Organization Of Laminin Research Articles

Inhibition of mmp13a during zebrafish fin regeneration disrupts fin growth, osteoblasts differentiation, and Laminin organization.

Matrix metalloproteinases 13 (MMP13) is a potent endopeptidase that regulate cell growth, migration, and extracellular matrix remodeling. However, its role in fin regeneration remains unclear. mmp13a expression is strongly upregulated during blastema formation and persists in the distal blastema. mmp13a knockdown via morpholino electroporation impairs regenerative outgrowth by decreasing cell proliferation, which correlates with a downregulation of fgf10a and sall4 expression in the blastema. Laminin distribution in the basement membrane is also affected in mmp13a MO-injected rays. Another impact of mmp13a knockdown is observed in the skeletal elements of the fin rays. Expression of two main components of actinotrichia, Collagen II and Actinodin 1 is highly reduced in mmp13a MO-injected rays leading to highly disorganized actinotrichia pattern. Inhibition of mmp13a strongly affects bone formation as shown by a reduction of Zns5 and sp7 expression and of bone matrix mineralization in rays. These defects are accompanied by a significant increase in apoptosis in mmp13a MO-injected fin regenerates. Defects of expression of this multifunctional proteinase drastically affects osteoblast differentiation, bone and actinotrichia formation as well as Laminin distribution in the basement membrane of the fin regenerate, suggesting the important role of Mmp13 during the regenerative process.

Draxin alters laminin organization during basement membrane remodeling to control cranial neural crest EMT

Premigratory neural crest cells arise within the dorsal neural tube and subsequently undergo an epithelial-to-mesenchymal transition (EMT) to leave the neuroepithelium and initiate migration. Draxin is a Wnt modulator that has been shown to control the timing of cranial neural crest EMT. Here we show that this process is accompanied by three stages of remodeling of the basement membrane protein laminin, from regression to expansion and channel formation. Loss of Draxin results in blocking laminin remodeling at the regression stage, whereas ectopic maintenance of Draxin blocks remodeling at the expansion stage. The latter effect is rescued by addition of Snail2, previously shown to be downstream of Draxin. Our results demonstrate an essential function for the Wnt modulator Draxin in regulating basement membrane remodeling during cranial neural crest EMT.

Tmem2 regulates cell-matrix interactions that are essential for muscle fiber attachment

Skeletal muscle morphogenesis depends upon interactions between developing muscle fibers and the extracellular matrix (ECM) that anchors fibers to the myotendinous junction (MTJ). The pathways that organize the ECM and regulate its engagement by cell-matrix adhesion complexes (CMACs) are therefore essential for muscle integrity. Here, we demonstrate the impact of transmembrane protein 2 (tmem2) on cell-matrix interactions during muscle morphogenesis in zebrafish. Maternal-zygotic tmem2 mutants (MZtmem2) exhibit muscle fiber detachment, in association with impaired laminin organization and ineffective fibronectin degradation at the MTJ. Similarly, disorganized laminin and fibronectin surround MZtmem2 cardiomyocytes, which could account for their hindered movement during cardiac morphogenesis. In addition to ECM defects, MZtmem2 mutants display hypoglycosylation of α-dystroglycan within the CMAC, which could contribute to the observed fiber detachment. Expression of the Tmem2 ectodomain can rescue aspects of the MZtmem2 phenotype, consistent with a possible extracellular function of Tmem2. Together, our results suggest that Tmem2 regulates cell-matrix interactions by affecting both ECM organization and CMAC activity. These findings evoke possible connections between the functions of Tmem2 and the etiologies of congenital muscular dystrophies, particularly dystroglycanopathies.

Tmem2 regulates cell-matrix interactions that are essential for muscle fiber attachment.

Skeletal muscle morphogenesis depends upon interactions between developing muscle fibers and the extracellular matrix (ECM) that anchors fibers to the myotendinous junction (MTJ). The pathways that organize the ECM and regulate its engagement by cell-matrix adhesion complexes (CMACs) are therefore essential for muscle integrity. Here, we demonstrate the impact of transmembrane protein 2 (tmem2) on cell-matrix interactions during muscle morphogenesis in zebrafish. Maternal-zygotic tmem2 mutants (MZtmem2) exhibit muscle fiber detachment, in association with impaired laminin organization and ineffective fibronectin degradation at the MTJ. Similarly, disorganized laminin and fibronectin surround MZtmem2 cardiomyocytes, which could account for their hindered movement during cardiac morphogenesis. In addition to ECM defects, MZtmem2 mutants display hypoglycosylation of α-dystroglycan within the CMAC, which could contribute to the observed fiber detachment. Expression of the Tmem2 ectodomain can rescue aspects of the MZtmem2 phenotype, consistent with a possible extracellular function of Tmem2. Together, our results suggest that Tmem2 regulates cell-matrix interactions by affecting both ECM organization and CMAC activity. These findings evoke possible connections between the functions of Tmem2 and the etiologies of congenital muscular dystrophies, particularly dystroglycanopathies.

