Requirements for sulfate transport and the diastrophic dysplasia sulfate transporter in fibronectin matrix assembly

Diastrophic dysplasia sulfate transporter (DTDST) is a sulfate/chloride antiporter whose function is impaired in several human chondrodysplasias. We show that DTDST is upregulated by dexamethasone stimulation of HT1080 fibrosarcoma cells and is required for fibronectin (FN) extracellular matrix deposition by these cells. DTDST imports sulfate for the modification of glycosaminoglycans. We find that N-sulfation of these chains is important for FN matrix assembly and that sulfation of cell surface proteoglycans is reduced in the absence of DTDST. Of the candidate HT1080 cell surface proteoglycans, only loss of syndecan-2 compromises FN assembly, as shown by syndecan-2 small interfering RNA knockdown. DTDST is both necessary and sufficient to induce FN matrix assembly in HT1080 cells. Knockdown of DTDST ablates FN matrix, whereas its overexpression increases assembly without dexamethasone stimulation. These results identify a previously unrecognized regulatory pathway for matrix assembly via modulation of a sulfate transporter and proteoglycan sulfation. These data raise the possibility that FN assembly defects contribute to chondrodysplasias

Organogenesis: Cutting to the Chase

AbstractGonad morphogenesis in Caenorhabditis elegans requires two secreted proteases. Recent studies show that alterations of the extracellular matrix component fibulin-1 rescue gonadogenesis in the absence of these proteases. This finding is a critical step toward understanding the role of extracellular matrix in organogenesis

Tenascin-C Suppresses Rho Activation

Cell binding to extracellular matrix (ECM) components changes cytoskeletal organization by the activation of Rho family GTPases. Tenascin-C, a developmentally regulated matrix protein, modulates cellular responses to other matrix proteins, such as fibronectin (FN). Here, we report that tenascin-C markedly altered cell phenotype on a three-dimensional fibrin matrix containing FN, resulting in suppression of actin stress fibers and induction of actin-rich filopodia. This distinct morphology was associated with complete suppression of the activation of RhoA, a small GTPase that induces actin stress fiber formation. Enforced activation of RhoA circumvented the effects of tenascin. Effects of active Rho were reversed by a Rho inhibitor C3 transferase. Suppression of GTPase activation allows tenascin-C expression to act as a regulatory switch to reverse the effects of adhesive proteins on Rho function. This represents a novel paradigm for the regulation of cytoskeletal organization by ECM

A Single Cysteine, Cys-64, Is Essential for Assembly of Tenascin-C Hexabrachions

Tenascin-C is a large, multimeric extracellular matrix protein that is found in a variety of tissues and can have profound effects on cell adhesion. It is secreted from cells as a hexamer of six identical chains called a hexabrachion. Disulfide bonding among tenascin subunits mediates intracellular assembly into hexamers. The amino-terminal assembly domain consists of heptad repeats and at least six cysteine residues (Cys-64, -111, -113, -140, -146, -147) that could be involved in multimerization. We have now determined the requirements for these cysteine residues during hexamer assembly. Our results show that only Cys-64 is required to form the hexameric structure. Mutation of Cys-64 to glycine resulted in release of trimer intermediates, which probably form via the heptad repeats, but no hexamers were secreted. In contrast, individual or pairs of mutations of each of the other cysteines had no effect on tenascin hexamer formation, and inclusion of any other cysteine mutations along with C64G did not further disrupt the multimer pattern. However, when all six cysteines were mutated, monomers were the major extracellular form. Together, these results show that trimers are an intermediate of tenascin-C assembly and that Cys-64 is essential for formation of hexabrachions

Endoplasmic spreading requires coalescence of vimentin intermediate filaments at force-bearing adhesions

10.1091/mbc.E12-05-0377Molecular Biology of the Cell24121-30MBCE

Differential Regulation of Neurite Outgrowth and Growth Cone Morphology by 3D Fibronectin and Fibronectin-Collagen Extracellular Matrices

