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Laminin polymerization is receptor-facilitated and is defective in dystrophic dy2j mice
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Abstracts
Laminin Polymerization is Receptor-Facilitated and is Defective in Dystrophic dy2~Mice
Regulation of Fibronectin Matrix Assembly K. A. Brenner, S. A. Corbett and J. E. Schwarzbauer
H. Colognato and P. D. Yurchenco Robert Wood Johnson Medical School, Piscataway N,I 08854 Laminin-2 (LN-2) is required for the function of mature skeletal muscle, as evidenced by -1/2 of autosomal congenital muscular dystrophies that show abhsence of or abnormalities in the laminin (LN) c~2 chain. A similar pathology is seen in dy -'l mice, which express LN ct2 chain with a deletion in N-terminal domain VI, yet is secreted to the sarcolemmal basement membrane (BM). Here we compare normal LNs-1 and -2 and dy 2-1 mouse LN-2 in order to understand how LN, in particular domain VI, contributes to muscle stability and function. The majority of dy 21 skeletal muscle LN-2 was retained in the BM solely through linkage via entactin to the collagen IV network. An assay for self-assembly revealed that dy-'l-LN-2 was defective in its ability to form LN polymer bonds. Using LN proteolytic fragments and recombinant proteins, we found that mouse myoblasts and myotubes did not interact with the N-terminal c~-chain, but mediated binding to LN through its C-terminal long arm. LN polymerized at concentrations more than 100-fold lower than its solution free critical concentration while engaged to muscle surface receptors. This receptor-facilitated assembly (RFA) was not observed using dy -'1 mouse LN-2, and RFA of normal LN was inhibited by polymer-inhibiting reagents. Disruption of interactions between the muscle surface and the two most C-terminal subunits of c~-chain G domain surprisingly affected the formation and distribution of LN surface polymer, whereas an additional blockade of 0~7 or ]31 integrin subunits completely abolished LN binding to the surface. Surface-bound LN polymer progressively co-localized with dystroglycan and the 0t7[31 integrin. Furthermore, the location and organization of these receptors was altered by the addition of polymer-forming LN. In summary, RFA can occur on the muscle surface, requiring both receptor interactions and polymer bonds, and this process results in reorganization of receptors such as ct7]31 integrin and dystroglycan. Although it is retained in the BM and its receptor interactions are preserved, dy2J LN is unable to mediate RF self-assembly, suggesting that this process may be required for maintenance of stable cytoskeletal-BM linkages in the muscle sarcolemma.
Department of Molecular Biology, Princeton University, Princeton, NJ 08544 Fibronectin matrix assembly in the HT1080 human fibrosarcoma cell line is regulated by the glucocorticoid dexamethasone. Overnight incubation in dexamethasone induces fibronectin to assemble into fibrils. This induction is inhibited by cycloheximide, showing a requirement for new protein synthesis. However integrin levels do not change in treated cells. Dexamethasonetreated cells show increased actin organization into stress fibers. Integrins link the actin cytoskeleton to the extracellular matrix and cytoskeletal organization may induce a change in the activation state of the integrins while leaving their levels unchanged. Certain divalent cations and anti-integrin antibodies have been shown to activate integrins. Cells were treated with either manganese or the ]31 stimulatory antibody TS2/16 in the absence of dexamethasone. Each independently increased matrix assembly in HT1080 cells. To determine how the cytoskeleton ~night affect integrin function we examined caldesmon, an actin and calcium binding protein. Caldesmon localizes along stress fibers in dexamethasone-treated cells, whereas in the untreated cells it is visible only at plasma membrane protrusions. Furthermore, caldesmon shows a modest increase in expression with dexamethasone treatment. We propose that dexamethasone is inducing cytoskeletal reorganization, possibly mediated by caldesmon, which leads to integrin activation and upregulated matrix assembly.