In vivo generation of a mature and functional artificial skeletal muscle.

Extensive loss of skeletal muscle tissue results in mutilations and severe loss of function. In vitro-generated artificial muscles undergo necrosis when transplanted in vivo before host angiogenesis may provide oxygen for fibre survival. Here, we report a novel strategy based upon the use of mouse or human mesoangioblasts encapsulated inside PEG-fibrinogen hydrogel. Once engineered to express placental-derived growth factor, mesoangioblasts attract host vessels and nerves, contributing to in vivo survival and maturation of newly formed myofibres. When the graft was implanted underneath the skin on the surface of the tibialis anterior, mature and aligned myofibres formed within several weeks as a complete and functional extra muscle. Moreover, replacing the ablated tibialis anterior with PEG-fibrinogen-embedded mesoangioblasts also resulted in an artificial muscle very similar to a normal tibialis anterior. This strategy opens the possibility for patient-specific muscle creation for a large number of pathological conditions involving muscle tissue wasting.

Contractility of Neonatal Cardiomyocytes is Altered with Different Densities of Laminin Covalently Attached to Microposts

Heart disease is the number one killer in the USA. The collective contractility of the muscle cells of the myocardium - cardiomyocytes - generates the necessary force for the function of a healthy beating heart. Laminin interacts in vivo with cardiomyocytes. Changes in the extracellular concentration and organization of laminin relate to different types of heart disease. Arrays of polydimethylsiloxane (PDMS) microposts measure forces generated by adhesive mammalian cells and were here used to characterize the contractility of single neonatal cardiomyocytes. We used two types of organosilanes to bind laminin to the surface of PDMS microposts: 3-glycidoxypropyltrimethoxysilane and 3-aminopropyltriethoxysilane. We acquired videos of contracting cardiomyocytes at two different days after cells started to beat and functionally characterized the contractility of single cells. More specifically, we calculated generated forces, beating rate, time of contractions and speeds of contraction and relaxation. These parameters varied in time as a function of organosilane surface stability and cardiomyocyte biological changes when cultured in vitro. Higher forces are generated by cardiomyocytes cultured on laminin covalently attached to PDMS microposts relative to laminin physisorbed to oxidized PDMS. We obtained higher laminin density with 3-glycidoxypropyltrimethoxysilane, which correlated to higher generated forces. We also observed higher beating rate at the day 1 and a considerable decrease at day 2. Compared to 3-glycidoxypropyltrimethoxysilane, higher stability of laminin covalent attachment was observed with 3-aminopropyltriethoxysilane. The beating rate and speeds of contraction and relaxation increased and time of contractions decreased at day 2 for neonatal cardiomyocytes cultured on these PDMS micropost surfaces. Our results shed light on the potential of in vitro biomechanical systems to model extracellular disease conditions of heart pathologies. Future work will test the contractility of cardiomyocytes with mutations known to originate cardiomyopathies.

NAD+ Biosynthesis Ameliorates a Zebrafish Model of Muscular Dystrophy

Author SummaryA variety of diseases, both inherited and acquired, affect muscle tissues in humans. Critical to muscle homeostasis is the anchoring of muscle fibers to their surrounding microenvironment through cell adhesion complexes that help to resist the repeated stress experienced during muscle contraction. Genetic mutations in these complexes weaken this mechanical attachment, making fibers more susceptible to damage and death. The resulting increased fiber degeneration can eventually lead to progressive muscle-wasting diseases, known collectively as muscular dystrophies. Although clinical trials are ongoing, there is presently no way to cure the loss of muscle structure and function associated with these diseases. We identified a novel cell adhesion pathway involving integrin alpha6 that promotes adhesion of muscle cells to their microenvironment. Here, we show that activation of this pathway not only significantly reduces muscle degeneration but also improves the swimming ability of dystrophic zebrafish. We explore the likely benefits and limitations of this pathway in treating symptoms of congenital muscular dystrophies. Our findings suggest that activation of this pathway (for example, by boosting levels of NAD+) has the potential to ameliorate loss of muscle structure and function in multiple muscular dystrophies.

