatin decompression, gene transcription regulation, and cell fate determination. But studies on F-actin and aging are still absence. It is unclear whether nuclear F-actin is present during cellular senescence. Here, by confocal optical sectioning and time-lapse imaging, we found acitn chrommobody-TagGFP2-NLS shows the beneficial on investigating senescent human fibroblast IMR-90 cells. To induce the nuclear F-actin assembly in single cell, we uesd Latrunculin B (latB) which a cytoplasmic F-actin polymerization inhibitor. It is currently unknown whether the nuclear F-actin cytoskeleton in young and senescent cells responds differently to latB treatment. Here, latB application induces distinct nuclear F-actin patterns and dynamics in young and senescent cells. Thus, after analyzing the results of actin dynamic we demonstrate a diverse effect of latB on the nuclear F-actin cytoskeleton in young and senescent cells.

The Spatiotemporal Expression Patterns of Mechanical-Stress Related Regulatory Proteins in Mouse Molar Development

In order to better understand the role of mechanical stress in early tooth development, we examined the spatiotemporal expression patterns of mechanical-stress related regulatory protein (actin filament, or F-actin), non-muscle myosin ⅡB (NMⅡB) and vinculin at different stages of tooth development in mice. Mouse first mandible molars were used as the research model. Immunofluorescence staining was performed to detect the expression patterns of F-actin, NMⅡB and Vinculin, the key molecules constituting the chemical mechanical system, at bud, cap, early bell and late bell stages of tooth. F-actin, NMⅡB and vinculin were all expressed in the tooth epithelium in an extensive or a limited way at different stages of tooth development, while F-actin was also expressed steadily in the mesenchymal cells. The quantitative analysis of fluorescence intensity showed that F-actin and NMⅡB exhibited significantly increase in the early stage of tooth development, but then dropped to their basal levels at the end of the late bell stage and the early bell stage respectively, with the differences of expression changes between successive developmental stages showing statistically significance ( P<0.05). Vinculin expression, however, remained at a relatively constant level except for the late bell stage when vinculin expression was slightly elevated compared to that of the early bell stage ( P<0.05). The findings suggest that mechanical stress is involved in early tooth development. F-actin may have an important role in dispersing and transmitting mechanical stress while NMⅡB may participate in tooth epithelial invagination and cusps formation. The findings also suggest that vinculin can respond to the mechanical stimuli and its interaction with cell adhesion molecules may play a role in tooth development. The mechanism of how actomyosin and cell adhesion interact with each other in regulating tooth development still needs further investigation.

Neuronal activity remodels the F-actin based submembrane lattice in dendrites but not axons of hippocampal neurons

The nanoscale organization of the F-actin cytoskeleton in neurons comprises membrane-associated periodical rings, bundles, and longitudinal fibers. The F-actin rings have been observed predominantly in axons but only sporadically in dendrites, where fluorescence nanoscopy reveals various patterns of F-actin arranged in mixed patches. These complex dendritic F-actin patterns pose a challenge for investigating quantitatively their regulatory mechanisms. We developed here a weakly supervised deep learning segmentation approach of fluorescence nanoscopy images of F-actin in cultured hippocampal neurons. This approach enabled the quantitative assessment of F-actin remodeling, revealing the disappearance of the rings during neuronal activity in dendrites, but not in axons. The dendritic F-actin cytoskeleton of activated neurons remodeled into longitudinal fibers. We show that this activity-dependent remodeling involves text {Ca}^{2+} and NMDA receptor-dependent mechanisms. This highly dynamic restructuring of dendritic F-actin based submembrane lattice into longitudinal fibers may serve to support activity-dependent membrane remodeling, protein trafficking and neuronal plasticity.

Imaging flow cytometry and confocal microscopy-based examination of F-actin and phosphoinositide dynamics during leukocyte immune-type receptor-mediated phagocytic events

