Polymerisation of fibrin αC-domains promotes endothelial cell migration and proliferation.

Upon conversion of fibrinogen into fibrin, fibrinogen αC-domains containing the RGD recognition motif form ordered αC polymers. Our previous study revealed that polymerisation of these domains promotes integrin-dependent adhesion and spreading of endothelial cells, as well as integrin-mediated activation of the FAK and ERK1/2 signalling pathways. The major goal of this study was to test the impact of αC-domain polymerisation on endothelial cell migration and proliferation during wound healing, and to clarify the mechanism underlying superior activity of αC polymers toward endothelial cells. In an in vitro wound healing assay, confluent endothelial cell monolayers on tissue culture plates coated with the αC monomer or αC polymers were wounded by scratching and wound closure was monitored by time-lapse videomicroscopy. Although the plates were coated with equal amounts of αC species, as confirmed by ELISA, wound closure by the cells occurred much faster on αC polymers, indicating that αC-domain polymerisation promotes cell migration and proliferation. In agreement, endothelial cell proliferation was also more efficient on αC polymers, as revealed by cell proliferation assay. Wound closure on both types of substrates was equally inhibited by the integrin-blocking GRGDSP peptide and a specific antagonist of the ERK1/2 signalling pathway. In contrast, blocking the FAK signaling pathway by a specific antagonist decreased wound closure only on αC polymers. These results indicate that polymerisation of the αC-domains enhances integrin-dependent endothelial cell migration and proliferation mainly through the FAK signalling pathway. Furthermore, clustering of integrin-binding RGD motifs in αC polymers is the major mechanism triggering these events.

Cell-surface transglutaminase promotes fibronectin assembly via interaction with the gelatin-binding domain of fibronectin: a role in TGFbeta-dependent matrix deposition.

Assembly of fibronectin into a fibrillar matrix is critical for regulation of cell growth and migration, embryogenesis and wound healing. We have previously shown that cell-surface tissue transglutaminase serves as an integrin-binding adhesion coreceptor for fibronectin. Here we report that transglutaminase strongly promotes fibronectin assembly mediated by alpha5beta1 integrin. This effect is independent from transglutaminase-mediated enzymatic crosslinking of fibronectin and separate from the ability of transglutaminase to stimulate cell spreading. Surface transglutaminase increases the binding of fibronectin to cells via interaction with its gelatin-binding domain that contains modules I6II1,2I7-9 and lacks integrin-binding motifs. The gelatin-binding fragment of fibronectin binds to surface transglutaminase on cells in suspension but does not interact with cell monolayers where surface transglutaminase is occupied by fibronectin. Surface transglutaminase colocalizes with growing fibronectin fibrils at early timepoints of matrix formation and remains codistributed with fibronectin matrices thereafter. The observed stimulation of matrix assembly by transglutaminase is blocked by the gelatin-binding fragment of fibronectin, but is not strongly perturbed by its N-terminal fragment consisting of modules I1-5. These results implicate an interaction between transglutaminase and the gelatin-binding domain of fibronectin in matrix assembly and suggest its role in initiation of fibrillogenesis. However, blocking antibodies against alpha5beta1 integrin or the cell-binding fragment of fibronectin that contains modules III2-11 most strongly suppress matrix formation and abolish the effects of transglutaminase. Hence, transglutaminase cooperates with but can not substitute for alpha5beta1 integrin in fibronectin assembly. Treatment of fibroblasts with transforming growth factor beta (TGFbeta) significantly increases surface expression of transglutaminase and its association with beta1 integrins, but not with alphaVbeta3 integrin. TGFbeta enhances the binding of fibronectin to the cell surface and elevates matrix formation, whereas antibody against transglutaminase or the gelatin-binding fragment of fibronectin suppresses these effects, indicating an involvement of transglutaminase in TGFbeta-dependent fibronectin assembly. Therefore, TGFbeta-induced fibronectin matrix deposition during normal wound healing or fibrotic disorders may depend on upregulation of integrin-associated surface transglutaminase.

Cell surface tissue transglutaminase is involved in adhesion and migration of monocytic cells on fibronectin.

