Rotenone inhibits embryonic chick myogenesis in a ROS-dependent mechanism.

Skeletal muscle function is highly dependent on the energy supply provided by mitochondria. Besides ATP production, mitochondria have several other roles, such as calcium storage, heat production, cell death signaling, autophagy regulation and redox state modulation. Mitochondrial function is crucial for skeletal muscle fiber formation. Disorders that affect mitochondria have a major impact in muscle development and function. Here we studied the role of mitochondria during chick skeletal myogenesis. We analyzed the intracellular distribution of mitochondria in myoblasts, fibroblasts and myotubes using Mitotracker labeling. Mitochondrial respiration was investigated in chick muscle cells. Our results show that (i) myoblasts and myotubes have more mitochondria than muscle fibroblasts; (ii) mitochondria are organized in long lines within the whole cytoplasm and around the nuclei of myotubes, while in myoblasts they are dispersed in the cytoplasm; (iii) the area of mitochondria in myotubes increases during myogenesis, while in myoblasts and fibroblasts there is a slight decrease; (iv) mitochondrial length increases in the three cell types (myoblasts, fibroblasts and myotubes) during myogenesis; (v) the distance of mitochondria to the nucleus increases in myoblasts and myotubes during myogenesis; (vi) Rotenone inhibits muscle fiber formation, while FCCP increases the size of myotubes; (vii) N-acetyl cysteine (NAC), an inhibitor of ROS formation, rescues the effects of Rotenone on muscle fiber size; and (viii) Rotenone induces the production of ROS in chick myogenic cells. The collection of our results suggests a role of ROS signaling in mitochondrial function during chick myogenesis.

Manganese Exposure Induces Cellular Aggregates and the Accumulation of ß-Catenin in Skin of Zebrafish Embryos.

The effects of manganese (Mn) toxicity in different organs and tissues in humans and other vertebrates have been studied since the beginning of the past century, but most of its cellular effects remain largely unknown. In this study, we studied the effects of Mn in zebrafish, at the cellular level, due to the transparent nature of zebrafish larvae that enables a powerful analysis under the light microscope. The collection of our results shows that environmental concentrations of 0.5 mg/L affect swim bladder inflation; at concentration of 50 and 100 mg/L Mn (1) induces alterations in viability, swim bladder, heart, and size of zebrafish larvae, (2) induces an increase in melanocyte area and the formation of cellular aggregates in the skin, and (3) induces an accumulation of ß-Catenin in mesenchymal cells in the caudal fin of zebrafish larvae. Our data suggest that increased levels of Mn induce cell aggregate formation in the skin and the presence of more melanocytes in the zebrafish caudal fin. Interestingly, the adhesion protein ß-Catenin was activated in mesenchymal cells near the cell aggregates. These results open important new questions on the role of Mn toxicity on cellular organization and ß-Catenin responses in fishes.

Blood meal digestion and changes in lipid reserves are associated with the post-ecdysis development of the flight muscle and ovary in young adults of Rhodnius prolixus.

Rhodnius prolixus is a hemimetabolous, hematophagous insect, and both nymphs and adults feed exclusively on blood. The blood feeding triggers the molting process and, after five nymphal instar stages, the insect reaches the winged adult form. After the final ecdysis, the young adult still has a lot of blood in the midgut and, thus, we have investigated the changes in protein and lipid contents that are observed in the insect organs as digestion continues after molting. Total midgut protein content decreased during the days after the ecdysis, and digestion was finished fifteen days later. Simultaneously, proteins and triacylglycerols present in the fat body were mobilized, and their contents decreased, whereas they increased in both the ovary and the flight muscle. In order to evaluate the activity of de novo lipogenesis of each organ, the fat body, ovary and flight muscle were incubated in the presence of radiolabeled acetate, and the fat body showed the highest efficiency rate (around 47%) to convert the taken up acetate into lipids. The levels of de novo lipid synthesis in the flight muscle and ovary were very low. When 3H-palmitate was injected into the young females, its incorporation by the flight muscle was higher than by the ovary or the fat body. In the flight muscle, the 3H-palmitate was similarly distributed amongst triacylglycerols, phospholipids, diacylglycerols and free fatty acids, while in the ovary and fat body it was mostly found in triacylglycerols and phospholipids. The flight muscle was not fully developed after the molt, and at day two no lipid droplets were observed. At day five, very small lipid droplets were present, and they increased in size up to day fifteen. The diameter of the muscle fibers also increased from day two to fifteen, as well as the internuclear distance, indicating that muscle hypertrophy occurred along these days. The lipid droplets from the fat body showed a different pattern, and their diameter decreased after day two, but started to increase again at day ten. The data presented herein describes the development of the flight muscle after the final ecdysis, and modifications that occur regarding lipid stores. We show that, after molting, substrates that are present in the midgut and fat body are mobilized and directed to the ovary and flight muscle, for the adults of R. prolixus to be ready to feed and reproduce.

