The mechanotransduction machinery at work at adherens junctions.

The shaping of a multicellular body, and the maintenance and repair of adult tissues require fine-tuning of cell adhesion responses and the transmission of mechanical load between the cell, its neighbors and the underlying extracellular matrix. A growing field of research is focused on how single cells sense mechanical properties of their micro-environment (extracellular matrix, other cells), and on how mechanotransduction pathways affect cell shape, migration, survival as well as differentiation. Within multicellular assemblies, the mechanical load imposed by the physical properties of the environment is transmitted to neighboring cells. Force imbalance at cell-cell contacts induces essential morphogenetic processes such as cell-cell junction remodeling, cell polarization and migration, cell extrusion and cell intercalation. However, how cells respond and adapt to the mechanical properties of neighboring cells, transmit forces, and transform mechanical signals into chemical signals remain open questions. A defining feature of compact tissues is adhesion between cells at the specialized adherens junction (AJ) involving the cadherin super-family of Ca(2+)-dependent cell-cell adhesion proteins (e.g., E-cadherin in epithelia). Cadherins bind to the cytoplasmic protein ß-catenin, which in turn binds to the filamentous (F)-actin binding adaptor protein α-catenin, which can also recruit vinculin, making the mechanical connection between cell-cell adhesion proteins and the contractile actomyosin cytoskeleton. The cadherin-catenin adhesion complex is a key component of the AJ, and contributes to cell assembly stability and dynamic cell movements. It has also emerged as the main route of propagation of forces within epithelial and non-epithelial tissues. Here, we discuss recent molecular studies that point toward force-dependent conformational changes in α-catenin that regulate protein interactions in the cadherin-catenin adhesion complex, and show that α-catenin is the core mechanosensor that allows cells to locally sense, transduce and adapt to environmental mechanical constrains.

Immobilized dimers of N-cadherin-Fc chimera mimic cadherin-mediated cell contact formation: contribution of both outside-in and inside-out signals.

Cell adhesion receptors of the cadherin family are involved in various developmental processes, affecting cell adhesion and migration, and also cell proliferation and differentiation. In order to dissect the molecular mechanisms of cadherin-based cell-cell adhesion and subsequent signal transduction to the cytoskeleton and/or cytoplasm leading to adapted cell responses, we developed an approach allowing us to mimic and control cadherin activation. We produced a dimeric N-cadherin-Fc chimera (Ncad-Fc) which retains structural and functional properties of cadherins, including glycosylation, Ca(2+)-dependent trypsin sensitivity and the ability to mediate Ca(2+)-dependent self-aggregation of covered microbeads. Beads covered with either Ncad-Fc or anti-N-cadherin antibodies specifically bound to N-cadherin expressing cells. Both types of beads induced the recruitment of N-cadherin, beta-catenin, alpha-catenin and p120, by lateral mobilization of preexisting cell membrane complexes. Furthermore, cadherin clustering elicited by Ncad-Fc beads triggered local accumulations of tyrosine phosphorylated proteins, a recruitment and redistribution of actin filaments, as well as local membrane remodeling. These results support a model where the adhesion of cadherin ectodomains is followed by clustering of cadherin/catenin complexes allowing signal transduction affecting both cytoskeletal reorganization and cytoplasmic signal mobilization (outside-in signaling). Interestingly, bead-cell binding was altered by agents promoting microfilament and microtubule depolymerization or tyrosine phosphorylation, indicating a possible regulation of the adhesive properties of the extracellular domain of N-cadherin by intracellular factors (inside-out signaling).

Cadherins M, 11, and 6 expression patterns suggest complementary roles in mouse neuromuscular axis development.

