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The formation of a vertebrate skeletal muscle fiber involves a series of sequential and interdependent events that occurs during embryogenesis. One of these events is myoblast fusion which has been widely studied, yet not completely understood. It was previously shown that during myoblast fusion there is an increase in the expression of Na+/K+-ATPase. This fact prompted us to search for a role of the enzyme during chick in vitro skeletal myogenesis. Chick myogenic cells were treated with the Na+/K+-ATPase inhibitor ouabain in four different concentrations (0.01-10 μM) and analyzed. Our results show that 0.01, 0.1 and 1 μM ouabain did not induce changes in cell viability, whereas 10 μM induced a 45% decrease. We also observed a reduction in the number and thickness of multinucleated myotubes and a decrease in the number of myoblasts after 10 μM ouabain treatment. We tested the involvement of MEK-ERK and p38 signaling pathways in the ouabain-induced effects during myogenesis, since both pathways have been associated with Na+/K+-ATPase. The MEK-ERK inhibitor U0126 alone did not alter cell viability and did not change ouabain effect. The p38 inhibitor SB202190 alone or together with 10 μM ouabain did not alter cell viability. Our results show that the 10 μM ouabain effects in myofiber formation do not involve the MEK-ERK or the p38 signaling pathways, and therefore are probably related to the pump activity function of the Na+/K+-ATPase.

Colon cancer is one of the most prevalent types of cancer in the world and is one of the leading causes of cancer death. The anti-metabolite 5- fluorouracil (5-FU) is widely used in the treatment of patients with colon cancer and other cancer types. 5-FU-based chemotherapy has been shown to be very efficient in the improvement of overall survival of the patients and for the eradication of the disease. Unfortunately, common side effects of 5-FU include severe alterations in the motility of the gastrointestinal tissues. Nevertheless, the molecular and cellular effects of 5-FU in smooth muscle cells are poorly understood. Primary smooth muscle cell cultures are an important tool for studies of the biological consequences of 5-FU at the cellular level. The avian gizzard is one of the most robust organs of smooth muscle cells. Here we studied the molecular and cellular effects of the chemotherapic drug 5-FU in a primary culture of chick gizzard smooth muscle cells. We found that treatment of smooth muscle cells with 5-FU inhibits cell proliferation by the arrest of cells in the G1 phase of cell cycle and induce apoptosis. 5-FU induced a decrease in the percentage of histone H3-positive cells. Treatment of cells with 5-FU induced changes in cellular and nuclear morphology, a decrease in the number of stress fibers and a major decrease in the number of caveolin-3 positive cells. Our results suggest that the disorganization of the actin cytoskeleton and the reduction of caveolin-3 expression could explain the alterations in contractility observed in patients treated with 5-FU. These findings might have an impact in the understanding of the cellular effects of 5-FU in smooth muscle tissues and might help the improvement of new therapeutic protocols for the treatment of colon cancer.

In Laurencia dendroidea, halogenated secondary metabolites are primarily located in the vacuole named the corps en cerise (CC). For chemical defence at the surface level, these metabolites are intracellularly mobilised through vesicle transport from the CC to the cell periphery for posterior exocytosis of these chemicals. The cell structures involved in this specific vesicle traffic as well as the cellular structures related to the positioning and anchoring of the CC within the cell are not well known. Here, we aimed to investigate the role of cytoskeletal elements in both processes. Cellular and molecular assays were conducted to i) determine the ultrastructural apparatus involved in the vesicle traffic, ii) localise cytoskeletal filaments, iii) evaluate the role of different cytoskeletal filaments in the vesicle transport, iv) identify the cytoskeletal filaments responsible for the positioning and anchoring of the CC, and v) identify the transcripts related to cytoskeletal activity and vesicle transport. Our results show that microfilaments are found within the connections linking the CC to the cell periphery, playing an essential role in the vesicle traffic at these connections, which means a first step of the secondary metabolites transport to the cell surface. After that, the microtubules work in the positioning of the vesicles along the cell periphery towards specific regions where exocytosis takes place, which corresponds to the second step of the secondary metabolites transport to the cell surface. In addition, microtubules are involved in anchoring and positioning the CC to the cell periphery. Transcriptomic analysis revealed the expression of genes coding for actin filaments, microtubules, motor proteins and cytoskeletal accessory proteins. Genes related to vesicle traffic, exocytosis and membrane recycling were also identified. Our findings show, for the first time, that actin microfilaments and microtubules play an underlying cellular role in the chemical defence of red algae.