Cells of the innate immune system rapidly detect and eliminate invading microbes using surface-expressed immunoregulatory receptors that translate extracellular binding events into potent effector responses. Channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) are a family of immunoregulatory proteins that have been shown to regulate several innate immune cell effector responses including the phagocytic process. The mechanisms by which these receptors regulate phagocytosis are not entirely understood but we have previously shown that different IpLITR-types use ITAM-dependent as well as ITAM-independent pathways for controlling target engulfment. The main objective of this study was to develop and use imaging flow cytometry and confocal microscopy-based assays to further examine both F-actin and phosphoinositide dynamics that occur during the different IpLITR-mediated phagocytic pathways. Results show that the ITAM-dependent IpLITR-induced phagocytic response promotes canonical changes in F-actin polymerization and PI(4,5)P2 redistributions. However, the ITAM-independent IpLITR phagocytic response induced unique patterns of F-actin and PI(4,5)P2 redistributions, which are likely due to its ability to regulate alternative signaling pathways. Additionally, both IpLITR-induced phagocytic pathways induced target internalization into PI(3)P-enriched phagosomes indicative of a maturing phagosome compartment. Overall, this imaging-based platform can be further applied to monitor the recruitment and distribution of signaling molecules during IpLITR-mediated phagocytic processes and may serve as a useful strategy for functional examinations of other immunoregulatory receptor-types in fish.

A Wnt-planar polarity pathway instructs neurite branching by restricting F-actin assembly through endosomal signaling.

Spatial arrangement of neurite branching is instructed by both attractive and repulsive cues. Here we show that in C. elegans, the Wnt family of secreted glycoproteins specify neurite branching sites in the PLM mechanosensory neurons. Wnts function through MIG-1/Frizzled and the planar cell polarity protein (PCP) VANG-1/Strabismus/Vangl2 to restrict the formation of F-actin patches, which mark branching sites in nascent neurites. We find that VANG-1 promotes Wnt signaling by facilitating Frizzled endocytosis and genetically acts in a common pathway with arr-1/β-arrestin, whose mutation results in defective PLM branching and F-actin patterns similar to those in the Wnt, mig-1 or vang-1 mutants. On the other hand, the UNC-6/Netrin pathway intersects orthogonally with Wnt-PCP signaling to guide PLM branch growth along the dorsal-ventral axis. Our study provides insights for how attractive and repulsive signals coordinate to sculpt neurite branching patterns, which are critical for circuit connectivity.

Role of Wasp and the small GTPases RhoA, RhoB, and Cdc42 during capacitation and acrosome reaction in spermatozoa of English guinea pigs.

Cytoskeleton remodeling is necessary for capacitation and the acrosome reaction in spermatozoa. F-actin is located in the acrosome and equatorial region during capacitation, but is relocated in the post-acrosomal region during the acrosome reaction in spermatozoa from bull, rat, mice, and guinea pig. Actin polymerization and relocalization are generally regulated by small GTPases that activate Wasp protein, which coordinates with Arp2/3, profilin I, and profilin II to complete cytoskeletal remodeling. This sequence of events is not completely described in spermatozoa, though. Therefore, the aim of this study was to determine if Wasp interacts with small GTPases (RhoA, RhoB, and Cdc42) and proteins (Arp2/3, profilin I, and profilin II) that co-localize with F-actin during capacitation and the acrosome reaction in English guinea pig spermatozoa obtained from the vas deferens. The spermatozoa were capacitated in calcium-free medium, incubated with an activator or an inhibitor of GTPases, and then induced to acrosome react using calcium. The distribution patterns of F-actin were compared to the patterns of Wasp and its putative interaction partners: Wasp and RhoB, but not RhoA or Cdc42, localization overlap with F-actin during capacitation and the acrosome reaction. Activation of small GTPases localized RhoB to the post-acrosomal region whereas their inhibition prevented acrosome exocytosis. Arp2/3 and profilin II appear to interact with Wasp in the post-acrosomal region and flagellum, while profilin I and Wasp could be found in the equatorial region. Thus, Wasp and F-actin distribution overlap during capacitation and acrosome reaction, and small GTPases play an important role in cytoskeleton remodeling during these processes in spermatozoa. Mol. Reprod. Dev. 83: 927-937, 2016 © 2016 Wiley Periodicals, Inc.

Effect of Leflunomide on the Abnormal Expression of Lipid Rafts and F-Actin in B Lymphocytes from Patients with Systemic Lupus Erythematosus.