As the result of a systematic search for cell adhesion molecules of the cadherin family expressed in the developing mouse neuromuscular system, we obtained cDNAs coding for eight molecules of the family, including cadherins M, 11, and 6. Northern blot and in situ hybridization analysis in the mouse embryo revealed a complementary expression of these transcripts. M-cadherin is found in embryonic somitic and nonsomitic striated muscles. As far as the hypaxial musculature is concerned, M-cadherin is expressed in committed but not in migratory precursor cells. Cadherin-11 is detected in mesodermal and conjunctive tissues and transiently in the ependymal germinative layer and in the motoneuron columns of the spinal cord. Cadherin-6 is found in embryonic spinal motoneuron columns and in Schwann cell precursors. In vitro experiments confirmed the muscular, glial, and fibroblastic origins of cadherins M, 11, and 6 transcripts, respectively. Altogether, these results suggest that various cadherins are differentially involved in muscle cell, Schwann cell, and motoneuron interactions and differentiation during neuromuscular development.

Distinct location and prevalence of alpha-, beta-catenins and gamma-catenin/plakoglobin in developing and denervated skeletal muscle.

We studied the distribution of alpha-catenin, beta-catenin and gamma-catenin/plakoglobin in developing, adult and denervated mouse skeletal muscle. During primary myogenesis, all three catenins present a subsarcolemmal distribution within primary myotubes. During secondary myogenesis they accumulate at myotube-myotube contacts. In contrast to the other catenins, gamma-catenin is strongly expressed in the sarcoplasm. In adult muscle, all three catenins are localized on the presynaptic elements of the neuromuscular junction. In denervated muscles, alpha- and beta-catenins are upregulated like N- and M-cadherin, while the levels of gamma-catenin/plakoglobin remain unchanged. The developmental changes in localization and regulation of alpha- and beta-catenins in muscle compared to gamma-catenin/plakoglobin are suggestive of a privileged association of alpha- and beta-catenins with N- and M-cadherins, while gamma-catenin/plakoglobin appears to be expressed quite independently and must assume a different role during myogenesis.

[Cadherins, the development and regeneration of the neuromuscular axis]. / Les cadhérines, la mise en place et la régénération de l'axe neuromusculaire.

Various cell adhesion molecules of the cadherin family characterize the neuromuscular system. During development, cadherins N and M are sequentially expressed by myogenic cells during the two waves of myoblast fusion. Two other cadherins, called 6 and 11, are also expressed during the embryonic musculature development. In adult muscle, cadherins N and M, whose expression is suppressed by muscle activity, persist only at the neuromuscular junction and are reexpressed at the surface of denervated fibers. Cadherins N, M and E are also expressed in adult peripheral nerves. Their differential localization at Schmidt-Lanterman clefts, Ranvier nodes and neuromuscular junctions suggest that these molecules contribute to the stabilization of specialized intercellular contacts. In conclusion, a combination of cadherins, the expression of which is spatially and temporally regulated, participates in the differentiation and maintenance of the organization of the various cellular and tissular components of the neuromuscular system.

Localized deposition of M-cadherin in the glomeruli of the granular layer during the postnatal development of mouse cerebellum.

M-cadherin is a Ca2+-dependent cell adhesion molecule of the cadherin family, initially localized at the areas of contact between myotubes during myogenesis, but also detected in the peripheral nerve and at the adult neuromuscular junction. In this study, searching for the expression of M-cadherin in the adult mouse brain, we observed a restricted expression of M-cadherin in one of the three layers of the cerebellar cortex: the granular layer. M-cadherin was accumulated in structures rich in synapses and other intercellular junctions where mossy fibers connect granule cell dendrites, the glomeruli. This molecule was not expressed in the cerebellum during the first steps of postnatal cerebellar neurogenesis: granule cell proliferation and migration and Purkinje cell alignment. M-cadherin expression was first detected at postnatal day (P) 11, after the establishment of the synaptic connections between mossy fibers and granule cell dendrites. It then accumulated in glomeruli during their phase of maturation which is characterized by the formation of puncta adherentia between granule cell dendrites. M-cadherin was undetectable in the cerebella of the weaver and staggerer mutants, lacking granule cells, and therefore mature glomeruli and puncta adherentia. Furthermore, other components classically associated with intercellular junctions, i.e., alpha-caterin, beta-catenin and actin filaments, closely paralleled M-cadherin appearance and colocalized with M-cadherin in the mature glomeruli. M-cadherin, which appears as a molecular marker of glomerulus maturation, might be implicated in the formation, and be the ligand, of adherens junctions encountered in this structure.

