The follicular thyroid cell line PCCL3 responds differently to laminin and to polylaminin, a polymer of laminin assembled in acidic pH.

The extracellular-matrix protein laminin forms polymers both in vivo and in vitro. Acidification of pH leads to the formation of an artificial polymer with biomimetic properties, named polylaminin (polyLM). Follicle cells in the thyroid are in close contact with laminin, but their response to this important extracellular signal is still poorly understood. PCCL3 thyroid follicular cells cultured on glass, on regular laminin (LM) or on laminin previously polymerized in acidic pH (polyLM) showed different cell morphologies and propensities to proliferate, as well as differences in the organization of their actin cytoskeleton. On polyLM, cells displayed a typical epithelial morphology and radially organized actin fibers; whereas on LM, they spread irregularly on the substrate, lost cell contacts, and developed thick actin fibers extending through the entire cytoplasm. Iodide uptake decreased similarly in response to both laminin substrates, in comparison to glass. On both the LM and polyLM substrates, the expression of the sodium iodide symporter (NIS) decreased slightly but not significantly. NIS showed dotted immunostaining at the plasma membrane in the cells cultured on glass; on polyLM, NIS was observed mainly in the perinuclear region, and more diffusely throughout the cytoplasm on the LM substrate. Additionally, polyLM specifically favored the maintenance of cell polarity in culture. These findings indicate that PCCL3 cells can discriminate between LM and polyLM and that they respond to the latter by better preserving the phenotype observed in the thyroid tissue.

The Wnt signaling pathway regulates Nalm-16 b-cell precursor acute lymphoblastic leukemic cell line survival and etoposide resistance.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common malignancy in children. The Wnt signaling pathway has been found to be extensively involved in cancer onset and progression but its role in BCP-ALL remains controversial. We evaluate the role of the Wnt pathway in maintenance of BCP-ALL cells and resistance to chemotherapy. Gene expression profile revealed that BCP-ALL cells are potentially sensitive to modulation of Wnt pathway. Nalm-16 and Nalm-6 cell lines displayed low levels of canonical activation, as reflected by the virtually complete absence of total beta-catenin in Nalm-6 and the beta-catenin cell membrane distribution in Nalm-16 cell line. Canonical activation with Wnt3a induced nuclear beta-catenin translocation and led to BCP-ALL cell death. Lithium chloride (LiCl) also induced a cytotoxic effect on leukemic cells. In contrast, both Wnt5a and Dkk-1 increased Nalm-16 cell survival. Also, Wnt3a enhanced the in vitro sensitivity of Nalm-16 to etoposide (VP-16) while treatment with canonical antagonists protected leukemic cells from chemotherapy-induced cell death. Overall, our results suggest that canonical activation of the Wnt pathway may exerts a tumor suppressive effect, thus its inhibition may support BCP-ALL cell survival.

Distribution of cytoskeletal and adhesion proteins in adult zebrafish skeletal muscle.

The organization of cytoskeletal and adhesion proteins in skeletal muscle is critical for its contractile function. Zebrafish has become a paramount model for studies of vertebrate biology, including muscle. However, only a few studies have been published using immunolabeling to specifically localize proteins in adult zebrafish muscle. To fully appreciate the distribution of cytoskeletal and adhesion proteins, and therefore to better correlate the adult muscle with its myogenesis, we used indirect immunofluorescence microscopy of frozen adult zebrafish skeletal muscle sections. Here we describe the fish muscle cytoskeletal architecture and location of the major myofibrillar proteins desmin, alpha-actinin, myosin, titin, troponin, tropomyosin and nebulin, the adhesion proteins vinculin and paxillin, and the extracellular matrix proteins laminin and fibronectin. Electron microscopical analysis in ultra-thin sections of adult zebrafish skeletal muscle showed bundles of collagen fibers and fibroblastic cells in the extracellular space of the myosepta.

Changes in cell shape and desmin intermediate filament distribution are associated with down-regulation of desmin expression in C2C12 myoblasts grown in the absence of extracellular Ca2+.