Improving quantification of myotube width and nuclear/cytoplasmic ratio in myogenesis research.

BACKGROUND AND OBJECTIVE: The culture of skeletal muscle cells is particularly relevant to basic biomedical research and translational medicine. The incubation of dissociated cells under controlled conditions has helped to dissect several molecular mechanisms associated with muscle cell differentiation, in addition to contributing for the evaluation of drug effects and prospective cell therapies for patients with degenerative muscle pathologies. The formation of mature multinucleated myotubes is a stepwise process involving well defined events of cell proliferation, commitment, migration, and fusion easily identified through optical microscopy methods including immunofluorescence and live cell imaging. The characterization of each step is usually based on muscle cell morphology and nuclei number, as well as the presence and intracellular location of specific cell markers. However, manual quantification of these parameters in large datasets of images is work-intensive and prone to researcher's subjectivity, mostly because of the extremely elongated cell shape of large myotubes and because myotubes are multinucleated. METHODS: Here we provide two semi-automated ImageJ macros aimed to measure the width of myotubes and the nuclear/cytoplasmic localization of molecules in fluorescence images. The width measuring macro automatically determines the best angle, perpendicular to most cells, to draw a profile plot and identify and measure individual myotubes. The nuclear/cytoplasmic ratio macro compares the intensity values along lines, drawn by the user, over cytoplasm and nucleus. RESULTS: We show that the macro measurements are more consistent than manual measurements by comparing with our own results and with the literature. CONCLUSIONS: By relying on semi-automated muscle specific ImageJ macros, we seek to improve measurement accuracy and to alleviate the laborious routine of counting and measuring muscle cell features.

Epithelial-to-mesenchymal transition as a learning paradigm of cell biology.

Epithelial-to-mesenchymal transition (EMT) is a complex biological process that occurs during normal embryogenesis and in certain pathological conditions, particularly in cancer. EMT can be viewed as a cell biology-based process, since it involves all the cellular components, including the plasma membrane, cytoskeleton and extracellular matrix, endoplasmic reticulum, Golgi apparatus, lysosomes, and mitochondria, as well as cellular processes, such as regulation of gene expression and cell cycle, adhesion, migration, signaling, differentiation, and death. Therefore, we propose that EMT could be used to motivate undergraduate medical students to learn and understand cell biology. Here, we describe and discuss the involvement of each cellular component and process during EMT. To investigate the density with which different cell biology concepts are used in EMT research, we apply a bibliometric approach. The most frequent cell biology topics in EMT studies were regulation of gene expression, cell signaling, cell cycle, cell adhesion, cell death, cell differentiation, and cell migration. Finally, we suggest that the study of EMT could be incorporated into undergraduate disciplines to improve cell biology understanding among premedical, medical and biomedical students.

The Wnt/Beta-Catenin Pathway and Cytoskeletal Filaments Are Involved in the Positioning, Size, and Function of Lysosomes during Chick Myogenesis.

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.

Discovery of Putative Dual Inhibitor of Tubulin and EGFR by Phenotypic Approach on LASSBio-1586 Homologs.