Purposes. To investigate the possible changes in B cell subsets and in B cell expression patterns of lipid rafts (LRs) and F-actin in patients with SLE and whether leflunomide treatment may have effect on these changes. Methods. The B cell subsets and LRs expression were determined by flow cytometry and confocal microscopy, and F-actin expression was examined by confocal microscopy. Results. CD27+IgD+ B cell subsets were significantly decreased while CD38+CD95+ B cell subsets increased in SLE patients. The LRs levels of B cells were remarkably increased and positively correlated with SLEDAI and anti-dsDNA titer in SLE patients. The expression level of LRs was significantly higher in CD38+ B cells than CD38− B cells and negatively correlated with C3 levels. The increased expression of LRs was associated with reduced expression of F-actin in the B cells from active SLE patients. Furthermore, in vitro treatment of the cells with A771726 reduced the expression level of LRs, attenuated the overaggregation of LRs, and normalized the distribution of F-actin. Conclusions. There were abnormalities in B cell subsets and LRs and F-actin expression of B cell from SLE patients. Modulation of B cell expression of LRs and F-actin by LEF could be a potential therapeutic target for SLE.

Investigating the role of F-actin in human immunodeficiency virus assembly by live-cell microscopy.

Human immunodeficiency virus type 1 (HIV-1) particles assemble at the plasma membrane, which is lined by a dense network of filamentous actin (F-actin). Large amounts of actin have been detected in HIV-1 virions, proposed to be incorporated by interactions with the nucleocapsid domain of the viral polyprotein Gag. Previous studies addressing the role of F-actin in HIV-1 particle formation using F-actin-interfering drugs did not yield consistent results. Filamentous structures pointing toward nascent HIV-1 budding sites, detected by cryo-electron tomography and atomic force microscopy, prompted us to revisit the role of F-actin in HIV-1 assembly by live-cell microscopy. HeLa cells coexpressing HIV-1 carrying fluorescently labeled Gag and a labeled F-actin-binding peptide were imaged by live-cell total internal reflection fluorescence microscopy (TIR-FM). Computational analysis of image series did not reveal characteristic patterns of F-actin in the vicinity of viral budding sites. Furthermore, no transient recruitment of F-actin during bud formation was detected by monitoring fluorescence intensity changes at nascent HIV-1 assembly sites. The chosen approach allowed us to measure the effect of F-actin-interfering drugs on the assembly of individual virions in parallel with monitoring changes in the F-actin network of the respective cell. Treatment of cells with latrunculin did not affect the efficiency and dynamics of Gag assembly under conditions resulting in the disruption of F-actin filaments. Normal assembly rates were also observed upon transient stabilization of F-actin by short-term treatment with jasplakinolide. Taken together, these findings indicate that actin filament dynamics are dispensable for HIV-1 Gag assembly at the plasma membrane of HeLa cells. Importance: HIV-1 particles assemble at the plasma membrane of virus-producing cells. This membrane is lined by a dense network of actin filaments that might either present a physical obstacle to the formation of virus particles or generate force promoting the assembly process. Drug-mediated interference with the actin cytoskeleton showed different results for the formation of retroviral particles in different studies, likely due to general effects on the cell upon prolonged drug treatment. Here, we characterized the effect of actin-interfering compounds on the HIV-1 assembly process by direct observation of virus formation in live cells, which allowed us to measure assembly rate constants directly upon drug addition. Virus assembly proceeded with normal rates when actin filaments were either disrupted or stabilized. Taken together with the absence of characteristic actin filament patterns at viral budding sites in our analyses, this indicates that the actin network is dispensable for HIV-1 assembly.

Knockdown of Both p120 Catenin and Kaiso Promotes Expansion of Human Corneal Endothelial Monolayers via RhoA-ROCK-Noncanonical BMP-NFκB Pathway

To determine the signaling pathway involved in expanding contact-inhibited human corneal endothelial cells (HCECs) using p120 and Kaiso small interfering RNAs (siRNAs). Expansion of HCEC monolayers on collagen IV in SHEM using p120 siRNA was optimized regarding various dosage, frequency, and starting date before being added Kaiso siRNA or various inhibitors of Rho, ROCK, NFκB, and TAK1. Phase contrast micrographs were used for monitoring cell shape, monolayer size, and cell density. Immunostaining was used to determine cytolocalization of BrdU, p120, pNFkB, F-actin, α-catenin, β-catenin, LEF1, Na+/K+-ATPase, N-cadherin, ZO-1, and S100A4. Western blotting was used to determine the protein level of RhoA and RhoA-guanosine-5'-triphosphate (GTP). The HCEC monolayer size in diameter was expanded from 2.1 ± 0.4 mm to 4.3 ± 0.3 mm (P < 0.05) by increasing p120 siRNA from 40 nM to 100 nM starting at day 7, to 5.0 ± 0.4 mm (P < 0.05) by adding 100 nM Kaiso siRNA, to 6.8 ± 0.3 mm by using one-fourth corneoscleral rim (P < 0.05), and to 8.1 ± 0.5 mm by using one-half corneoscleral rim (P < 0.05). Such proliferative effect required activation of RhoA-ROCK-noncanonical bone morphogenic protein (BMP) signaling and nuclear translocation of phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells (pNFκB). After withdrawal of siRNAs for 1 week, the resultant HCEC monolayer maintained a hexagonal shape, the average cell density of 2254 ± 87 mm(2) (n = 3), and normal expression patterns of F-actin, α-catenin, β-catenin, N-cadherin, ZO-1, and Na+/K+-ATPase without S100A4 and alpha-smooth muscle actin (α-SMA). The optimized knockdown with p120 and Kaiso siRNAs further expands the size of HCEC monolayers without endothelial mesenchymal transition (EMT) via selective activation of p120/Kaiso signaling that requires the RhoA-ROCK-noncanonical BMP-NFkB signaling.