Desmin is the main intermediate filament (IF) protein of muscle cells. In skeletal muscle, desmin IFs form a scaffold that interconnects the entire contractile apparatus with the subsarcolemmal cytoskeleton and cytoplasmic organelles. The interaction between desmin and the sarcolemma is mediated by a number of membrane proteins, many of which are Ca2+-sensitive. In the present study, we analyzed the effects of the Ca2+ chelator EGTA (1.75 mM) on the expression and distribution of desmin in C2C12 myoblasts grown in culture. We used indirect immunofluorescence microscopy and reverse transcription polymerase chain reaction (RT-PCR) to analyze desmin distribution and expression in C2C12 cells grown in the presence or absence of EGTA. Control C2C12 myoblasts showed a well-spread morphology after a few hours in culture and became bipolar when grown for 24 h in the presence of EGTA. Control C2C12 cells showed a dense network of desmin from the perinuclear region to the cell periphery, whereas EGTA-treated cells showed desmin aggregates in the cytoplasm. RT-PCR analysis revealed a down-regulation of desmin expression in EGTA-treated C2C12 cells compared to untreated cells. The present results suggest that extracellular Ca2+ availability plays a role in the regulation of desmin expression and in the spatial distribution of desmin IFs in myoblasts, and is involved in the generation and maintenance of myoblast cell shape.

Changes in cell shape and desmin intermediate filament distribution are associated with down-regulation of desmin expression in C2C12 myoblasts grown in the absence of extracellular Ca2+

Desmin is the main intermediate filament (IF) protein of muscle cells. In skeletal muscle, desmin IFs form a scaffold that interconnects the entire contractile apparatus with the subsarcolemmal cytoskeleton and cytoplasmic organelles. The interaction between desmin and the sarcolemma is mediated by a number of membrane proteins, many of which are Ca2+-sensitive. In the present study, we analyzed the effects of the Ca2+ chelator EGTA (1.75 mM) on the expression and distribution of desmin in C2C12 myoblasts grown in culture. We used indirect immunofluorescence microscopy and reverse transcription polymerase chain reaction (RT-PCR) to analyze desmin distribution and expression in C2C12 cells grown in the presence or absence of EGTA. Control C2C12 myoblasts showed a well-spread morphology after a few hours in culture and became bipolar when grown for 24 h in the presence of EGTA. Control C2C12 cells showed a dense network of desmin from the perinuclear region to the cell periphery, whereas EGTA-treated cells showed desmin aggregates in the cytoplasm. RT-PCR analysis revealed a down-regulation of desmin expression in EGTA-treated C2C12 cells compared to untreated cells. The present results suggest that extracellular Ca2+ availability plays a role in the regulation of desmin expression and in the spatial distribution of desmin IFs in myoblasts, and is involved in the generation and maintenance of myoblast cell shape.

Desmin: molecular interactions and putative functions of the muscle intermediate filament protein

Desmin is the intermediate filament (IF) protein occurring exclusively in muscle and endothelial cells. There are other IF proteins in muscle such as nestin, peripherin, and vimentin, besides the ubiquitous lamins, but they are not unique to muscle. Desmin was purified in 1977, the desmin gene was characterized in 1989, and knock-out animals were generated in 1996. Several isoforms have been described. Desmin IFs are present throughout smooth, cardiac and skeletal muscle cells, but can be more concentrated in some particular structures, such as dense bodies, around the nuclei, around the Z-line or in costameres. Desmin is up-regulated in muscle-derived cellular adaptations, including conductive fibers in the heart, electric organs, some myopathies, and experimental treatments with drugs that induce muscle degeneration, like phorbol esters. Many molecules have been reported to associate with desmin, such as other IF proteins (including members of the membrane dystroglycan complex), nebulin, the actin and tubulin binding protein plectin, the molecular motor dynein, the gene regulatory protein MyoD, DNA, the chaperone alphaB-crystallin, and proteases such as calpain and caspase. Desmin has an important medical role, since it is used as a marker of tumors' origin. More recently, several myopathies have been described, with accumulation of desmin deposits. Yet, after almost 30 years since its identification, the function of desmin is still unclear. Suggested functions include myofibrillogenesis, mechanical support for the muscle, mitochondrial localization, gene expression regulation, and intracellular signaling. This review focuses on the biochemical interactions of desmin, with a discussion of its putative functions.

Desmin: molecular interactions and putative functions of the muscle intermediate filament protein.