M-cadherin distribution in the mouse adult neuromuscular system suggests a role in muscle innervation.

M-cadherin belongs to the Ca(2+)-dependent cadherin family of cell adhesion molecules and was first isolated from a mouse muscle cell line cDNA library. It is specifically expressed in muscle tissue during development and is supposed to play an important role in secondary myogenesis. In the present study the expression of M-cadherin mRNA and protein and its localization were investigated in adult mouse skeletal muscle and peripheral nerve. The mRNA was abundant in embryonic legs from embryonic day (E)14 to E18. It remained expressed in new-born and adult muscles. In the adult muscle M-cadherin immunoreactivity was only detected at the neuromuscular junction, associated with perijunctional mononucleated cells and on intramuscular nerves. Peripheral nerves were also M-cadherin-positive. The molecule was found at the surface of myelinated nerve fibres where it was concentrated at the node of Ranvier. When a nerve was crushed and allowed to regenerate, M-cadherin was over-expressed at the site of nerve injury and in the distal stump. M-cadherin was also upregulated on the sarcolemma of denervated muscle fibres. Taken together, these observations point toward a much wider tissue distribution of M-cadherin than previously thought. M-cadherin might be involved not only in specific steps of myogenesis but also in some aspects of synaptogenesis, axon/Schwann cell interactions and node of Ranvier structural maintenance.

[Gallstone dissolution in an adult female population: trial with lovastatin therapy]. / Disolución de cálculos vesiculares en una población de mujeres adultas: ensayo de terapia con lovastatina.

BACKGROUND: There are no chilean reports on gallstone dissolution using oral medications. AIM: To measure the proportion of asymptomatic adult women eligible for gallstone dissolution therapy and to test the effectiveness and tolerance of lovastatin for this purpose. SUBJECTS AND METHODS: Three hundred fifty six women working at health care institutions were subjected to a gallbladder ultrasound examination. Non pregnant women with radiolucent gallstones of less than 2 cm phi were invited to receive lovastatin 20 mg od and were followed during six months. RESULTS: Twenty two women had gallstones and eight eligible women received lovastatin therapy. No reduction in stone size was observed in these women. CONCLUSIONS: Less than half of asymptomatic women with gallstones are eligible for oral dissolution therapy. Lovastatin did not reduce gallstone size after six months of therapy.

Growth and cell density-dependent expression of stathmin in C2 myoblasts in culture.

Stathmin is a 19-kDa, ubiquitous cytoplasmic phosphoprotein whose expression is strongly regulated during tissue development and maturation and which was proposed as a general relay integrating diverse intracellular signaling pathways. Since myoblasts tend to align and differentiate in vitro toward myotubes above a certain density in culture, we examined the expression of stathmin as a function of cell density in the C2 myogenic cell line. Whereas stathmin was hardly detectable in low-to medium-density cultures corresponding to less than 1 microgram soluble protein/cm2, it became expressed to a stable level above this threshold of cell density. This cell density effect on stathmin expression was not mediated by a diffusible factor, since myogenic C2 or fibroblastic 3T3 cells grown at low and high density within the same culture flask displayed the same pattern of density-dependent stathmin expression, significant stathmin levels being observed only in the dense moiety of the flask. Interestingly, culture conditions which indirectly perturb cell-cell contacts, such as low Ca2+ or incubation with cytoskeleton disrupting agents such as nocodazole or cytochalasin D, prevented the expression of stathmin in C2 cells even at high density. More directly, anti-E-cadherin immunoglobulins, interfering with direct cell-cell contacts of the E-cadherin expressing S180 sarcoma-derived 2B2 cells, also prevented the expression of stathmin in these cells even at high density. Altogether, our results indicate that cell-cell contacts, probably mediated by adhesion molecules such as cadherins, are responsible for the high-density-induced expression of stathmin, which might then participate, in particular in the case of myogenic cells, in the control of the proliferation of cells and of their entry into the differentiation process.