Ultrastructural localization of F-actin using phalloidin and quantum dots in HL-60 promyelocytic leukemia cell line after cell death induction by arsenic trioxide

Quantum dots (QDs) are fluorescent nanocrystals whose unique properties are fundamentally different from organic fluorophores. Moreover, their cores display sufficient electron density to be visible under transmission electron microscopy (TEM). Here, we report a technique for phalloidin-based TEM detection of F-actin. The ultrastructural reorganization of F-actin after arsenic trioxide (ATO) treatment was estimated using a combination of pre- and post-embedding techniques with biotinylated phalloidin and QD–streptavidin conjugates or colloidal gold (AU) conjugated to streptavidin. Ultrastructural studies showed ATO-induced apoptosis of HL-60 cells. Moreover, different patterns of QD-labeled F-actin after ATO treatment were seen. In the case of AU labeling, only a few gold particles were seen and it was impossible to see any difference in F-actin distribution. TEM imaging experiments using QDs and colloidal gold (AU) showed that the strategy of bioconjugation of nanoprobes is the most important factor in biotinylated phalloidin detection of F-actin using streptavidin-coated nanoparticles, especially at the ultrastructural level. Additionally, the results presented in present study confirm the essential role of F-actin in chromatin reorganization during cell death processes.

The neuromuscular system in continuously swimming cercariae from Belarus. II Echinostomata, Gymnocephala and Amphistomata

The neuromuscular system in cercariae of Moliniella anceps, Echinostoma revolutum, Cathaemasia hians, Psilochasmus oxyurus, Sphaeridiotrema globulus, Paramphistomum cervi and Diplodiscus subclavatus was studied with immunocytochemical methods and confocal scanning laser microscopy. The patterns of F-actin in the musculature, 5-HT immunoreactive (IR), FMRFamide-IR neuronal elements and α-tubulin-IR sensory receptors were investigated. The general patterns of musculature, 5-HT- and FMRFamide-IR neuronal elements in the 12 species studied here and in paper I are similar to those observed in other cercariae and reflect the morphology of the groups. The musculature of the tail shows variations which are related to the different strategies of host finding. In the Echinostomatoidea and Paramphistomoidea, the striated musculature of the tail is well developed compared to that in the Xiphidiocercariae. Specialized muscle fibres were found in S. globulus, which are able to change the shape of the tail. Nine of the species studied have seven paired 5-HT-IR neurons in the body, and two species have eight. No correlation between the body size and the number of 5-HT-IR neurons was observed. However, the size of the neurons followed the body size. The number of 5-HT-IR neurons in the brain ganglia increased from the primitive to the advanced forms. The number of FMRFamide-IR transverse commissures in the body correlates with the size of the cercariae. Regardless of the differences in the second intermediate host, the distribution of α-tubulin-IR sensory receptors shows a high degree of conformity in all species except in P. cervi, which encysts on plants.

The neuro-muscular system in continuously swimming cercariae from Belarus. I Xiphidiocercariae

The neuromuscular system (NMS) in cercariae of Neoastiotrema trituri, Plagiorchis elegans, Omphalometra flexuosa, Skrjabinoeces similis and Prosthogonimus ovatus was studied with immunocytochemical methods and confocal scanning laser microscopy. The patterns of F-actin in the musculature, 5-HT immunoreactive (IR), FMRFamide-IR neuronal elements and α-tubulin-IR sensory receptors were investigated, and they were found to be rather similar in all the cercariae studied. Four species have seven paired 5-HT-IR neurons in the body, and P. elegans has eight. N. trituri has three 5-HT-IR neurons in each brain ganglion, while the other species have four. A high degree of conformity in the structure of the NMS was observed, probably reflecting the close phylogenetic relationship and the similar strategy of host finding.