Desmin is the intermediate filament (IF) protein occurring exclusively in muscle and endothelial cells. There are other IF proteins in muscle such as nestin, peripherin, and vimentin, besides the ubiquitous lamins, but they are not unique to muscle. Desmin was purified in 1977, the desmin gene was characterized in 1989, and knock-out animals were generated in 1996. Several isoforms have been described. Desmin IFs are present throughout smooth, cardiac and skeletal muscle cells, but can be more concentrated in some particular structures, such as dense bodies, around the nuclei, around the Z-line or in costameres. Desmin is up-regulated in muscle-derived cellular adaptations, including conductive fibers in the heart, electric organs, some myopathies, and experimental treatments with drugs that induce muscle degeneration, like phorbol esters. Many molecules have been reported to associate with desmin, such as other IF proteins (including members of the membrane dystroglycan complex), nebulin, the actin and tubulin binding protein plectin, the molecular motor dynein, the gene regulatory protein MyoD, DNA, the chaperone alphaB-crystallin, and proteases such as calpain and caspase. Desmin has an important medical role, since it is used as a marker of tumors' origin. More recently, several myopathies have been described, with accumulation of desmin deposits. Yet, after almost 30 years since its identification, the function of desmin is still unclear. Suggested functions include myofibrillogenesis, mechanical support for the muscle, mitochondrial localization, gene expression regulation, and intracellular signaling. This review focuses on the biochemical interactions of desmin, with a discussion of its putative functions.

Changes in cell shape, cytoskeletal proteins and adhesion sites of cultured cells after extracellular Ca2+ chelation

Although much is known about the molecules involved in extracellular Ca2+ regulation, the relationship of the ion with overall cell morphology is not understood. The objective of the present study was to determine the effect of the Ca2+ chelator EGTA on the major cytoskeleton components, at integrin-containing adhesion sites, and their consequences on cell shape. Control mouse cell line C2C12 has a well-spread morphology with long stress fibers running in many different directions, as detected by fluorescence microscopy using rhodamine-phalloidin. In contrast, cells treated with EGTA (1.75 mM in culture medium) for 24 h became bipolar and showed less stress fibers running in one major direction. The adhesion plaque protein alpha5-integrin was detected by immunofluorescence microscopy at fibrillar adhesion sites in both control and treated cells, whereas a dense labeling was seen only inside treated cells. Microtubules shifted from a radial arrangement in control cells to a longitudinal distribution in EGTA-treated cells, as analyzed by immunofluorescence microscopy. Desmin intermediate filaments were detected by immunofluorescence microscopy in a fragmented network dispersed within the entire cytoplasm in EGTA-treated cells, whereas a dense network was seen in the whole cytoplasm of control cells. The present results suggest that the role of extracellular Ca2+ in the regulation of C2C12 cell shape can be mediated by actin-containing stress fibers and microtubules and by intermediate filament reorganization, which may involve integrin adhesion sites

Cytoskeletal and cellular adhesion proteins in zebrafish (Danio rerio) myogenesis

The current myogenesis and myofibrillogenesis model has been based mostly on in vitro cell culture studies, and, to a lesser extent, on in situ studies in avian and mammalian embryos. While the more isolated artificial conditions of cells in culture permitted careful structural analysis, the actual in situ cellular structures have not been described in detail because the embryos are more difficult to section and manipulate. To overcome these difficulties, we used the optically clear and easy to handle embryos of the zebrafish Danio rerio. We monitored the expression of cytoskeletal and cell-adhesion proteins (actin, myosin, desmin, alpha-actinin, troponin, titin, vimentin and vinculin) using immunofluorescence microscopy and video-enhanced, background-subtracted, differential interference contrast of 24- to 48-h zebrafish embryos. In the mature myotome, the mononucleated myoblasts displayed periodic striations for all sarcomeric proteins tested. The changes in desmin distribution from aggregates to perinuclear and striated forms, although following the same sequence, occurred much faster than in other models. All desmin-positive cells were also positive for myofibrillar proteins and striated, in contrast to that which occurs in cell cultures. Vimentin appeared to be striated in mature cells, while it is developmentally down-regulated in vitro. The whole connective tissue septum between the somites was positive for adhesion proteins such as vinculin, instead of the isolated adhesion plaques observed in cell cultures. The differences in the myogenesis of zebrafish in situ and in cell culture in vitro suggest that some of the previously observed structures and protein distributions in cultures could be methodological artifacts

Changes in cell shape, cytoskeletal proteins and adhesion sites of cultured cells after extracellular Ca2+ chelation.