Cardiac cells are organized in vivo in a complex tridimensional structural organization that is crucial for heart function. While in vitro studies can reveal details about cardiac cell biology, usually cells are grown on simplified two-dimensional (2D) environments. To address these differences, we established a cardiac cell culture composed of both 2D and three-dimensional (3D)-organized cells. Our results shows significant differences between the two culture contexts in relation to the overall morphology of the cells, contraction ability, proliferation rate, presence of intercellular adhesion structures, organization of myofibrils, mitochondria morphology, endoplasmic reticulum contents, cytoskeletal filaments and extracellular matrix distribution, and expression of markers of cardiac differentiation. Cardiac cells grown in 2D-context displayed a flattened and well spread shape, were mostly isolated and their cytoplasm was filled with a large network of microfilaments and microtubules. In contrast, 3D-cells were smaller in size, were always in close contact with each other with several cellular junctions, and displayed a less conspicuous cytoskeletal network. 3D-cells had more mitochondria and myofibrils and these cells contract spontaneously more often than 2D-cells. On the other hand, endoplasmic reticulum membranes were present in higher amounts in 2D-cells when compared to 3D-cells. The expression of desmin, cadherin and alpha-actinin was higher in 3D-aggregates compared to 2D-spread cells. These findings indicate that the tridimensional environment in which the cardiac cells are grown influence several aspects of cardiac differentiation, including cell adhesion, cell shape, myofibril assembly, mitochondria contents and protein expression. We suggest that the use of this cardiac culture model, with 2D and 3D-context cells, could be useful for studies on the effects of different drugs, or growth factors, giving valuable information on the biological response of cells grown in different spatial organizations.

BACKGROUND Streptococcus agalactiae capsular type III strains are a leading cause of invasive neonatal infections. Many pathogens have developed mechanisms to escape from host defense response using the host membrane microdomain machinery. Lipid rafts play an important role in a variety of cellular functions and the benefit provided by interaction with lipid rafts can vary from one pathogen to another. OBJECTIVES This study aims to evaluate the involvement of membrane microdomains during infection of human endothelial cell by S. agalactiae. METHODS The effects of cholesterol depletion and PI3K/AKT signaling pathway activation during S. agalactiae-human umbilical vein endothelial cells (HUVEC) interaction were analysed by pre-treatment with methyl-β-cyclodextrin (MβCD) or LY294002 inhibitors, immunofluorescence and immunoblot analysis. The involvement of lipid rafts was analysed by colocalisation of bacteria with flotillin-1 and caveolin-1 using fluorescence confocal microscopy. FINDINGS In this work, we demonstrated the importance of the integrity of lipid rafts microdomains and activation of PI3K/Akt pathway during invasion of S. agalactiae strain to HUVEC cells. Our results suggest the involvement of flotillin-1 and caveolin-1 during the invasion of S. agalactiae strain in HUVEC cells. CONCLUSIONS The collection of our results suggests that lipid microdomain affects the interaction of S. agalactiae type III belonging to the hypervirulent ST-17 with HUVEC cells through PI3K/Akt signaling pathway.

Simvastatin is one of the most common medicines prescribed to treat human hypercholesterolemia. Simvastatin acts through the inhibition of cholesterol synthesis. Unfortunately, simvastatin causes unwanted side effects on muscles, such as soreness, tiredness, or weakness. Therefore, to understand the mechanism of action of simvastatin, it is important to study its physiological and structural impacts on muscle in varied animal models. Here we report on the effects of simvastatin on two biological models: zebrafish embryos and chicken muscle culture. In the last years, our group and others showed that simvastatin treatment in zebrafish embryos reduces fish movements and induces major structural alterations in skeletal muscles. We also showed that simvastatin and membrane cholesterol depletion induce major changes in proliferation and differentiation of muscle cells in chick muscle cultures. Here, we review and discuss these observations considering reported data on the use of simvastatin as a potential therapy for Duchenne muscular dystrophy.

