Some distinctive features of zebrafish myogenesis based on unexpected distributions of the muscle cytoskeletal proteins actin,myosin, desmin,alpha-actinin,troponin and titin

The current myofibrillogenesis model is based mostly on in vitro cell cultures and on avian and mammalian embryos in situ. We followed the expression of actin, myosin, desmin, alpha-actinin, titin, and troponin using immunofluorescence microscopy of zebrafish (Danio rerio) embryos. We could see young mononucleated myoblasts with sharp striations. The striations were positive for all the sarcomeric proteins. Desmin distribution during muscle maturation changes from dispersed aggregates to a perinuclear concentration to striated afterwards. We could not observe desmin-positive, myofibrillar-proteins-negative cells, and we could not find any non-striated distribution of sarcomeric proteins, such as stress fiber-like structures. Some steps, like fusion before striation, seem to be different in the zebrafish when compared with the previously described myogenesis sequences.     

Structural Analysis of Alterations in Zebrafish Muscle Differentiation Induced by Simvastatin and Their Recovery with Cholesterol

In vitro studies show that cholesterol is essential to myogenesis. We have been using zebrafish to overcome the limitations of the in vitro approach and to study the sub-cellular structures and processes involved during myogenesis. We use simvastatin—a drug widely used to prevent high levels of cholesterol and cardiovascular disease—during zebrafish skeletal muscle formation. Simvastatin is an efficient inhibitor of cholesterol synthesis that has various myotoxic consequences. Here, we employed simvastatin concentrations that cause either mild or severe morphological disturbances to observe changes in the cytoskeleton (intermediate filaments and microfilaments), extracellular matrix and adhesion markers by confocal microscopy. With low-dose simvastatin treatment, laminin was almost normal, and alpha-actinin was reduced in the myofibrils. With high simvastatin doses, laminin and vinculin were reduced and appeared discontinuous along the septa, with almost no myofibrils, and small amounts of desmin accumulating close to the septa. We also analyzed sub-cellular alterations in the embryos by electron microscopy, and demonstrate changes in embryo and somite size, septa shape, and in myofibril structure. These effects could be reversed by the addition of exogenous cholesterol. These results contribute to the understanding of the mechanisms of action of simvastatin in muscle cells in particular, and in the study of myogenesis in general.     

Dystonin-Deficient Mice Exhibit an Intrinsic Muscle Weakness and an Instability of Skeletal Muscle Cytoarchitecture

Dystonia musculorum (dt) was originally described as a hereditary sensory neurodegeneration syndrome of the mouse. The gene defective in dt encodes a cytoskeletal linker protein, dystonin, that is essential for maintaining neuronal cytoskeletal integrity. In addition to the nervous system, dystonin is expressed in a variety of other tissues, including muscle. We now show that dystonin cross-links actin and desmin filaments and that its levels are increased during myogenesis, coinciding with the progressive reorganization of the intermediate filament network. A disorganization of cytoarchitecture in skeletal muscle from dt/dt mice was observed in ultrastructural studies. Myoblasts from dt/dt mice fused to form myotubes in culture; however, terminally differentiated myotubes contained incompletely assembled myofibrils. Another feature observed in dt/dt myotubes in culture and in skeletal muscle in situ was an accumulation and abnormal distribution of mitochondria. The diaphragm muscle from dt/dt mice was weak in isometric contractility measurements in vitro and was susceptible to contraction-induced sarcolemmal damage. Altogether, our data indicate that dystonin is a cross-linker of actin and desmin filaments in muscle and that it is essential for establishing and maintaining proper cytoarchitecture in mature muscle.     