Expression and localization of RAP1 proteins during myogenic differentiation.

The RAP1 subfamily of small GTPases has been involved in various differentiation programs. In skeletal muscle, several lines of evidence suggest that various small GTPases could be implicated in muscle development. This raised the question of whether the RAP1 proteins (RAP1A and/or RAP1B) could be involved in myogenesis. In the present study, we report on the regulation of RAP1 transcripts and proteins during myogenic differentiation. Northern blot analysis performed with differentiated and undifferentiated C2 myogenic cells pointed out that both genes undergo specific regulation during myogenesis in vitro since differentiation of C2 cells was accompanied by a down-regulation of RAP1B gene transcription and continuous expression of the RAP1A mRNA. In addition, immunofluorescence experiments revealed the accumulation of the RAP1 proteins in differentiated C2 cells and in primary culture of mouse myotubes. Investigation of the intracellular location of RAP1 proteins in undifferentiated and differentiated C2 cells showed that the proteins were associated with the late endocytic compartments. To verify that the build-up of RAP1 proteins had a relevance for developmental mechanisms in vivo, we studied their expression and localization at different stages of skeletal muscle development. We found that RAP1 proteins accumulated in specialized muscle cell domains undergoing important modifications during early and late myogenesis: these were the neuromuscular and myotendinous junctions, respectively. Altogether, our data indicate that RAP1 proteins are regulated during myogenic differentiation.

Cadherin-dependent cell aggregation is affected by decapeptide derived from rat extracellular super-oxide dismutase.

A synthetic HAV-containing decapeptide homologous to the amino acid sequence 44R-Q53 in rat extracellular superoxide dismutase B affects cadherin-dependent cell aggregation. Cell lines, some of them transfected, expressing different types of cadherins were tested using in vitro cell aggregation and cell dissociation assays. A concentration-dependent inhibition of aggregation by the EC-SOD-derived HAV-containing peptide was detected only in N-cadherin expressing cells. These results suggest the localisation and possible protective role of EC-SOD B for cells expressing N-cadherin.

M-cadherin localization in developing adult and regenerating mouse skeletal muscle: possible involvement in secondary myogenesis.

In this work, we investigated the distribution of the Ca(2+)-dependent cell adhesion molecule, M-cadherin, in mouse limb muscle during normal development and regeneration. Using two unrelated anti-M-cadherin peptide antibodies, we found scarce M-cadherin immunostaining during primary myogenesis (E12-E14) with no accumulation at areas of cell-cell contact. In contrast, the staining sharply increased in intensity at E16, remained high during secondary myogenesis (E16-P0) but disappeared soon after birth. During secondary myogenesis, M-cadherin was specifically accumulated at the characteristic sites of insertion of secondary myotubes in neighbouring primary myotubes. M-cadherin was also accumulated at the areas of contact between fusing secondary myoblasts and myotubes in vitro. In the adult normal and regenerating muscle, we did not detect M-cadherin accumulations at the surface of myofibres. All together, these observations suggest that M-cadherin is specifically involved in secondary myogenesis.

[Association of cholesterolosis and cholelithiasis: pathogenic implications and effects of the natural history of cholelithiasis]. / Asociación de colesterolosis y litíasis de la vesícula biliar: implicancia patogénica y efecto sobre la historia natural de la colelitiasis.