Although much is known about the molecules involved in extracellular Ca2+ regulation, the relationship of the ion with overall cell morphology is not understood. The objective of the present study was to determine the effect of the Ca2+ chelator EGTA on the major cytoskeleton components, at integrin-containing adhesion sites, and their consequences on cell shape. Control mouse cell line C2C12 has a well-spread morphology with long stress fibers running in many different directions, as detected by fluorescence microscopy using rhodamine-phalloidin. In contrast, cells treated with EGTA (1.75 mM in culture medium) for 24 h became bipolar and showed less stress fibers running in one major direction. The adhesion plaque protein alpha 5-integrin was detected by immunofluorescence microscopy at fibrillar adhesion sites in both control and treated cells, whereas a dense labeling was seen only inside treated cells. Microtubules shifted from a radial arrangement in control cells to a longitudinal distribution in EGTA-treated cells, as analyzed by immunofluorescence microscopy. Desmin intermediate filaments were detected by immunofluorescence microscopy in a fragmented network dispersed within the entire cytoplasm in EGTA-treated cells, whereas a dense network was seen in the whole cytoplasm of control cells. The present results suggest that the role of extracellular Ca2+ in the regulation of C2C12 cell shape can be mediated by actin-containing stress fibers and microtubules and by intermediate filament reorganization, which may involve integrin adhesion sites.

Cytoskeletal and cellular adhesion proteins in zebrafish (Danio rerio) myogenesis.

The current myogenesis and myofibrillogenesis model has been based mostly on in vitro cell culture studies, and, to a lesser extent, on in situ studies in avian and mammalian embryos. While the more isolated artificial conditions of cells in culture permitted careful structural analysis, the actual in situ cellular structures have not been described in detail because the embryos are more difficult to section and manipulate. To overcome these difficulties, we used the optically clear and easy to handle embryos of the zebrafish Danio rerio. We monitored the expression of cytoskeletal and cell-adhesion proteins (actin, myosin, desmin, alpha-actinin, troponin, titin, vimentin and vinculin) using immunofluorescence microscopy and video-enhanced, background-subtracted, differential interference contrast of 24- to 48-h zebrafish embryos. In the mature myotome, the mononucleated myoblasts displayed periodic striations for all sarcomeric proteins tested. The changes in desmin distribution from aggregates to perinuclear and striated forms, although following the same sequence, occurred much faster than in other models. All desmin-positive cells were also positive for myofibrillar proteins and striated, in contrast to that which occurs in cell cultures. Vimentin appeared to be striated in mature cells, while it is developmentally down-regulated in vitro. The whole connective tissue septum between the somites was positive for adhesion proteins such as vinculin, instead of the isolated adhesion plaques observed in cell cultures. The differences in the myogenesis of zebrafish in situ and in cell culture in vitro suggest that some of the previously observed structures and protein distributions in cultures could be methodological artifacts.

Induction of the lipocyte phenotype in murine hepatic stellate cells: reorganisation of the actin cytoskeleton.

Hepatic stellate cells (HSCs) are intralobular connective tissue cells presenting myofibroblast or lipocyte phenotypes. They participate in the homeostasis of liver extracellular matrix, repair, regeneration and fibrosis under the former phenotype, and control retinol metabolism, storage and release under the latter one. Responding to systemic or local demands, they can convert into the required phenotype with deep modifications of their structures. Using immunofluorescence microscopy and Western blots, we investigated the expression and organisation of actin filaments and of two actin-binding proteins, alpha-actinin and tropomyosin, in the cloned GRX cell line representative of murine HSCs. GRX cells expressing the myofibroblast phenotype showed typical well-organised actin stress-fibres, anchored at the focal adhesions located at the cell periphery. Retinol treatment induced active reorganisation of the cytoskeleton. The major stress fibres were reduced in length, and frequently formed a polygonal meshwork. Subsequently, they fragmented and generated diffuse or granular actin in the perinuclear area, a thin continuous layer around lipid droplets and, in fully converted lipocytes, a peripheral layer of thin actin fibres. alpha-Actinin and tropomyosin were present only in lipocytes, co-distributed with actin in a granular form. Since the cytoskeleton reorganisation preceded lipid accumulation, we conclude that the induction of the lipocyte phenotype represents a full reprogramming of cell gene expression and function. We consider that both the lipocyte and the myofibroblast phenotypes should be considered "activated states" of HSCs, each responding to specific physiological or pathological modifications of liver functions.