Identification of the cytoskeletal proteins in lens-forming cells, a special epithelioid cell type

Proteins of contractile and cytoskeletal elements have been studied in bovine lens-forming cells growing in culture as well as in bovine and murine lenses grown in situ by immunofluorescence microscopy using antibodies to the following proteins: actin, myosin, tropomyosin, α-actinin, tubulin, prekeratin, vimentin, and desmin. Lens-forming cells contain actin, myosin, tropomyosin, and α-actinin which in cells grown in culture are enriched in typical cable-like structures, i.e. microfilament bundles. Antibodies to tubulin stain normal, predominantly radial arrays of microtubules. In the epithelioid lens-forming cells of both monolayer cultures grown in vitro and lens tissue grown in situ intermediate-sized filaments of the vimentin type are abundant, whereas filaments containing prekeratin-like proteins (‘cytokeratins’) and desmin filaments have not been found. The absence of cytokeratin proteins observed by immunological methods is supported by gel electrophoretic analyses of cytoskeletal proteins, which show the prominence of vimentin and the absence of detectable amounts of cytokeratins and desmin. This also correlates with electron microscopic observations that typical desmosomes and tonofilament bundles are absent in lens-forming cells, as opposed to a high density of vimentin filaments. Our observations show that the epithelioid lens-forming cells have normal arrays of (i) microfilament bundles containing proteins of contractile structures; (ii) microtubules; and (iii) vimentin filaments, but differ from most true epithelial cells by the absence of cytokeratins, tonofilaments and typical desmosomes. The question of their relationship to other epithelial tissues is discussed in relation to lens differentiation during embryogenesis. We conclude that the lens-forming cells either represent an example of cell differentiation of non-epithelial cells to epithelioid morphology, or represent a special pathway of epithelial differentiation characterized by the absence of cytokeratin filaments and desmosomes. Thus two classes of tissue with epithelia-like morphology can be distinguished: those epithelia which contain desmosomes and cytokeratin filaments and those epithelioid tissues which do not contain these structures but are rich in vimentin filaments (lens cells, germ epithelium of testis, endothelium).     

Selective association of the tyrosine kinases Src, Fyn, and Lyn with integrin-rich actin cytoskeletons of activated, nonaggregated platelets.

Integrin-mediated interactions between cytoskeletal proteins and extracellular fibrinogen are required for platelet adhesion. We have previously demonstrated that the major platelet integrin, alpha(IIb)beta(3), becomes incorporated into the actin cytoskeleton of platelets in an activation-dependent, aggregation-independent manner. To determine if regulatory molecules are also associated with these integrin-rich cytoskeletal complexes, we examined actin cytoskeletons for the presence of kinases and phosphoproteins. Western immunoblot analysis revealed that the tyrosine kinases Src, Fyn, and Lyn are specifically associated with actin cytoskeletons of activated, nonaggregated platelets. However, as noted by others, the cytoskeletal association of focal adhesion kinase depends on platelet aggregation. Actin cytoskeletons isolated from (32)P-labeled platelets also contain a number of phosphorylated proteins. Interestingly, an approximately 18-kDa phosphoprotein was uniquely present in activated platelet cytoskeletons. Collectively, our results demonstrate that actin cytoskeletons of activated, nonaggregated platelets contain not only integrins, but also kinases and phosphoproteins that could regulate platelet adhesion and transmembrane communication.     

Distribution of cytoskeletal proteins, integrins, leukocyte adhesion molecules and extracellular matrix proteins in plastic-embedded human and rat kidneys.

OBJECTIVE: To study the distribution of cytoskeletal proteins (actin, alpha-actinin, vinculin, beta-tubulin, keratin, vimentin, desmin), adhesion molecules for cell-matrix interations (very later antigens [VLA1-61, beta1, beta2 [CD18], vitronectin receptor [alphavbeta3], CD 11b), leukocyte adhesion molecules (ICAM-1) and extracellular matrix proteins (collagen IV, fibronectin, laminin, vitronectin) in human and rat kidneys by using a superior processing and immunohistochemical staining technique. STUDY DESIGN: Human and rat kidneys were fixed in 2% paraformaldehyde, dehydrated in acetone and processed in a new, low-toxic glycol, methacrylate mixture, especially developed for immunohistochemistry. Both the glomeruli and the interstitial areas were carefully examined and scored semiquantitatively. RESULTS: Immunostained plastic sections showed excellent morphology combined with remarkably well preserved antigenicity. CONCLUSION: The above mentioned provides an excellent tool for the accurate localization of a wide variety of antigens at the light microscopic level.     