We studied 793 patients subjected to cholecystectomy to determine a) the relative frequency of cholesterolosis and cholelithiasis, b) the effects of their association on the natural history of biliary disease, c) the characteristics of gallstones associated to cholesterolosis and d) factors potentially associated to their pathogenesis. The gallbladders of all patients were examined and in 289 subjects a preoperative clinical history was taken. We observed that cholesterolosis is associated to earlier clinical manifestations of biliary disease and cholecystectomy, to a greater frequency of single calculus and to a higher weight/height index. It is concluded that there are relationships between the pathogenesis of cholesterolosis and cholelithiasis and that their association favors the development of clinical manifestations.

Is intercellular communication via gap junctions required for myoblast fusion?

Fusion of myoblasts to form syncitial muscle cells results from a complex series of sequential events including cell alignment, cell adhesion and cell communication. The aim of the present investigation was to assess whether intercellular communication through gap junctions would be required for subsequent membrane fusion. The presence of the gap junction protein connexin 43 at areas of contact between prefusing rat L6 myoblasts was established by immunofluorescent staining. These myoblasts were dye-coupled, as demonstrated by the use of the scrape-loading/dye transfer technique. L6 myoblast dye coupling was reversibly blocked by heptanol in short term experiments as well as after chronic treatment. After a single addition of 3.5 mM heptanol, gap junctions remained blocked for up to 8 hours, then this inhibitory effect decreased gradually, likely because the alcohol was evaporated. Changing heptanol solutions every 8 hours during the time course of L6 differentiation resulted in a lasting drastic inhibition of myoblast fusion. We further investigated the effect of heptanol and of other uncoupling agents on the differentiation of primary cultures of embryonic chicken myoblasts. These cells are transiently coupled by gap junctions before myoblast fusion and prolonged application of heptanol, octanol and 18-beta-glycyrrhetinic acid also inhibited their fusion. The effect of heptanol and octanol was neither due to a cytotoxic effect nor to a modification of cell proliferation. Moreover, heptanol treatment did not alter myoblast alignment and adhesion. Taken together these observations suggest that intercellular communication might be a necessary step for myoblast fusion.

[Cholecystectomy in young women from the south-oriental area of Santiago: comparison between 2 periods]. / Colecistectomía en mujeres jovenes del area suroriente de Santiago: comparación entre dos períodos.

The aim of this study was to know if cholecystectomy rates have decreased in young women, considering that these rates have decreased in the last years in the general chilean population. The frequency of previous cholecystectomy was compared in 1582 women aged 23.9 +/- 5.8 years admitted to a maternity for delivery between 1985 and 1986 and 4943 women aged 24.6 +/- 5.9 years admitted between 1989 and 1990 for the same reason. There was a reduction in cholecystectomy frequency from 4.7 to 2.5% specially among women 21 to 35 years old. Cholecystectomy was performed at a mean age of 23 years in both groups and 42.6% of the procedures were done before the first pregnancy. An unexpected finding was a lower body weight among women studied in the second period (62.5 +/- 9.1 vs 67.7 +/- 8.4 k).

Cadherin expression is required for the spread of Shigella flexneri between epithelial cells.

Shigella flexneri, a gram-negative pathogen, invades the human colonic epithelium. After entering epithelial cells, bacteria escape into the cytoplasm, move intracellularly, and pass from cell to cell. The bacterium diverts actin and associated actin-binding proteins to generate a cytoskeleton-based motor that pushes forward the bacterium. As the moving bacterium reaches the inner face of the host-cell cytoplasmic membrane, a protrusion forms that allows passage of this bacterium into a neighboring cell. We show here that components of the intermediate junction are used by the bacterium to allow this passage. Using S180, a mouse fibroblastic sarcoma cell line that does not produce cell adhesion molecules (CAM), and S180L and S180cadN, the same cell line transfected with L-CAM and N-cadherin cDNA, respectively, we demonstrate that expression of a cadherin is required for cell-to-cell spread to occur.

N-cadherin expression in developing, adult and denervated chicken neuromuscular system: accumulations at both the neuromuscular junction and the node of Ranvier.