Intermediate filaments modulation in an in vitro model of the hepatic stellate cell activation or conversion into the lipocyte phenotype.

Hepatic stellate cells are intralobular connective tissue cells expressing the myofibroblast or the lipocyte phenotypes. They participate in homeostasis of the liver extracellular matrix, repair, regeneration, and fibrosis under the former phenotype, and control the retinol metabolism, storage, and release under the latter one. They are heterogeneous in terms of their tissue distribution, function, and expression of cytoskeletal proteins. We have studied the expressions of intermediate filaments in the cloned GRX cell line representative of murine hepatic stellate cells, by immunolabeling, reverse transcription polymerase chain reaction (RT-PCR), immunoprecipitation and Western blots. GRX cells expressed vimentin, desmin, glial fibrillary acidic protein (GFAP), and smooth muscle alpha actin (SM-alphaA). Vimentin, desmin, and SMN-alphaA were expressed in all cultures. GFAP showed a heterogeneous intensity of expression and did not form a filamentous cytoskeletal network, showing a distinct punctuate cytoplasmic distribution. When activated by inflammatory mediators, GRX cells increased expression of desmin and GFAP. Retinol-mediated induction of the lipocyte phenotype elicited a strong decrease of intermediate filament protein expression and the collapse of the filamentous structure of the cytoskeleton. Quiescent hepatic stellate precursors can respond to physiologic or pathologic stimuli, expressing activated myofibroblast or lipocyte phenotypes with distinct patterns of cytoskeleton structure, metabolic function, and interaction with the tissue environment.

Mast cells can revert dexamethasone-mediated down-regulation of stem cell factor.

Mast cell hyperplasia can be causally related with chronic inflammation and liver fibrosis. Their survival and proliferation is dependent upon locally produced growth factors, the major one being the stem cell factor (SCF). Glucocorticoids can decrease mastocytosis, down-regulating the mast cell production of pro-inflammatory factors or inhibiting the expression of SCF in stroma. We compared dexamethasone effect on SCF expression in co-cultures of mast cells with NIH/3T3 fibroblasts or with primary cultures of activated hepatic stellate cells. Dexamethasone abrogated the NIH/3T3 stroma capacity to sustain mast cell proliferation, but not of hepatic stellate cells, at the post-transcriptional level. Mast cells reverted completely dexamethasone effect on hepatic stellate cells, increasing their SCF synthesis and transport. In both models, dexamethasone inhibited the mast cell spreading on the stroma, which was thus not required for mast cell survival and proliferation. Liver pathologies associated with mast cell hyperplasia are not expected to be sensitive to glucocorticoid treatments.

Volumetric quantification of the gastric emptying: computer-based method for generation of volumetric index from fluoroscopic images.

We developed an automated software-based procedure for estimation of the volume variation of the stomach using videofluoroscopic analysis of the gastric emptying. We used radiological images with postero-anterior incidence of eight healthy volunteers and in vitro experimental tests, with different volumes and concentrations of the contrast medium. This computational method generates an index that measures, in the three dimensions, the dynamic behaviour of gastric emptying. Using adequate contrast concentration (barium sulphate solution), it is possible to determine volume behaviour from density variations. This software can automate this computation, facilitating the amount of work, avoiding mistakes and improving reproducibility.

Costimulatory action of glycoinositolphospholipids from Trypanosoma cruzi: increased interleukin 2 secretion and induction of nuclear translocation of the nuclear factor of activated T cells 1.