2D and 3D-organized cardiac cells shows differences in cellular morphology, adhesion junctions, presence of myofibrils and protein expression

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.     

Cytoskeletal features of alveolar myofibroblasts and pericytes in normal human and rat lung.

Frozen or paraffin-embedded human and rat lung specimens were stained with antibodies against total actin, alpha-smooth muscle (SM) actin, vimentin, desmin, or gelsolin. Alveolar interstitial myofibroblasts [i.e., contractile interstitial cells (CIC)] were labeled by total actin antibody but not by alpha-SM actin antibody. They stained for vimentin and gelsolin and, in rat lungs, most of them for desmin. Pericytes located around venules at the junction of three alveolar septa were always positive for alpha-SM actin and never for desmin. Tissue samples were also immunostained by an alpha-SM actin antibody and studied by electron microscopy. With this technique we confirmed that cells, identified as pericytes on the basis of their location, were intensely labeled by alpha-SM actin antibodies, whereas alveolar myofibroblasts were not. We conclude that in the lung interstitium pericytes and alveolar myofibroblasts have distinct cytoskeletal features, alpha-SM actin antibody staining being a simple method to distinguish between them. Furthermore, it appears that alveolar myofibroblasts have a peculiar pattern of cytoskeletal protein composition which, in the rat, is similar to that previously described for stromal cells in uterine submucosa, liver sinusoids (Ito cells), or the core of intestinal villi.     

The Drosophila spectraplakin Short stop regulates focal adhesion dynamics by cross-linking microtubules and actin

The spectraplakin family of proteins includes ACF7/MACF1 and BPAG1/dystonin in mammals, VAB-10 in Caenorhabditis elegans, Magellan in zebrafish, and Short stop (Shot), the sole Drosophila member. Spectraplakins are giant cytoskeletal proteins that cross-link actin, microtubules, and intermediate filaments, coordinating the activity of the entire cytoskeleton. We examined the role of Shot during cell migration using two systems: the in vitro migration of Drosophila tissue culture cells and in vivo through border cell migration. RNA interference (RNAi) depletion of Shot increases the rate of random cell migration in Drosophila tissue culture cells as well as the rate of wound closure during scratch-wound assays. This increase in cell migration prompted us to analyze focal adhesion dynamics. We found that the rates of focal adhesion assembly and disassembly were faster in Shot-depleted cells, leading to faster adhesion turnover that could underlie the increased migration speeds. This regulation of focal adhesion dynamics may be dependent on Shot being in an open confirmation. Using Drosophila border cells as an in vivo model for cell migration, we found that RNAi depletion led to precocious border cell migration. Collectively, these results suggest that spectraplakins not only function to cross-link the cytoskeleton but may regulate cell–matrix adhesion.     

Integrin-linked kinase (ILK) and its interactors: a new paradigm for the coupling of extracellular matrix to actin cytoskeleton and signaling complexes.

How intracellular cytoskeletal and signaling proteins connect and communicate with the extracellular matrix (ECM) is a fundamental question in cell biology. Recent biochemical, cell biological, and genetic studies have revealed important roles of cytoplasmic integrin-linked kinase (ILK) and its interactive proteins in these processes. Cell adhesion to ECM is an important process that controls cell shape change, migration, proliferation, survival, and differentiation. Upon adhesion to ECM, integrins and a selective group of cytoskeletal and signaling proteins are recruited to cell matrix contact sites where they link the actin cytoskeleton to the ECM and mediate signal transduction between the intracellular and extracellular compartments. In this review, we discuss the molecular activities and cellular functions of ILK, a protein that is emerging as a key component of the cell-ECM adhesion structures.     