Interactive properties of human glioblastoma cells with brain neurons in culture and neuronal modulation of glial laminin organization

The harmonious development of the central nervous system depends on the interactions of the neuronal and glial cells. Extracellular matrix elements play important roles in these interactions, especially laminin produced by astrocytes, which has been shown to be a good substrate for neuron growth and axonal guidance. Glioblastomas are the most common subtypes of primary brain tumors and may be astrocytes in origin. As normal laminin-producing glial cells are the preferential substrate for neurons, and glial tumors have been shown to produce laminin, we questioned whether glioblastoma retained the same normal glial–neuron interactive properties with respect to neuronal growth and differentiation. Then, rat neurons were co-cultured onto rat normal astrocytes or onto three human glioblastoma cell lines obtained from neurosurgery. The co-culture confirmed that human glioblastoma cells as well as astrocytes maintained the ability to support neuritogenesis, but non-neural normal or tumoral cells failed to do so. However, glioblastoma cells did not distinguish embryonic from post-natal neurons in relation to neurite pattern in the co-cultures, as normal astrocytes did. Further, the laminin organization on both normal and tumoral glial cells was altered from a filamentous arrangement to a mixed punctuate/filamentous pattern when in co-culture with neurons. Together, these results suggest that glioblastoma cells could identify neuronal cells as partners, to support their growth and induce complex neurites, but they lost the normal glia property to distinguish neuronal age. In addition, our results show for the first time that neurons modulate the organization of astrocytes and glioblastoma laminin on the extracellular matrix.

β1-Integrin Orients Epithelial Polarity via Rac1 and Laminin

Epithelial cells polarize and orient polarity in response to cell-cell and cell-matrix adhesion. Although there has been much recent progress in understanding the general polarizing machinery of epithelia, it is largely unclear how this machinery is controlled by the extracellular environment. To explore the signals from cell-matrix interactions that control orientation of cell polarity, we have used three-dimensional culture systems in which Madin-Darby canine kidney (MDCK) cells form polarized, lumen-containing structures. We show that interaction of collagen I with apical beta1-integrins after collagen overlay of a polarized MDCK monolayer induces activation of Rac1, which is required for collagen overlay-induced tubulocyst formation. Cysts, comprised of a monolayer enclosing a central lumen, form after embedding single cells in collagen. In those cultures, addition of a beta1-integrin function-blocking antibody to the collagen matrix gives rise to cysts that have defects in the organization of laminin into the basement membrane and have inverted polarity. Normal polarity is restored by either expression of activated Rac1, or the inclusion of excess laminin-1 (LN-1). Together, our results suggest a signaling pathway in which the activation of beta1-integrins orients the apical pole of polarized cysts via a mechanism that requires Rac1 activation and laminin organization into the basement membrane.

LARGE can functionally bypass alpha-dystroglycan glycosylation defects in distinct congenital muscular dystrophies.

Several congenital muscular dystrophies caused by defects in known or putative glycosyltransferases are commonly associated with hypoglycosylation of alpha-dystroglycan (alpha-DG) and a marked reduction of its receptor function. We have investigated changes in the processing and function of alpha-DG resulting from genetic manipulation of LARGE, the putative glycosyltransferase mutated both in Large(myd) mice and in humans with congenital muscular dystrophy 1D (MDC1D). Here we show that overexpression of LARGE ameliorates the dystrophic phenotype of Large(myd) mice and induces the synthesis of glycan-enriched alpha-DG with high affinity for extracellular ligands. Notably, LARGE circumvents the alpha-DG glycosylation defect in cells from individuals with genetically distinct types of congenital muscular dystrophy. Gene transfer of LARGE into the cells of individuals with congenital muscular dystrophies restores alpha-DG receptor function, whereby glycan-enriched alpha-DG coordinates the organization of laminin on the cell surface. Our findings indicate that modulation of LARGE expression or activity is a viable therapeutic strategy for glycosyltransferase-deficient congenital muscular dystrophies.