Combretastatin A-4 (CA-4, 1) is an antimicrotubule agent used as a prototype for the design of several synthetic analogues with anti-tubulin activity, such as LASSBio-1586 (2). A series of branched and unbranched homologs of the lead-compound 2, and vinyl, ethinyl and benzyl analogues, were designed and synthesized. A comparison between the cytotoxic effect of these homologs and 2 on different human tumor cell lines was performed from a cell viability study using MTT with 48 h and 72 h incubations. In general, the compounds were less potent than CA-4, showing CC50 values ranging from 0.030 µM to 7.53 µM (MTT at 72 h) and 0.096 µM to 8.768 µM (MTT at 48 h). The antimitotic effect of the target compounds was demonstrated by cell cycle analysis through flow cytometry, and the cellular mechanism of cytotoxicity was determined by immunofluorescence. While the benzyl homolog 10 (LASSBio-2070) was shown to be a microtubule stabilizer, the lead-compound 2 (LASSBio-1586) and the methylated homolog 3 (LASSBio-1735) had microtubule destabilizing behavior. Molecular docking studies were performed on tubulin protein to investigate their binding mode on colchicine and taxane domain. Surprisingly, the benzyl homolog 10 was able to modulate EGFR phosphorylate activity in a phenotypic model. These data suggest LASSBio-2070 (10) as a putative dual inhibitor of tubulin and EGFR. Its binding mode with EGFR was determined by molecular docking and may be useful in lead-optimization initiatives.

Simvastatin and Muscle: Zebrafish and Chicken Show that the Benefits are not Worth the Damage.

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.

What does desmin do: A bibliometric assessment of the functions of the muscle intermediate filament.

Intermediate filaments were first described in muscle in 1968, and desmin was biochemically identified about 10 years afterwards. Its importance grew after the identification of desminopathies and desmin mutations that cause mostly cardiopathies. Since its characterization until recently, different functions have been attributed to desmin. Here, we use bibliometric tools to evaluate the articles published about desmin and to assess its several putative functions. We identified the most productive authors and the relationships between research groups. We studied the more frequent words among 9734 articles (September 2021) containing "desmin" on the title and abstract, to identify the major research focus. We generated an interactive spreadsheet with the 934 papers that contain "desmin" only on the title that can be used to search and quantify terms in the abstract. We further selected the articles that contained the terms "function" or "role" from the spreadsheet, which we then classified according to type of function, organelle, or tissue involved. Based on the bibliographic analysis, we assess comparatively the putative functions, and we propose an alternative explanation for the desmin function.

The perinuclear region concentrates disordered proteins with predicted phase separation distributed in a 3D network of cytoskeletal filaments and organelles.

Membraneless organelles have emerged during the evolution of eukaryotic cells as intracellular domains in which multiple proteins organize into complex structures to perform specialized functions without the need of a lipid bilayer compartment. Here we describe the perinuclear space of eukaryotic cells as a highly organized network of cytoskeletal filaments that facilitates assembly of biomolecular condensates. Using bioinformatic analyses, we show that the perinuclear proteome is enriched in intrinsic disorder with several proteins predicted to undergo liquid-liquid phase separation. We also analyze immunofluorescence and transmission electron microscopy images showing the association between the nucleus and other organelles, such as mitochondria and lysosomes, or the labeling of specific proteins within the perinuclear region of cells. Altogether our data support the existence of a perinuclear dense sub-micron region formed by a well-organized three-dimensional network of structural and signaling proteins, including several proteins containing intrinsically disordered regions with phase behavior. This network of filamentous cytoskeletal proteins extends a few micrometers from the nucleus, contributes to local crowding, and organizes the movement of molecular complexes within the perinuclear space. Our findings take a key step towards understanding how membraneless regions within eukaryotic cells can serve as hubs for biomolecular condensates assembly, in particular the perinuclear space. Finally, evaluation of the disease context of the perinuclear proteins revealed that alterations in their expression can lead to several pathological conditions, and neurological disorders and cancer are among the most frequent.