Interstitial cell migration: integrin-dependent and alternative adhesion mechanisms

Adhesion and migration are integrated cell functions that build, maintain and remodel the multicellular organism. In migrating cells, integrins are the main transmembrane receptors that provide dynamic interactions between extracellular ligands and actin cytoskeleton and signalling machineries. In parallel to integrins, other adhesion systems mediate adhesion and cytoskeletal coupling to the extracellular matrix (ECM). These include multifunctional cell surface receptors (syndecans and CD44) and discoidin domain receptors, which together coordinate ligand binding with direct or indirect cytoskeletal coupling and intracellular signalling. We review the way that the different adhesion systems for ECM components impact cell migration in two- and three-dimensional migration models. We further discuss the hierarchy of these concurrent adhesion systems, their specific tasks in cell migration and their contribution to migration in three-dimensional multi-ligand tissue environments.     

Pak1 regulates focal adhesion strength, myosin IIA distribution, and actin dynamics to optimize cell migration

Cell motility requires the spatial and temporal coordination of forces in the actomyosin cytoskeleton with extracellular adhesion. The biochemical mechanism that coordinates filamentous actin (F-actin) assembly, myosin contractility, adhesion dynamics, and motility to maintain the balance between adhesion and contraction remains unknown. In this paper, we show that p21-activated kinases (Paks), downstream effectors of the small guanosine triphosphatases Rac and Cdc42, biochemically couple leading-edge actin dynamics to focal adhesion (FA) dynamics. Quantitative live cell microscopy assays revealed that the inhibition of Paks abolished F-actin flow in the lamella, displaced myosin IIA from the cell edge, and decreased FA turnover. We show that, by controlling the dynamics of these three systems, Paks regulate the protrusive activity and migration of epithelial cells. Furthermore, we found that expressing Pak1 was sufficient to overcome the inhibitory effects of excess adhesion strength on cell motility. These findings establish Paks as critical molecules coordinating cytoskeletal systems for efficient cell migration.     

Focal adhesion kinase regulates actin nucleation and neuronal filopodia formation during axonal growth

The establishment of neural circuits depends on the ability of axonal growth cones to sense their surrounding environment en route to their target. To achieve this, a coordinated rearrangement of cytoskeleton in response to extracellular cues is essential. Although previous studies have identified different chemotropic and adhesion molecules that influence axonal development, the molecular mechanism by which these signals control the cytoskeleton remains poorly understood. Here, we show that in vivo conditional ablation of the focal adhesion kinase gene (Fak) from mouse hippocampal pyramidal cells impairs axon outgrowth and growth cone morphology during development, which leads to functional defects in neuronal connectivity. Time-lapse recordings and in vitro FRAP analysis indicate that filopodia motility is altered in growth cones lacking FAK, probably owing to deficient actin turnover. We reveal the intracellular pathway that underlies this process and describe how phosphorylation of the actin nucleation-promoting factor N-WASP is required for FAK-dependent filopodia formation. Our study reveals a novel mechanism through which FAK controls filopodia formation and actin nucleation during axonal development.     

Activation of ROCK by RhoA is regulated by cell adhesion, shape, and cytoskeletal tension

Adhesion to the extracellular matrix regulates numerous changes in the actin cytoskeleton by regulating the activity of the Rho family of small GTPases. Here, we report that adhesion and the associated changes in cell shape and cytoskeletal tension are all required for GTP-bound RhoA to activate its downstream effector, ROCK. Using an in vitro kinase assay for endogenous ROCK, we found that cells in suspension, attached on substrates coated with low density fibronectin, or on spreading-restrictive micropatterned islands all exhibited low ROCK activity and correspondingly low myosin light chain phosphorylation, in the face of high levels of GTP-bound RhoA. In contrast, allowing cells to spread against substrates rescued ROCK and myosin activity. Interestingly, inhibition of tension with cytochalasin D or blebbistatin also inhibited ROCK activity within 20 min. The abrogation of ROCK activity by cell detachment or inhibition of tension could not be rescued by constitutively active RhoA-V14. These results suggest the existence of a feedback loop between cytoskeletal tension, adhesion maturation, and ROCK signaling that likely contributes to numerous mechanochemical processes.     