The effects of the glycoinositolphospholipids (GIPLs) fromTrypanosoma cruzi on T lymphocyte activation were investigated in a mouse T cell hybridoma (DO-11.10). Purified GIPLs from T. cruzi strains Y and G markedly increased IL-2 mRNA transcripts and IL-2 secretion induced by mitogenic anti-CD3 and anti-Thy1 mAbs. This costimulatory function was also revealed by the induction of IL-2 secretion after the simultaneous addition of the T. cruzi GIPLs and either the calcium ionophore A23187 or phorbol ester. The capacity of the GIPL molecule to induce an increase in cytoplasmic calcium levels was also demonstrated. After exposure of T cell hybridoma to GIPL, the nuclear transcription factor NFAT1 became partially dephosphorylated, and its nuclear localization was demonstrated both in the T cell hybridoma and in Balb/c CD3(+) cells. These results demonstrate that T. cruzi GIPL molecules are capable of signaling to T cells and therefore could be valuable tools for the study of T cell activation, besides playing a potential role in subverting the T lymphocyte immune response during T. cruzi infection.

Distribution of F-actin, alpha-actinin, tropomyosin, tubulin and organelles in Euglena gracilis by immunofluorescence microscopy.

Euglena gracilis, a unicellular flagellated alga, can display numerous shape changes. These changes are most probably caused by a pellicle and an internal cytoskeleton. In this paper we studied the distribution of the cytoskeletal proteins actin, alpha-actinin, tropomyosin and tubulin in dark-adapted Euglena, using immunofluorescence microscopy. We found that F-actin, alpha-actinin, tropomyosin and tubulin have a distribution that is coincident in the plasma membrane and, in addition, alpha-actinin and tropomyosin are seen in small patches in the cytoplasm, and tubulin in the flagella. We have also studied the distribution of the endoplasmic reticulum, nucleus, and Golgi apparatus of these cells, using fluorescent probes. Both the endoplasmic reticulum and the Golgi apparatus have a meshwork pattern distributed throughout the cytoplasm, and the nucleus has a chromatin evenly distributed in the nucleoplasm.

Differences in the isodesmin pattern between the electric organs of Electrophorus electricus L.

Desmin, the intermediate filament protein of muscle, is present in the electric organs of Electrophorus electricus L. as five isovariants, instead of the one to two isovariants found in muscle. We analyzed the isodesmin pattern in the three different electric organs using densitometry of Coomassie blue-stained bands in electrofocusing polyacrylamide gel electrophoresis. We were able to compare the relative amount of each of the five desmin isovariants in an isodesmin pattern characteristic of each electric organ. These patterns proved to be, in some cases, statistically different. Desmin in each electric organ could have slightly different functions in order to correlate with the organ-specific isovariant patterns.

Desmin and actin filaments in membrane-cytoskeletal preparations of the electric tissue of Electrophorus electricus, L.

The electrocyte of the electric organ of the electric eel, Electrophorus electricus, L was investigated by light and electron microscopy as well as immuno-electron microscopy, in order to clarify the fine structures and distribution of cytoskeleton filaments and their relations to proteins, especially desmin and actin. Cytoskeleton-enriched fractions of the electrocytes were analysed with SDS-PAGE. It was verified that a meshwork of filaments was distributed in the electrocytes, more abundantly in the anterior than in the posterior part of the cell, and that this could be associated with membrane invaginations. Desmin and actin were the components of this meshwork, suggesting that desmin intermediate filaments and actin filaments might play a role in the maintenance of the morphology of electrocytes and, as an intracellular filamentous meshwork, they may contribute to the organization of the components of membranes and papillae formation on the anterior face of the electrocytes.

Desmin filaments in the electrocytes of the electric organ of the electric eel Electrophorus electricus.

Desmin protein is an abundant constituent of the intermediate filaments in the electrocytes of the electric organ of the electric eel Electrophorus electricus. Polyclonal antibodies were raised against purified desmin from the electric organ and used for immunolabeling of the protein in reconstituted filaments. In thick sections of the main electric organ that has been stained with fluorescein-labeled desmin-specific antibodies, light microscope revealed a diffuse meshwork of desmin filaments dispersed in the cytoplasm of electrocytes. In the region under the membrane, the immunostaining was slightly more intense than elsewhere. The meshwork of intermediate filaments composed of desmin was examined by electron microscopy of the main electric organ. Immuno-gold labeling demonstrated a widespread meshwork of desmin filaments in the cytoplasm and in close association with the plasma membrane. These observations suggest that intermediate filaments play a role in the maintenance of the morphology of electrocytes and, as an intracellular meshwork spanning the width of the cell, they may contribute to the organization of the intracellular compartments.