Activation of YAP regulates muscle fiber size in a PKC-dependent mechanism during chick in vitro myogenesis.

The formation of skeletal muscle fibers is an intricate process controlled by a multitude of signaling pathways, including Wnt, Shh, and FGF. However, the role of the Hippo pathway during vertebrate myofiber formation has conflicting reports, which we decided to address in chick muscle cultures. We found that the transcriptional regulator Yes-associated protein (YAP) was highly concentrated within the nuclei of myoblasts. As cells differentiate into myotubes, YAP localization shifted to the cell cytoplasm in more mature myotubes. Treatment of cultures with XMU-MP-1 (XMU), a MST1/2 inhibitor, stimulated the nuclear localization of YAP in myoblasts and in myotubes, upregulated myogenin, and promoted myoblast fusion, ultimately resulting in the formation of large and fully striated multinucleated myotubes. The XMU-induced phenotype was blocked by the protein kinase C (PKC) inhibitor calphostin, which raises the possibility that the Hippo pathway controls the growth of skeletal muscle fibers through a PKC-dependent mechanism.

New Findings on LMO7 Transcripts, Proteins and Regulatory Regions in Human and Vertebrate Model Organisms and the Intracellular Distribution in Skeletal Muscle Cells.

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.

Dact1 is expressed during chicken and mouse skeletal myogenesis and modulated in human muscle diseases.

Vertebrate skeletal muscle development and repair relies on the precise control of Wnt signaling. Dact1 (Dapper/Frodo) is an important modulator of Wnt signaling, interacting with key components of the various Wnt transduction pathways. Here, we characterized Dact1 mRNA and protein expression in chicken and mouse fetal muscles in vivo and during the differentiation of chick primary and mouse C2C12 myoblasts in vitro. We also performed in silico analysis to investigate Dact1 gene expression in human myopathies, and evaluated the Dact1 protein structure to seek an explanation for the accumulation of Dact1 protein aggregates in the nuclei of myogenic cells. Our results show for the first time that in both chicken and mouse, Dact1 is expressed during myogenesis, with a strong upregulation as cells engage in terminal differentiation, cell cycle withdrawal and cell fusion. In humans, Dact1 expression was found to be altered in specific muscle pathologies, including muscular dystrophies. Our bioinformatic analyses of Dact1 proteins revealed long intrinsically disordered regions, which may underpin the ability of Dact1 to interact with its many partners in the various Wnt pathways. In addition, we found that Dact1 has strong propensity for liquid-liquid phase separation, a feature that explains its ability to form nuclear aggregates and points to a possible role as a molecular 'on'-'off' switch. Taken together, our data suggest Dact1 as a candidate, multi-faceted regulator of amniote myogenesis with a possible pathophysiological role in human muscular diseases.

The Role of Embryonic Chick Muscle Cell Culture in the Study of Skeletal 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.

Adenosine Diphosphate Improves Wound Healing in Diabetic Mice Through P2Y12 Receptor Activation.

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-ß (TGF-ß) 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.

Persistent mdx diaphragm alterations are accompanied by increased expression and activity of calcium and muscle-specific proteins.

The mdx mouse model of Duchenne Muscular Dystrophy (DMD) presents sarcolemma instability and develops a mild multi-stage dystrophinopathy characterized by intense myonecrosis with inflammatory infiltrate at 4-weeks; muscular regeneration at 12-weeks and persistent fibrosis onwards. Mdx diaphragm muscle has a more severe phenotype with structural and functional deterioration that closely resembles the diaphragm impairment responsible for DMD human patients' morbidity. Herein, we compared calcium deposits, activity of calcium-related proteases, and expression of muscle-specific proteins in mdx diaphragm at 4-weeks and 12-weeks. We found increased calcium deposits mainly at 12-weeks, concomitant with high activity of calpains and matrix metalloprotease-9, but decreased expression of Myh4 (Myhc IIb) and Atp2a1 (SERCA1), and high expression of the myogenic regulatory factors Myod1 and Myog. Our results suggest that increased calcium deposits and persistent activity of calcium dependent proteases throughout the disease are involved in the degeneration and regeneration processes in the mdx diaphragm.