Cytoskeleton structure and adhesion properties of human stromal precursors under conditions of simulated microgravity

During spaceflight and in simulated microgravity (SMG), cytoskeleton rearrangements were observed in lymphocytes, glial cells and osteoblasts. One potential mechanism for the cytoskeletal gravisensitivity of cells is the disruption of the extracellular matrix and integrin interactions. We investigated the effect of SMG on the structure of the actin cytoskeleton, distribution of cellular vinculin, the expression of some integrin subtypes and cellular adhesion molecules in cultured mesenchymal stem cells (hMSCs) derived from human bone marrow in vitro. Simulated microgravity was produced by desktop RPM equipment (Dutch Space, Netherlands). Cells were exposed to simulated microgravity for 30 min to 120 h. The results showed that the actin cytoskeleton was reorganized very quickly (30 min). Later (6, 24, and 48 h), the number of cells with disrupted actin cytoskeletons was increased; however, after 120 h of exposure, cells partly regained their F-actin structures. RPM exposure augmented the number of cells that express integrin-α2. We also observed a decrease in the number of VCAM-1-positive cells and changes in the expression of ICAM-1. Our findings indicate that SMG induces reversible microfilament reorganization in hMSCs and alters their adhesion properties.     

Roles of nectins in cell adhesion, migration and polarization

Nectins are Ca2+-independent immunoglobulin (Ig)-like cell-cell adhesion molecules, which comprise a family consisting of four members. Nectins have five activities: (1) they show Ca2+-independent cell-cell adhesion activity by homo- and hetero-trans-interactions through their extracellular regions; (2) they bind afadin, an actin filament (F-actin)-binding protein, through their cytoplasmic tails and are connected to the actin cytoskeleton; (3) they induce activation of Cdc42 and Rac small G proteins through their cytoplasmic tails; (4) they bind Par-3, a cell polarity protein, through their cytoplasmic tails; and (5) they heterophilically trans-interact with Necls, nectin-like molecules, through their extracellular regions. Through these activities, nectins regulate a variety of cellular functions, including adhesion, migration, and polarization. Here we describe these activities and functions of nectins.     

miR-24 triggers epidermal differentiation by controlling actin adhesion and cell migration

During keratinocyte differentiation and stratification, cells undergo extensive remodeling of their actin cytoskeleton, which is important to control cell mobility and to coordinate and stabilize adhesive structures necessary for functional epithelia. Limited knowledge exists on how the actin cytoskeleton is remodeled in epithelial stratification and whether cell shape is a key determinant to trigger terminal differentiation. In this paper, using human keratinocytes and mouse epidermis as models, we implicate miR-24 in actin adhesion dynamics and demonstrate that miR-24 directly controls actin cable formation and cell mobility. miR-24 overexpression in proliferating cells was sufficient to trigger keratinocyte differentiation both in vitro and in vivo and directly repressed cytoskeletal modulators (PAK4, Tks5, and ArhGAP19). Silencing of these targets recapitulated the effects of miR-24 overexpression. Our results uncover a new regulatory pathway involving a differentiation-promoting microribonucleic acid that regulates actin adhesion dynamics in human and mouse epidermis.     

Beta1 integrins are distributed in adhesion structures with fibronectin and caveolin and in coated pits.