Cloning and characterization of the actin gene from Puccinia striiformis f. sp. tritici

The fungus Puccinia striiformis f. sp. tritici, the causal agent of wheat stripe rust, is an obligate biotrophic basidiomycete. Urediniospores are the most common spore type involved in the epidemiology of this disease. Tip growth of germ tubes of germinated urediniospores is a key step during infection of wheat, but few studies have investigated it so far. Recent research has found that actin is closely associated with hyphal tip growth. In this study, we have cloned and obtained the full-length actin cDNA from P. striiformis f. sp. tritici and characterized its expression. Furthermore, actin filament (F-actin) patterns were visualized microscopically during germ tube formation. The most conspicuous actin-containing structures were actin patches. They were mainly concentrated near the hyphal tip and scattered throughout the cortex. By using cytochalasin B, we observed that depolymerization of F-actin greatly reduced the germination rate of urediniospores and disrupted the transport of vesicles to the germ tube tip, indicating that F-actin played a key role in the tip growth of P. striiformis f. sp. tritici. This work helps us to understand the tip growth mechanism of P. striiformis f. sp. tritici, and may provide a theoretical framework for designing novel pesticides.

The neuromuscular system in freshwater furcocercaria from Belarus. II Diplostomidae, Strigeidae, and Cyathocotylidae

The neuromuscular system (NMS) in cercariae of Diplostomum pseudospathaceum, Cotylurus szidati, Australapatemon burti, Holostephanus volgensis, and Paracoenogonimus ovatus was studied with immunocytochemical methods and confocal scanning laser microscopy. The patterns of F-actin in the musculature, 5-HT immunoreactive (-IR), FMRF-amide-IR neuronal elements, and α-tubulin-IR in sensory receptors were investigated. The NMS in the five species studied were compared with each other and with three species of Schistosomatidae studied earlier (Bilharziella polonica, Trichobilharzia szidati, and Trichobilharzia franki). No major structural differences in the musculature, the 5-HT-IR or FMRF-IR neuronal elements were noticed between the cercariae. The minor variations observed in the musculature were related to the size and organization of the muscle fibers. The checked pattern formed by the transverse muscle fibers in the tail stems of D. pseudospathaceum, C. szidati, A. burti, H. volgensis, and P. ovatus was not observed in B. polonica, T. szidati, and T. franki. A trend in the differentiation of the longitudinal muscle fibers in the furca from evenly distributed fibers in H. volgensis and P. ovatus to many bundles in D. pseudospathaceum and two well-organized lateral bundles in C. szidati, A. burti, and Trichobilharzia spp. was observed. The transverse muscle fibers in the furca follow the same trend. The number of 5-HT-IR neurons in the cercarial bodies varied between 10 and 16. In cercariae of H. volgensis and P. ovatus, the central nervous system (CNS) was less centralized compared to the CNS in the other species studied, with only two 5-HT-IR marker neurons in each brain ganglion and the other neurons distributed evenly along the main cords. In the tails of H. volgensis and P. ovatus, many transverse 5-HT-IR comissures were found. In the tails of higher strigeidid cercariae, only a few crosslinks were observed. The number and distribution of sensory receptors on the bodies and tails of the cercarial species differed from each other. A trend in the differentiation of the sensory receptors in the tails was discerned. A process of grouping and decrease in number of ciliated receptors in the stem and in the furca from H. volgensis and P. ovatus to Schistosomatid cercariae took place.

The neuro-muscular system in fresh-water furcocercaria from Belarus. I Schistosomatidae

The neuro-muscular system (NMS) in cercariae of the family Schistosomatidae from Belarus was studied with immunocytochemical methods and confocal scanning laser microscopy. The specimens of Bilharziella polonica were compared with Trichobilharzia szidati and Trichobilharzia franki. The patterns of F-actin in the musculature, 5-HT-immunoreactive (IR), FMRFamide-IR neuronal elements and α-tubulin-IR in sensory receptors and nerves were investigated. No indications of structural differences in the musculature, the 5-HT-IR, FMRF-IR neuronal elements and the general distribution of sensory receptors were noticed between cercariae of Trichobilharzia spp. The number of 5-HT-IR neurons in the cercarial bodies is 16. In cercaria B. polonica, the tail musculature is weaker than in Trichobilharzia spp. A detailed schematic picture of the NMS in the tail of Trichobilharzia spp. cercaria is given. The function of NMS elements in the tail is discussed.