Regulation of G1 Cell Cycle Progression by Assembly of a Fibronectin Matrix J. L. Sechler and J. E. Schwarzbauer Department of Molecular Biology, Princeton University, Princeton NJ 08544 Assembly of a fibronectin (FN) matrix is a multistep process requiring binding to integrin receptors and specific interactions between individual FN molecules. We have generated recombinant FNs (recFN) containing various mutations and have shown that the rate of assembly and cytoskeletal re-organization is altered in re-
Abstracts
Laminin Polymerization is Receptor-Facilitated and is Defective in Dystrophic dy2~Mice
Regulation of Fibronectin Matrix Assembly K. A. Brenner, S. A. Corbett and J. E. Schwarzbauer
H. Colognato and P. D. Yurchenco Robert Wood Johnson Medical School, Piscataway N,I 08854 Laminin-2 (LN-2) is required for the function of mature skeletal muscle, as evidenced by -1/2 of autosomal congenital muscular dystrophies that show abhsence of or abnormalities in the laminin (LN) c~2 chain. A similar pathology is seen in dy -'l mice, which express LN ct2 chain with a deletion in N-terminal domain VI, yet is secreted to the sarcolemmal basement membrane (BM). Here we compare normal LNs-1 and -2 and dy 2-1 mouse LN-2 in order to understand how LN, in particular domain VI, contributes to muscle stability and function. The majority of dy 21 skeletal muscle LN-2 was retained in the BM solely through linkage via entactin to the collagen IV network. An assay for self-assembly revealed that dy-'l-LN-2 was defective in its ability to form LN polymer bonds. Using LN proteolytic fragments and recombinant proteins, we found that mouse myoblasts and myotubes did not interact with the N-terminal c~-chain, but mediated binding to LN through its C-terminal long arm. LN polymerized at concentrations more than 100-fold lower than its solution free critical concentration while engaged to muscle surface receptors. This receptor-facilitated assembly (RFA) was not observed using dy -'1 mouse LN-2, and RFA of normal LN was inhibited by polymer-inhibiting reagents. Disruption of interactions between the muscle surface and the two most C-terminal subunits of c~-chain G domain surprisingly affected the formation and distribution of LN surface polymer, whereas an additional blockade of 0~7 or ]31 integrin subunits completely abolished LN binding to the surface. Surface-bound LN polymer progressively co-localized with dystroglycan and the 0t7[31 integrin. Furthermore, the location and organization of these receptors was altered by the addition of polymer-forming LN. In summary, RFA can occur on the muscle surface, requiring both receptor interactions and polymer bonds, and this process results in reorganization of receptors such as ct7]31 integrin and dystroglycan. Although it is retained in the BM and its receptor interactions are preserved, dy2J LN is unable to mediate RF self-assembly, suggesting that this process may be required for maintenance of stable cytoskeletal-BM linkages in the muscle sarcolemma.
Department of Molecular Biology, Princeton University, Princeton, NJ 08544 Fibronectin matrix assembly in the HT1080 human fibrosarcoma cell line is regulated by the glucocorticoid dexamethasone. Overnight incubation in dexamethasone induces fibronectin to assemble into fibrils. This induction is inhibited by cycloheximide, showing a requirement for new protein synthesis. However integrin levels do not change in treated cells. Dexamethasonetreated cells show increased actin organization into stress fibers. Integrins link the actin cytoskeleton to the extracellular matrix and cytoskeletal organization may induce a change in the activation state of the integrins while leaving their levels unchanged. Certain divalent cations and anti-integrin antibodies have been shown to activate integrins. Cells were treated with either manganese or the ]31 stimulatory antibody TS2/16 in the absence of dexamethasone. Each independently increased matrix assembly in HT1080 cells. To determine how the cytoskeleton ~night affect integrin function we examined caldesmon, an actin and calcium binding protein. Caldesmon localizes along stress fibers in dexamethasone-treated cells, whereas in the untreated cells it is visible only at plasma membrane protrusions. Furthermore, caldesmon shows a modest increase in expression with dexamethasone treatment. We propose that dexamethasone is inducing cytoskeletal reorganization, possibly mediated by caldesmon, which leads to integrin activation and upregulated matrix assembly.
Regulation of G1 Cell Cycle Progression by Assembly of a Fibronectin Matrix J. L. Sechler and J. E. Schwarzbauer Department of Molecular Biology, Princeton University, Princeton NJ 08544 Assembly of a fibronectin (FN) matrix is a multistep process requiring binding to integrin receptors and specific interactions between individual FN molecules. We have generated recombinant FNs (recFN) containing various mutations and have shown that the rate of assembly and cytoskeletal re-organization is altered in re-