Integrins are found in adhesion structures, which link the extracellular matrix to cytoskeletal proteins. Here, we attempt to further define the distribution of beta1 integrins in the context of their association with matrix proteins and other cell surface molecules relevant to the endocytic process. We find that beta1 integrins colocalize with fibronectin in fibrillar adhesion structures. A fraction of caveolin is also organized along these adhesion structures. The extracellular matrix protein laminin is not concentrated in these structures. The alpha4beta1 integrin exhibits a distinct distribution from other beta1 integrins after cells have adhered for 1 h to extracellular matrix proteins but is localized in adhesion structures after 24 h of adhesion. There are differences between the fibronectin receptors: alpha5beta1 integrins colocalize with adaptor protein-2 in coated pits, while alpha4beta1 integrins do not. This parallels our earlier observation that of the two laminin receptors, alpha1beta1 and alpha6beta1, only alpha1beta1 integrins colocalize with adaptor protein-2 in coated pits. Calcium chelation or inhibition of mitogen-activated protein kinase kinase, protein kinase C, or src did not affect localization of alpha1beta1 and alpha5beta1 integrins in coated pits. Likewise, the integrity of coated-pit structures or adhesion structures is not required for integrin and adaptor protein-2 colocalization. This suggests a robust and possibly constitutive interaction between these integrins and coated pits.     

c-kit receptor signaling through its phosphatidylinositide-3'-kinase-binding site and protein kinase C: role in mast cell enhancement of degranulation, adhesion, and membrane ruffling.

In bone marrow-derived mast cells (BMMCs), the Kit receptor tyrosine kinase mediates diverse responses including proliferation, survival, chemotaxis, migration, differentiation, and adhesion to extracellular matrix. In connective tissue mast cells, a role for Kit in the secretion of inflammatory mediators has been demonstrated as well. We recently demonstrated a role for phosphatidylinositide-3' (PI 3)-kinase in Kit-ligand (KL)-induced adhesion of BMMCs to fibronectin. Herein, we investigated the mechanism by which Kit mediates enhancement of Fc epsilon RI-mediated degranulation, cytoskeletal rearrangements, and adhesion in BMMCs. Wsh/Wsh BMMCs lacking endogenous Kit expression, were transduced to express normal and mutant Kit receptors containing Tyr-->Phe substitution at residues 719 and 821. Although the normal Kit receptor fully restored KL-induced responses in Wsh/Wsh BMMCs, Kit gamma 719F, which fails to bind and activate PI 3-kinase, failed to potentiate degranulation and is impaired in mediating membrane ruffling and actin assembly. Inhibition of PI 3-kinase with wortmannin or LY294002 also inhibited secretory enhancement and cytoskeletal rearrangements mediated by Kit. In contrast, secretory enhancement and adhesion stimulated directly through protein kinase C (PKC) do not require PI 3-kinase. Calphostin C, an inhibitor of PKC, blocked Kit-mediated adhesion to fibronectin, secretory enhancement, membrane ruffling, and filamentous actin assembly. Although cytochalasin D inhibited Kit-mediated filamentous actin assembly and membrane ruffling, secretory enhancement and adhesion to fibronectin were not affected by this drug. Therefore, Kit-mediated cytoskeletal rearrangements that are dependent on actin polymerization can be uncoupled from the Kit-mediated secretory and adhesive responses. Our results implicate receptor-proximal PI 3-kinase activation and activation of a PKC isoform in Kit-mediated secretory enhancement, adhesion, and cytoskeletal reorganization.     

Integrin alpha3 subunit participates in myoblast adhesion and fusion in vitro

Satellite cells are myogenic precursor cells, participating in growth, and regeneration of skeletal muscles. The proteins that play a role in myogenesis are integrins. In this report, we show that the integrin alpha3 subunit is expressed in quiescent satellite cells and activated myoblasts. We also find that in myoblasts the integrin alpha3 subunit is localized at cell-cell and cell-extracellular matrix contacts. We notice that increase in protein and mRNA encoding the integrin alpha3 subunit accompanies myoblast differentiation. Using double immunofluorescence and immunoprecipitation experiments, we demonstrate that the integrin alpha3 subunit co-localizes with actin, and binds the integrin beta1 subunit and ADAM12, suggesting that the complex alpha3beta1/ADAM12 is probably involved in myoblast fusion. Importantly, overexpression of the full-length integrin alpha3 subunit increases myoblast fusion whereas an antibody against its extracellular domain inhibits fusion. These data demonstrate that the integrin alpha3 subunit may contribute to satellite cell activation and then myoblast adhesion and fusion.