The extracellular matrix (ECM) plays a critical role in development, homeostasis, and regeneration of tissue structures and functions. Cell interactions with the ECM are dynamic and cells respond to ECM remodeling by changes in morphology and motility. During nerve regeneration, the ECM facilitates neurite outgrowth and guides axons with target specificity. Decellularized ECMs retain structural, biochemical, and biomechanical cues of native ECM and have the potential to replace damaged matrix to support cell activities during tissue repair. To determine the ECM components that contribute to nerve regeneration, we analyzed neuron-ECM interactions on two types of decellularized ECM. One matrix was composed primarily of fibronectin (FN) fibrils, and the other FN-rich ECM also contained significant numbers of type I collagen (COL I) fibrils. Using primary neurons dissociated from superior cervical ganglion (SCG) explants, we found that neurites were extended on both matrices without a significant difference in average neurite length after 24 h. The most distinctive features of neurites on the FN matrix were numerous short actin-filled protrusions and longer branches extending from neurite shafts. Very few protrusions and branches were detected on FN-COL matrix. Growth cone morphologies also differed with mostly filopodial growth cones on FN matrix whereas on FN-COL matrix, equivalent numbers of filopodial and slender growth cones were formed. Our work provides new information about how changes in major components of the ECM during tissue repair modulate neuron and growth cone morphologies and helps to define the contributions of neuron-ECM interactions to nerve development and regeneration

α integrin cytoplasmic tails have tissue-specific roles during C. elegans development

Integrin signaling impacts many developmental processes. The complexity of these signals increases when multiple, unique integrin heterodimers are expressed during a single developmental event. Since integrin heterodimers have different signaling capabilities, the signals originating at each integrin type must be separated in the cell. C. elegans have two integrin heterodimers, α INA-1/β PAT-3 and α PAT-2/β PAT-3, which are expressed individually or simultaneously, based on tissue type. We used chimeric α integrins to assess the role of α integrin cytoplasmic tails during development. Chimeric integrin ina-1 with the pat-2 cytoplasmic tail rescued lethality and maintained neuron fasciculation in an ina-1 mutant. Interestingly, the pat-2 tail was unable to completely restore distal tip cell migration and vulva morphogenesis. Chimeric integrin pat-2 with the ina-1 cytoplasmic tail had a limited ability to rescue a lethal mutation in pat-2, with survivors showing aberrant muscle organization, yet normal distal tip cell migration. In a wild type background, α integrin pat-2 with the ina-1 cytoplasmic tail had a dominant negative effect which induced muscle disorganization, cell migration defects and lethality. These results show the α integrin cytoplasmic tails impact unique cellular behaviors that vary by tissue type during development

Gonad morphogenesis and distal tip cell migration in the Caenorhabditis elegans hermaphrodite

Cell migration and morphogenesis are key events in tissue development and organogenesis. In Caenorhabditis elegans, the migratory path of the distal tip cells determines the morphology of the hermaphroditic gonad. The distal tip cells undergo a series of migratory phases interspersed with turns to form the gonad. A wide variety of genes have been identified as crucial to this process, from genes that encode components and modifiers of the extracellular matrix to signaling proteins and transcriptional regulators. The connections between extracellular and transmembrane protein functions and intracellular pathways are essential for distal tip cell migration, and the integration of this information governs gonad morphogenesis and determines gonad size and shape

Tenascin-C Modulates Matrix Contraction via Focal Adhesion Kinase– and Rho-mediated Signaling Pathways

A provisional matrix consisting of fibrin and fibronectin (FN) is deposited at sites of tissue damage and repair. This matrix serves as a scaffold for fibroblast migration into the wound where these cells deposit new matrix to replace lost or damaged tissue and eventually contract the matrix to bring the margins of the wound together. Tenascin-C is expressed transiently during wound repair in tissue adjacent to areas of injury and contacts the provisional matrix in vivo. Using a synthetic model of the provisional matrix, we have found that tenascin-C regulates cell responses to a fibrin-FN matrix through modulation of focal adhesion kinase (FAK) and RhoA activation. Cells on fibrin-FN+tenascin-C redistribute their actin to the cell cortex, downregulate focal adhesion formation, and do not assemble a FN matrix. Cells surrounded by a fibrin-FN+tenascin-C matrix are unable to induce matrix contraction. The inhibitory effect of tenascin-C is circumvented by downstream activation of RhoA. FAK is also required for matrix contraction and the absence of FAK cannot be overcome by activation of RhoA. These observations show dual requirements for both FAK and RhoA activities during contraction of a fibrin-FN matrix. The effects of tenascin-C combined with its location around the wound bed suggest that this protein regulates fundamental processes of tissue repair by limiting the extent of matrix deposition and contraction to fibrin-FN-rich matrix in the primary wound area