Lysosomes are highly dynamic organelles involved in the breakdown and recycling of macromolecules, cell cycle, cell differentiation, and cell death, among many other functions in eukaryotic cells. Recently, lysosomes have been identified as cellular hubs for the modulation of intracellular signaling pathways, such as the Wnt/beta-catenin pathway. Here we analyzed morphological and functional characteristics of lysosomes in muscle and non-muscle cells during chick myogenesis, as well as their modulation by the Wnt/beta-catenin pathway. Our results show that (i) muscle and non-muscle cells show differences in lysosomal size and its distribution, (ii) lysosomes are found in spherical structures in myoblasts and fibroblasts and tubular structures in myotubes, (iii) lysosomes are found close to the plasma membrane in fibroblasts and close to the nucleus in myoblasts and myotubes, (iv) lysosomal distribution and size are dependent on the integrity of microtubules and microfilaments in myogenic cells, (v) alterations in lysosomal function, in the expression of LAMP2, and in Wnt/beta-catenin pathway affect the distribution and size of lysosomes in myogenic cells, (vi) the effects of the knockdown of LAMP2 on myogenesis can be rescued by the activation of the Wnt/beta-catenin pathway, and (vii) the chloroquine Lys05 is a potent inhibitor of both the Wnt/beta-catenin pathway and lysosomal function. Our data highlight the involvement of the Wnt/beta-catenin pathway in the regulation of the positioning, size, and function of lysosomes during chick myogenesis.

The mechanisms involved in the development of skeletal muscle fibers have been studied in the last 70 years and yet many aspects of this process are still not completely understood. A myriad of in vivo and in vitro invertebrate and vertebrate animal models has been used for dissecting the molecular and cellular events involved in muscle formation. Among the most used animal models for the study of myogenesis are the rodents rat and mouse, the fruit fly Drosophila, and the birds chicken and quail. Here, we describe the robustness and advantages of the chick primary muscle culture model for the study of skeletal myogenesis. In the myoblast culture obtained from embryonic chick pectoralis muscle it is possible to analyze all the steps involved in skeletal myogenesis, such as myoblast proliferation, withdrawal from cell cycle, cell elongation and migration, myoblast alignment and fusion, the assembly of striated myofibrils, and the formation of multinucleated myotubes. The fact that in vitro chick myotubes can harbor hundreds of nuclei, whereas myotubes from cell lines have only a dozen nuclei demonstrates the high level of differentiation of the autonomous chick myogenic program. This striking differentiation is independent of serum withdrawal, which points to the power of the model. We also review the major pro-myogenic and anti-myogenic molecules and signaling pathways involved in chick myogenesis, in addition to providing a detailed protocol for the preparation of embryonic chick myogenic cultures. Moreover, we performed a bibliometric analysis of the articles that used this model to evaluate which were the main explored topics of interest and their contributors. We expect that by describing the major findings, and their advantages, of the studies using the embryonic chick myogenic model we will foster new studies on the molecular and cellular process involved in muscle proliferation and differentiation that are more similar to the actual in vivo condition than the muscle cell lines.

Chronic wounds are a public health problem worldwide, especially those related to diabetes. Besides being an enormous burden to patients, it challenges wound care professionals and causes a great financial cost to health system. Considering the absence of effective treatments for chronic wounds, our aim was to better understand the pathophysiology of tissue repair in diabetes in order to find alternative strategies to accelerate wound healing. Nucleotides have been described as extracellular signaling molecules in different inflammatory processes, including tissue repair. Adenosine-5’-diphosphate (ADP) plays important roles in vascular and cellular response and is immediately released after tissue injury, mainly from platelets. However, despite the well described effect on platelet aggregation during inflammation and injury, little is known about the role of ADP on the multiple steps of tissue repair, particularly in skin wounds. Therefore, we used the full-thickness excisional wound model to evaluate the effect of local ADP application in wounds of diabetic mice. ADP accelerated cutaneous wound healing, improved new tissue formation, and increased both collagen deposition and transforming growth factor-β (TGF-β) production in the wound. These effects were mediated by P2Y12 receptor activation since they were inhibited by Clopidogrel (Clop) treatment, a P2Y12 receptor antagonist. Furthermore, P2Y1 receptor antagonist also blocked ADP-induced wound closure until day 7, suggesting its involvement early in repair process. Interestingly, ADP treatment increased the expression of P2Y12 and P2Y1 receptors in the wound. In parallel, ADP reduced reactive oxygen species (ROS) formation and tumor necrosis factor-α (TNF-α) levels, while increased IL-13 levels in the skin. Also, ADP increased the counts of neutrophils, eosinophils, mast cells, and gamma delta (γδ) T cells (Vγ4+ and Vγ5+ cells subtypes of γδ+ T cells), although reduced regulatory T (Tregs) cells in the lesion. In accordance, ADP increased fibroblast proliferation and migration, myofibroblast differentiation, and keratinocyte proliferation. In conclusion, we provide strong evidence that ADP acts as a pro-resolution mediator in diabetes-associated skin wounds and is a promising intervention target for this worldwide problem.