FAK Deficiency in Cells Contributing to the Basal Lamina Results in Cortical Abnormalities Resembling Congenital Muscular Dystrophies

Targeted deletion of focal adhesion kinase ( fak) in the developing dorsal forebrain resulted in local disruptions of the cortical basement membrane located between the neuroepithelium and pia-meninges. At disruption sites, clusters of neurons invaded the marginal zone. Retraction of radial glial endfeet, midline fusion of brain hemispheres, and gliosis also occurred, similar to type II cobblestone lissencephaly as seen in congenital muscular dystrophy. Interestingly, targeted deletion of fak in neurons alone did not result in cortical ectopias, indicating that fak deletion from glia is required for neuronal mislocalization. Unexpectedly, fak deletion specifically from meningeal fibroblasts elicited similar cortical ectopias in vivo and altered laminin organization in vitro. These observations provide compelling evidence that FAK plays a key signaling role in cortical basement membrane assembly and/or remodeling. In addition, FAK is required within neurons during development because neuron-specific fak deletion alters dendritic morphology in the absence of lamination defects.

Undersulfation of proteoglycans and proteins alter C6 glioma cells proliferation, adhesion and extracellular matrix organization

Proteoglycans are considered to be important molecule in cell–microenvironment interactions. They are overexpressed in neoplastic cells modifying their growth and migration in hosts. In this work we verified that undersulfation of proteoglycans and other sulfated molecules, induced by sodium chlorate treatment, inhibited C6 glioma cells proliferation in a dose-dependent way. This effect was restored by the addition of exogenous heparin. We could not detect significant cell mortality in our culture condition. The treatment also impaired in a dose-dependent manner, C6 cell adhesion to extracellular matrix (ECM) proteins (collagen IV, laminin and fibronectin). In addition, sodium chlorate treatment altered C6 glioma cell morphology, from the fibroblast-like to a more rounded one. This effect was accompanied by increased synthesis of fibronectin and alterations in its extracellular network organization. However, we could not observe modifications on laminin organization and synthesis. The results suggest an important connection between sulfation degree with important tumor functions, such as proliferation and adhesion. We suggest that proteoglycans may modulate the glioma microenvironment network during tumor cell progression and invasion.

Dystroglycan Expression in Hepatic Stellate Cells: Role in Liver Fibrosis

Dystroglycan is a membrane component of the dystrophin-glycoprotein transmembrane complex. Its expression is required for the spatial organization of laminin on the cell surface and for basement membrane assembly. In view of the constitution of a perisinusoidal basement membrane during liver fibrosis, we studied dystroglycan expression in liver fibrosis. Dystroglycan mRNA and protein expression were investigated by immunofluorescence, Western blot, and quantitative RT-PCR (TaqMan) in normal human and rat liver and in isolated rat hepatic stellate cells. On Western blot, a 43-kd band corresponding to β-dystroglycan was observed in protein extracted from normal and fibrotic human and rat livers. The specific 43-kd protein was also detected in lysates from rat hepatic stellate cells but not from hepatocytes. By immunofluorescence, patchy deposits of β-dystroglycan were detected on membrane of hepatic stellate cells in culture. On Western blot and quantitative RT-PCR, an up-regulation of β-dystroglycan was shown during spontaneous activation of hepatic stellate cells in culture. Direct evidence for the role of dystroglycan in laminin-hepatic stellate cell interaction was shown because specific antibody directed against α-dystroglycan inhibited partially hepatic stellate cell adhesion on laminin-coated plates. This mechanism was calcium dependent because EDTA inhibited cell/laminin adhesion, an effect reversed by addition of Ca2+. This study shows that dystroglycan is expressed on the membrane of hepatic stellate cells and is up-regulated during liver fibrosis. Dystroglycan interaction with laminin should be implicated in the concentration of pericellular laminin and in the constitution of a perisinusoidal basement membrane during liver fibrosis.

Distinct roles for dystroglycan, β1 integrin and perlecan in cell surface laminin organization

Dystroglycan (DG) is a cell surface receptor for several extracellular matrix (ECM) molecules including laminins, agrin and perlecan. Recent data indicate that DG function is required for the formation of basement membranes in early development and the organization of laminin on the cell surface. Here we show that DG-mediated laminin clustering on mouse embryonic stem (ES) cells is a dynamic process in which clusters are consolidated over time into increasingly more complex structures. Utilizing various null-mutant ES cell lines, we define roles for other molecules in this process. In beta1 integrin-deficient ES cells, laminin-1 binds to the cell surface, but fails to organize into more morphologically complex structures. This result indicates that beta1 integrin function is required after DG function in the cell surface-mediated laminin assembly process. In perlecan-deficient ES cells, the formation of complex laminin-1 structures is defective, implicating perlecan in the laminin matrix assembly process. Moreover, laminin and perlecan reciprocally modulate the organization of the other on the cell surface. Taken together, the data support a model whereby DG serves as a receptor essential for the initial binding of laminin on the cell surface, whereas beta1 integrins and perlecan are required for laminin matrix assembly processes after it binds to the cell.