The Actin Cytoskeleton Has an Active Role in the Electrotransfer of Plasmid DNA in Mammalian Cells

Electrotransfer of molecules is a well established technique which finds extensive use for gene transfer and holds great promise for anticancer treatment. Despite its widespread application, the mechanisms governing the entry of DNA into the cell and its intracellular trafficking are not yet known. The aim of this study is to unravel the role of the actin cytoskeleton during gene electrotransfer in cells. We performed single-cell level approaches to observe the organization of the actin cytoskeleton in Chinese hamster ovary (CHO) cells. In addition, we performed experiments at the multiple-cell level to evaluate the efficiency of DNA transfer after alteration of the actin cytoskeleton using the drug latrunculin B. Actin patches colocalizing with the DNA at the plasma membrane were observed with additional characteristics similar to those of the DNA aggregates in terms of time, number, and size. The disruption of the microfilaments reduces the DNA accumulation at the plasma membrane and the gene expression. This is the first direct experimental evidence of the participation of the actin cytoskeleton in DNA electrotransfer.

Immunocytochemical Analysis of a Three-dimensional Spheroidal Culture System

Three-dimensional (3D) cell cultures are expected to mimic in vivo environments. The NanoCulture plate (Scivax Corporation, Kawasaki) allows the formation of 3D spheroids. The cytoskeletal proteins are considered to be important regulators of cellular morphology and structure. Among the cytoskeletal proteins, fibrillar actin (F-actin) is a main component of microfilament proteins, and α-tubulin is the major protein forming microtubules in cells. Therefore, in this study, we used NanoCulture plates to examine the expression patterns of F-actin and α-tubulin in PANC-1, a human pancreatic cancer cell line. The cells were stained with polyclonal antibodies against α-tubulin antibody and with Alexa 568-labeled phalloidin to detect F-actin. Eighty-eight horizontal images (Fig. 1) were obtained at 0.25-μm intervals with a confocal laser microscope (TE2000-E, Nikon Instech Co., Ltd., Tokyo). In horizontal images, the expression of F-actin was observed at the periphery of cells (Fig. 2A― C, red), and strong F-actin expression was observed on the grids of the NanoCulture plate at the levels of cells nearest the plate (Fig. 2C, arrows and inset). The expression of α-tubulin was observed in the cytoplasm of cells in 3D cultures (Fig. 2A―C, green). Vertical images revealed that the cells piled up in 3D culture (Fig. 2D) and that cells formed actin-rich cell projections that attached to the surface (Fig. 2D arrows). The 3D images Correspondence to Zenya Naito, MD, PhD, Departments of Pathology, Integrative Oncological Pathology, Nippon Medical School, 1―1―5 Sendagi, Bunkyo-ku, Tokyo 113―8602, Japan

The neuro-muscular system in cercaria with different patterns of locomotion

The neuro-muscular system (NMS) of cercariae with different swimming patterns was studied with immunocytochemical methods and confocal scanning laser microscopy. Specimens of the continuously swimming Cercaria parvicaudata, Maritrema subdolum and Himasthla elongata were compared with specimens of the intermittently swimming Cryptocotyle lingua and the attached Podocotyle atomon. The patterns of F-actin in the musculature, 5-HT immunoreactive (-IR), FMRFamide-IR neuronal elements, α-tubulin-IR elements in the nervous and sensory systems and DAPI-stained nuclei were investigated. The general plan of the NMS was similar in all cercariae studied. No major structural differences in the patterns of muscle fibres were observed. However, in the tail of C. lingua, transverse muscle fibres connecting the bands of longitudinal muscles were found. No major structural differences in the 5-HT- or FMRFamide-IR nervous systems were observed. The number of 5-HT-IR neurones in the cercarial bodies varied between 12 and 14. The number and distribution of the α-tubulin-IR processes on the cercarial bodies and tails differed from each other. The relation between the number and structure of the α-tubulin-IR processes and the host finding strategy of the cercariae is discussed. A detailed schematic picture of the NMS in the tails of C. lingua and M. subdolum is presented.