Microscopy is the main technique to visualize and study the structure and function of cells. The impact of optical and electron microscopy techniques is enormous in all fields of biomedical research. It is possible that different research areas rely on microscopy in diverse ways. Here, we analyzed comparatively the use of microscopy in pharmacology and cell biology, among other biomedical sciences fields. We collected data from articles published in several major journals in these fields. We analyzed the frequency of use of different optical and electron microscopy techniques: bright field, phase contrast, differential interference contrast, polarization, conventional fluorescence, confocal, live cell imaging, super resolution, transmission and scanning electron microscopy, and cryoelectron microscopy. Our analysis showed that the use of microscopy has a distinctive pattern in each research area, and that nearly half of the articles from pharmacology journals did not use any microscopy method, compared to the use of microscopy in almost all the articles from cell biology journals. The most frequent microscopy methods in all the journals in all areas were bright field and fluorescence (conventional and confocal). Again, the pattern of use was different: while the most used microscopy methods in pharmacology were bright field and conventional fluorescence, in cell biology the most used methods were conventional and confocal fluorescence, and live cell imaging. We observed that the combination of different microscopy techniques was more frequent in cell biology, with up to 6 methods in the same article. To correlate the use of microscopy with the research theme of each article, we analyzed the proportion of microscopy figures with the use of cell culture. We analyzed comparatively the vocabulary of each biomedical sciences field, by the identification of the most frequent words in the articles. The collection of data described here shows a vast difference in the use of microscopy among different fields of biomedical sciences. The data presented here could be valuable in other scientific and educational contexts.

LMO7 is a multifunctional PDZ–LIM protein that can interact with different molecular partners and is found in several intracellular locations. The aim of this work was to shed light on LMO7 evolution, alternative transcripts, protein structure and gene regulation through multiple in silico analyses. We also explored the intracellular distribution of the LMO7 protein in chicken and zebrafish embryonic skeletal muscle cells by means of confocal fluorescence microscopy. Our results revealed a single LMO7 gene in mammals, sauropsids, Xenopus and in the holostean fish spotted gar while two lmo7 genes (lmo7a and lmo7b) were identified in teleost fishes. In addition, several different transcripts were predicted for LMO7 in human and in major vertebrate model organisms (mouse, chicken, Xenopus and zebrafish). Bioinformatics tools revealed several structural features of the LMO7 protein including intrinsically disordered regions. We found the LMO7 protein in multiple intracellular compartments in chicken and zebrafish skeletal muscle cells, such as membrane adhesion sites and the perinuclear region. Curiously, the LMO7 protein was detected within the nuclei of muscle cells in chicken but not in zebrafish. Our data showed that a conserved regulatory element may be related to muscle-specific LMO7 expression. Our findings uncover new and important information about LMO7 and open new challenges to understanding how the diverse regulation, structure and distribution of this protein are integrated into highly complex vertebrate cellular milieux, such as skeletal muscle cells.

Sponges have a high capacity for regeneration and this process improves biomass production in some species, thus contributing to a solution for the biomass supply problem for biotechnological applications. The aim of this work is to characterize the dynamics of cell behavior during the initial stages of sponge regeneration, using bright-field microscopy, confocal microscopy and SEM. We focused on the first 20 h of regeneration, during which blastema formation and epithelium initialization occur. An innovative sponge organotypic culture of the regenerating internal region is described and investigated by confocal microscopy, cell transplantation and vital staining. Cell-cell interaction and cell density are shown to affect events in morphogenesis such as epithelial/mesenchymal and mesenchymal/epithelial transitions as well as distinct cell movements required for regeneration. Extracellular matrix was organized according to the morphogenetic process observed, with evidence for cell-signaling instructions and remodeling. These data and the method of organotypic culture described here provide support for the development of viable sponge biomass production.