Thyroid Hormone Regulates the Extracellular Organization of Laminin on Astrocytes

Astrocytes produce laminin, a key extracellular matrix guidance molecule in the developing brain. Laminin is bound to transmembrane receptors on the surface of astrocytes known as integrins, which are, in turn, bound to the microfilament meshwork inside the astrocyte. Previous studies have shown that T4 regulates the pattern of integrin distribution in astrocytes by modulating the organization of the microfilaments. In this study, the effect of thyroid hormone on the secretion and topology of laminin in astrocytes was examined. Linear arrays of secreted laminin were observed on the surface of the T4-treated astrocytes within 10 h after seeding the cells onto poly-d-lysine-coated coverslips and became an organized meshwork by 24 h. In contrast, little if any laminin was identified on the surface of either hormone-deficient or T3-treated cells until 36 h after seeding and then was restricted to punctate deposits. Secretion of laminin into the medium by hormone-deficient and T3-treated cells was significantly greater than that by T4-treated cells. Conversely, deposition of laminin into the extracellular matrix was significantly greater in T4-treated cells than in hormone-deficient and T3-treated cells. Thyroid hormone had no effect on the production of laminin by astrocytes. These data show that T4 regulates the extracellular deposition and organization of laminin on the surface of astrocytes and provide a mechanism by which this morphogenic hormone can influence neuronal migration and axonal projection in the developing brain.

Thyroid hormone regulates the extracellular organization of laminin on astrocytes.

Astrocytes produce laminin, a key extracellular matrix guidance molecule in the developing brain. Laminin is bound to transmembrane receptors on the surface of astrocytes known as integrins, which are, in turn, bound to the microfilament meshwork inside the astrocyte. Previous studies have shown that T4 regulates the pattern of integrin distribution in astrocytes by modulating the organization of the microfilaments. In this study, the effect of thyroid hormone on the secretion and topology of laminin in astrocytes was examined. Linear arrays of secreted laminin were observed on the surface of the T4-treated astrocytes within 10 h after seeding the cells onto poly-D-lysine-coated coverslips and became an organized meshwork by 24 h. In contrast, little if any laminin was identified on the surface of either hormone-deficient or T3-treated cells until 36 h after seeding and then was restricted to punctate deposits. Secretion of laminin into the medium by hormone-deficient and T3-treated cells was significantly greater than that by T4-treated cells. Conversely, deposition of laminin into the extracellular matrix was significantly greater in T4-treated cells than in hormone-deficient and T3-treated cells. Thyroid hormone had no effect on the production of laminin by astrocytes. These data show that T4 regulates the extracellular deposition and organization of laminin on the surface of astrocytes and provide a mechanism by which this morphogenic hormone can influence neuronal migration and axonal projection in the developing brain.

Binding of the Cysteine Proteinases Papain and Cathepsin B-like to Coated Laminin: Use of Synthetic Peptides from Laminin and from the Laminin Binding Region of the β1Integrin Subunit to Characterize the Binding Site

Cysteine proteinases of the papain superfamily, i.e., papain and cathepsin B-like proteinase, were found to be able to bind to laminin-coated wells. When papain and cathepsin B-like proteinase were used, saturable binding curves were found. The characterization of the binding site was carried out using synthetic peptides which corresponded to the most relevant functional sites of laminin and an octapeptide from the laminin binding region of the β1integrin subunit. In binding experiments, the decapeptide RNIAEIIKDI and the pentapeptide YIGSR were able to displace papain and cathepsin B-like proteinase from coated laminin. Nevertheless, the integrin β1peptide DLYYLMDL was the most powerful in the same experimental system. From these results, the C-terminal region of this cross-shaped protein, i.e., the end of the long arm, and the region including the YIGSR sequence of the short arm of the β chain would be the cysteine proteinase binding site. This binding site is probably the result of the network organization of laminin which brings two regions, separated on a single laminin molecule, into proximity. In previous work, digestion of basement membranes has been found to be associated with the binding of cysteine proteinases to these supramolecular structures [N. Guinec, V. Dalet-Fumeron, and M. Pagano (1992)FEBS Lett.308, 305–308]. The present report demonstrates that laminin is the cysteine proteinase binding protein of basement membranes. This property of laminin could be associated with tumor invasion and other tissue remodeling processes linked to proteolysis of basement membranes and extracellular matrices.