MyoD converts primary dermal fibroblasts, chondroblasts, smooth muscle, and retinal pigmented epithelial cells into striated mononucleated myoblasts and multinucleated myotubes ( ABSTRACTShortly after their birth, postmitotic mononucleated myoblasts in myotomes, limb buds, and conventional muscle cultures elongate and assemble a cohort of myofibrillar proteins into definitively striated myofibrils. MyoD induces a number of immortalized and/or transformed nonmuscle cells to express desmin and several myofibrillar proteins and to fuse into myosacs. We now report that MyoD converts normal dermal fibroblasts, chondroblasts, gizzard smooth muscle, and pigmented retinal epithelial cells into elongated postmitotic mononucleated striated myoblasts. The sarcomeric localization of antibodies to desmin, a-actinin, titin, troponin-I, a-actin, myosin heavy chain, and myomesin in these converted myoblasts are indistinguishable from in vivo and in vitro normal myoblasts. Converted myoblasts fuse into typical anisodiametric multinucleated myotubes that often contract spontaneously. Conversion and subsequent expression of the skeletal myogenic program are autonomous events, occurring in four nonmuscle microenvironments consisting of different combinations of foreign extracellular matrix molecules. Early events associated with conversion by MyoD involve (i) withdrawal from the cell cycle, (ii) down-regulation of the subverted cell's ongoing differentiation program, and (iii) initiation of desmin synthesis in presumptive myoblasts and dramatic redistribution of microtubules and desmin intermediate filaments in postmitotic myoblasts.

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 αB-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.

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.

Neutrophil extracellular traps (NETs) have been associated with several steps of tumor progression, including primary growth and metastasis. One of the key features for the acquisition of the metastatic ability is the epithelial–mesenchymal transition (EMT), a complex cellular program. In this study, we evaluated the ability of isolated NETs in modulating the pro-metastatic phenotype of human breast cancer cells. Tumor cells were treated with isolated NETs and then samples were generated for cell migration, quantitative RT-PCR, western blotting, immunofluorescence, and flow cytometry assays. RNA-seq data from The Cancer Genome Atlas (TCGA) database were assessed. NETs changed the typical epithelial morphology of MCF7 cells into a mesenchymal phenotype, a process that was accompanied by enhanced migratory properties. Additional EMT traits were observed: increased expression of N-cadherin and fibronectin, while the E-cadherin expression was repressed. Notably, NETs positively regulated the gene expression of several factors linked to the pro-inflammatory and pro-metastatic properties. Analyses of TCGA data showed that samples from breast cancer patients exhibit a significant correlation between pro-tumoral and neutrophil signature gene expression, including several EMT and pro-metastatic factors. Therefore, NETs drive pro-metastatic phenotype in human breast cancer cells through the activation of the EMT program.

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Glycolytic enzymes reversibly associate with the human erythrocyte membrane (EM) as part of their regulatory mechanism. The site for this association has been described as the amino terminus of band 3, a transmembrane anion transporter. Binding of glycolytic enzymes to this site is recognized to inhibit glycolysis, since binding inhibits the catalytic activity of these enzymes, including the rate-limiting enzyme 6-phosphofructo-1-kinase (PFK). However, the existence of a putative stimulatory site for glycolytic enzymes within the EM has been proposed. PFK has been described as able to reversibly associate with other proteins, such as microtubules, which inhibit the enzyme, and filamentous actin, which activates the enzyme. Here, it is demonstrated that PFK also binds to actin filaments and its associated binding proteins in the protein meshwork that forms the erythrocyte cytoskeleton. Through fluorescence resonance energy transfer experiments using either confocal microscopy or fluorescence spectroscopy, we show that, within the EM, PFK and actin filaments containing its associated binding proteins are located close enough to propose binding between them. Moreover, specifically blocking PFK binding to band 3 results in an association of the enzyme with the EM that increases the enzyme's catalytic activity. Conversely, disruption of the association between PFK and actin filaments containing its associated binding proteins potentiates the inhibitory action of the EM on the enzyme. Furthermore, it is shown that insulin signaling increases the association of PFK to actin filaments and its associated binding proteins, revealing that this event may play a role on the stimulatory effects of insulin on erythrocyte glycolysis. In summary, the present work presents evidence that filamentous actin and its associated binding proteins are the stimulatory site for PFK within the EM.

The formation of a skeletal muscle fiber begins with the withdrawal of committed mononucleated precursors from the cell cycle. These myoblasts elongate while aligning with each other, guided by recognition between their membranes. This step is followed by cell fusion and the formation of long striated multinucleated myotubes. We used methyl-beta-cyclodextrin (MCD) in primary cultured chick skeletal muscle cells to deplete membrane cholesterol and investigate its role during myogenesis. MCD promoted a significant increase in the expression of troponin T, enhanced myoblast fusion, and induced the formation of large multinucleated myotubes with nuclei being clustered centrally and not aligned at the cell periphery. MCD myotubes were striated, as indicated by sarcomeric alpha-actinin staining, and microtubule and desmin filament distribution was not altered. Pre-fusion MCD-treated myoblasts formed large aggregates, with cadherin and beta-catenin being accumulated in cell adhesion contacts. We also found that the membrane microdomain marker GM1 was not present as clusters in the membrane of MCD-treated myoblasts. Our data demonstrate that cholesterol is involved in the early steps of skeletal muscle differentiation.

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