N-cadherin, a member of the Ca(2+)-dependent cell adhesion molecule family plays essential roles in morphogenesis and histogenesis. N-cadherin has been shown in vitro to promote myoblast fusion and neurite outgrowth. We report here the cellular localization of N-cadherin during development and regeneration of the chick neuromuscular system. N-cadherin was uniformly expressed along the surface of myoblasts and myotubes of E6 limb muscles. Later, as synaptogenesis and secondary myogenesis proceeded, N-cadherin expression was down-regulated and restricted to some large-diameter fibres, then to the areas of contact between few myofibres and subsequently disappeared by embryonic day 17, suggesting that this cadherin may be implicated predominantly in fusion of primary myoblasts and, at lower degree, of secondary myoblasts. The presence of N-cadherin in muscle during the period of nerve trunk ingrowth and its down-regulation after synaptogenesis suggests that this molecule might be implicated in both processes. N-cadherin became accumulated at the neuromuscular junction only a few days after the first synaptic contacts were established and remained at the adult neuromuscular junction, suggesting a role of this molecule in the stabilization of the mature neuromuscular junction. In sciatic nerve, the level of N-cadherin expression remained unchanged from hatching to adult life. N-cadherin was widely distributed on the surface of myelinated fibres and on myelinating Schwann cells: in addition, it was concentrated at the node of Ranvier. At the ultrastructural level, the molecule was detected inside, at the surface and in the basal lamina of Schwann cells and also associated with endoneurial collagen. These observations suggest a role of N-cadherin in the structuring and stabilization of the myelin sheaths. After nerve injury, N-cadherin continued to be expressed by proliferating Schwann cells in the distal stump providing a substratum for regenerating axons. N-cadherin reappeared at the surface of denervated muscle fibres without disappearing from the former synaptic sites. It was detected not only in the sarcoplasm and on sarcolemma of denervated muscle fibres, but also in the basal lamina and in the extracellular matrix. The reexpression of N-cadherin at the surface of denervated muscle fibres suggests a role for this molecule in muscle reinnervation. The presence of N-cadherin in basal lamina and its association with collagen fibres raise questions about the release of N-cadherin in the extracellular space and the existence of a putative heterophilic ligand for N-cadherin.

[Cell adhesion and development of skeletal muscle]. / Adhésion cellulaire et développement du muscle squelettique.

Cell adhesion is a cell autonomous property of pluricellular organisms at the basis of tissues and organs formation. Thus, adhesive processes must be considered as key features of the development of skeletal muscle as well as of other tissues. We present here the actual knowledge on cell adhesion molecules in skeletal muscle morphogenesis. The spatio-temporal expression patterns of N-CAM, N-cadherin, M-cadherin, VLA-4 and VCAM-1 during chicken and mouse myogenesis suggest that these cell adhesion molecules are differentially involved in myoblast-myoblast, myoblast-myotube and myotube-myotube interactions. These molecules link myogenic cells before they are separated by their basal laminae. They can potentially induce preferential cell adhesion and sorting-out as it has been described by Holtfreter. This differential adhesion may lead either to myoblast fusion or to preferential association between primary and secondary myotubes.

N-cadherin and N-CAM-mediated adhesion in development and regeneration of skeletal muscle.

In this review, the experimental evidence supporting the fact that the cell adhesion molecules N-CAM and N-cadherin are involved in myogenesis has been surveyed. In order to give access to the function of these molecules, a strategy of in vivo localization and in vitro perturbation of their adhesive function by interfering antibodies and peptides was applied. Both molecules are expressed at the surface of myogenic cells during myogenesis in vivo and in vitro. The blockade of the N-CAM adhesion function leads to a mild reduction of the rate of myoblast fusion, while the inhibition of the N-cadherin function induces a drastic inhibition of fusion suggesting that N-cadherin-mediated adhesion is a critical step in the process of myoblast fusion. Both molecules are re-expressed during muscle regeneration suggesting that adult myogenesis is under the control of the same adhesive systems as embryonic and foetal myogenesis.