A comparative study on the use of microscopy in pharmacology and cell biology research.

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.

Lipid Rafts from Olfactory Ensheathing Cells: Molecular Composition and Possible Roles.

Olfactory ensheathing cells (OECs) are specialized glial cells of the olfactory system, believed to play a role in the continuous production of olfactory neurons and ensheathment of their axons. Although OECs are used in therapeutic applications, little is known about the cellular mechanisms underlying their migratory behavior. Recently, we showed that OEC migration is sensitive to ganglioside blockage through A2B5 and Jones antibody in OEC culture. Gangliosides are common components of lipid rafts, where they participate in several cellular mechanisms, including cell migration. Here, we characterized OEC lipid rafts, analyzing the presence of specific proteins and gangliosides that are commonly expressed in motile neural cells, such as young neurons, oligodendrocyte progenitors, and glioma cells. Our results showed that lipid rafts isolated from OECs were enriched in cholesterol, sphingolipids, phosphatidylcholine, caveolin-1, flotillin-1, gangliosides GM1 and 9-O-acetyl GD3, A2B5-recognized gangliosides, CNPase, α-actinin, and ß1-integrin. Analysis of the actin cytoskeleton of OECs revealed stress fibers, membrane spikes, ruffled membranes and lamellipodia during cell migration, as well as the distribution of α-actinin in membrane projections. This is the first description of α-actinin and flotillin-1 in lipid rafts isolated from OECs and suggests that, together with ß1-integrin and gangliosides, membrane lipid rafts play a role during OEC migration. This study provides new information on the molecular composition of OEC membrane microdomains that can impact on our understanding of the role of OEC lipid rafts under physiological and pathological conditions of the nervous system, including inflammation, hypoxia, aging, neurodegenerative diseases, head trauma, brain tumor, and infection.

The scaffolding protein calpain-3 has multiple distributions in embryonic chick muscle cells and it is essential for the formation of muscle fibers.

CAPN3 is a muscle-specific and an intrinsically disordered protein. Thus, as a scaffolding protein CAPN3 could play a role during early stages of myogenesis. To test this hypothesis, we studied the distribution and function of CAPN3 during myogenesis using embryonic chick muscle cells grown in vitro. Super-resolution microscopy showed CAPN3 distribution in (i) amorphous patches in myoblasts, (ii) a region near the nuclei of myotubes; (iii) adhesion plaques in myotubes, (iv) stress fiber-like structures in myotubes, and (v) filaments in fibroblasts. Downregulation of CAPN3 induced a decrease in the number of muscle cells and in the size of myotubes formed. These data show a diverse intracellular distribution of CAPN3, compatible with a scaffolding protein, and suggest a multitude of different interactions of CAPN3 with other partners during muscle formation.

Acidic Compartment Size, Positioning, and Function during Myogenesis and Their Modulation by the Wnt/Beta-Catenin Pathway.

Lysosomes and acidic compartments are involved in breaking down of macromolecules, membrane recycling, and regulation of signaling pathways. Here, we analyzed the role of acidic compartments during muscle differentiation and the involvement of the Wnt/beta-catenin pathway in lysosomal function during myogenesis. Acridine orange was used to localize and quantify acidic cellular compartments in primary cultures of embryonic muscle cells from Gallus gallus. Our results show an increase in acidic compartment size and area, as well as changes in their positioning during the initial steps of myogenesis. The inhibition of lysosomal function by either the chloroquine Lys05 or the downregulation of LAMP-2 with siRNA impaired chick myogenesis, by inhibiting myoblast fusion. Two activators of the Wnt/beta-catenin pathway, BIO and Wnt3a, were able to rescue the inhibitory effects of Lys05 in myogenesis. These results suggest a new role for the Wnt/beta-catenin pathway in the regulation of acidic compartment size, positioning, and function in muscle cells.