Actin-based structures play fundamental roles in cellular functions. However it remains controversial how cells cope with the absence of F-actin structures. This report focuses on short- and long-term effects of cytochalasin B (CB) on actin-complexes in fibroblasts and myoblasts. Thirty min of CB treatment dispersed subplasma actin cortices, lamellipodia, ruffled membranes, stress fibers and adhesion plaques into actin patches in fibroblasts and muscle cells. In contrast, 72 hrs CB treatment showed distinct morphological effects. Fibroblasts became giant multinucleated-finger shaped with 5 to 10 protrusions, 3-8 μm in width, and >200 μm in length. They lacked cortical actin, stress fibers, adhesion plaques and ruffled membranes but contained immense lamelliopodia with abnormal adhesion plaque protein complexes. Muscle cells transformed into multinucleated globular-shaped but contained normal I-Z-I and A-bands, indicating that CB did not interfere with the assembly of myofibrils. Within 30 min after CB removal, finger-shaped fibroblasts returned to their original shape and actin-containing structures rapidly reappeared, whereas muscle cells respond slowly to form elongated myotubes following CB washout. The capacity to grow, complete several nuclear cycles, assemble intermediate filaments and microtubules without a morphologically recognizable actin cytoskeleton raises interesting issues related to the role of the actin compartments in eukaryotic cells.

Bone marrow stromal cells (BMSCs) are considered a promising tool for bone bioengineering. However, the mechanisms controlling osteoblastic commitment are still unclear. Osteogenic differentiation of BMSCs requires the activation of β-catenin signaling, classically known to be regulated by the canonical Wnt pathway. However, BMSCs treatment with canonical Wnts in vitro does not always result in osteogenic differentiation and evidence indicates that a more complex signaling pathway, involving cadherins, would be required to induce β-catenin signaling in these cells. Here we showed that Wnt3a alone did not induce TCF activation in BMSCs, maintaining the cells at a proliferative state. On the other hand, we verified that, upon BMSCs osteoinduction with dexamethasone, cadherins were cleaved by the PS1/γ-secretase complex at the plasma membrane, and this event was associated with an enhanced β-catenin translocation to the nucleus and signaling. When PS1/γ-secretase activity was inhibited, the osteogenic process was impaired. Altogether, we provide evidence that PS1/γ-secretase-mediated cadherin cleavage has as an important role in controlling β-catenin signaling during the onset of BMSCs osteogenic differentiation, as part of a complex signaling pathway responsible for cell fate decision. A comprehensive map of these pathways might contribute to the development of strategies to improve bone repair.

Myoblasts undergo a series of changes in the composition and dynamics of their plasma membranes during the initial steps of skeletal muscle differentiation. These changes are crucial requirements for myoblast fusion and allow the formation of striated muscle fibers. Membrane microdomains, or lipid rafts, have been implicated in myoblast fusion. Flotillins are scaffold proteins that are essential for the formation and dynamics of lipid rafts. Flotillins have been widely studied over the last few years, but still little is known about their role during skeletal muscle differentiation. In the present study, we analyzed the expression and distribution of flotillin-2 in chick, mice and human muscle cells grown in vitro. Primary cultures of chick myogenic cells showed a decrease in the expression of flotillin-2 during the first 72 hours of muscle differentiation. Interestingly, flotillin-2 was found to be highly expressed in chick myogenic fibroblasts and weakly expressed in chick myoblasts and multinucleated myotubes. Flotillin-2 was distributed in vesicle-like structures within the cytoplasm of chick myogenic fibroblasts, in the mouse C2C12 myogenic cell line, and in neonatal human muscle cells. Cryo-immunogold labeling revealed the presence of flotillin-2 in vesicles and in Golgi stacks in chick myogenic fibroblasts. Further, brefeldin A induced a major reduction in the number of flotillin-2 containing vesicles which correlates to a decrease in myoblast fusion. These results suggest the involvement of flotillin-2 during the initial steps of skeletal myogenesis.