Expression of tissue transglutaminase (transglutaminase II, tTG) was shown to increase drastically during monocyte differentiation into macrophages; however, its role in monocytic cells remains largely unknown. This study describes a novel function of cell surface tTG as an adhesion and migration receptor for fibronectin (Fn). Two structurally related transglutaminases, tTG and the A subunit of factor XIII (FXIIIA), are expressed on the surface of monocytic cells, whereas only surface tTG is associated with multiple integrins of the beta1 and beta3 subfamilies. Both surface levels of tTG and the amounts of integrin-bound tTG are sharply up-regulated during the conversion of monocytes into macrophages. In contrast, a reduction in biosynthesis and surface expression of FXIIIA accompanies monocyte differentiation. Cell surface tTG is colocalized with beta1- and beta3-integrins in podosomelike adhesive structures of macrophages adherent on Fn. Down-regulation of surface tTG by expression of antisense tTG construct or its inhibition by function-blocking antibodies significantly decreases adhesion and spreading of monocytic cells on Fn and, in particular, on the gelatin-binding fragment of Fn consisting of modules I6II1,2I7-9. Likewise, interfering with the adhesive function of surface tTG markedly reduces migration of myeloid cells on Fn and its gelatin-binding fragment. These data demonstrate that cell surface tTG serves as an integrin-associated adhesion receptor that might be involved in extravasation and migration of monocytic cells into tissues containing Fn matrices during inflammation.

Matrix-dependent proteolysis of surface transglutaminase by membrane-type metalloproteinase regulates cancer cell adhesion and locomotion.

Cell invasion requires cooperation between adhesion receptors and matrix metalloproteinases (MMPs). Membrane type (MT)-MMPs have been thought to be primarily involved in the breakdown of the extracellular matrix. Our report presents evidence that MT-MMPs in addition to the breakdown of the extracellular matrix may be engaged in proteolysis of adhesion receptors on tumor cell surfaces. Overexpression of MT1-MMP by glioma and fibrosarcoma cells led to proteolytic degradation of cell surface tissue transglutaminase (tTG) at the leading edge of motile cancer cells. In agreement, structurally related MT1-MMP, MT2-MMP, and MT3-MMP but not evolutionary distant MT4-MMP efficiently degraded purified tTG in vitro. Because cell surface tTG represents a ubiquitously expressed, potent integrin-binding adhesion coreceptor involved in the binding of cells to fibronectin (Fn), the proteolytic degradation of tTG by MT1-MMP specifically suppressed cell adhesion and migration on Fn. Reciprocally, Fn in vitro and in cultured cells protected its surface receptor, tTG, from proteolysis by MT1-MMP, thereby supporting cell adhesion and locomotion. In contrast, the proteolytic degradation of tTG stimulated migration of cells on collagen matrices. Together, our observations suggest both an important coreceptor role for cell surface tTG and a novel regulatory function of membrane-anchored MMPs in cancer cell adhesion and locomotion. Proteolysis of adhesion proteins colocalized with MT-MMPs at discrete regions on the surface of migrating tumor cells might be controlled by composition of the surrounding ECM.

Integrins as receptors for laminins.

Laminins are a family of trimeric glycoproteins present in the extracellular matrix and the major constituents of basement membranes. Integrins are alpha beta transmembrane receptors that play critical roles in both cell-matrix and cell-cell adhesion. Several members of the integrin family, including alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, alpha 6 beta 1, alpha 7 beta 1 and alpha 6 beta 4 heterodimers serve as laminin receptors on a variety of cell types. This review summarizes recent advances in understanding the involvement of individual integrins in cell interactions with laminins and the roles of laminin-binding integrins in adhesion-mediated events in vertebrates, including embryonic development, cell migration and tumor cell invasiveness, cell proliferation and differentiation, as well as basement membrane assembly. We discuss the regulation of integrin function via alternative splicing of cytoplasmic domains of alpha and beta subunits of the integrin receptors for laminins and present examples of functional collaboration between laminin-binding integrins and non-integrin laminin receptors. Advances in our understanding of the laminin-binding integrins continue to demonstrate the essential roles these receptors play in maintaining cell polarity and tissue architecture.

Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin.

The protein cross-linking enzyme tissue transglutaminase binds in vitro with high affinity to fibronectin via its 42-kD gelatin-binding domain. Here we report that cell surface transglutaminase mediates adhesion and spreading of cells on the 42-kD fibronectin fragment, which lacks integrin-binding motifs. Overexpression of tissue transglutaminase increases its amount on the cell surface, enhances adhesion and spreading on fibronectin and its 42-kD fragment, enlarges focal adhesions, and amplifies adhesion-dependent phosphorylation of focal adhesion kinase. These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII. Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins. Transglutaminase interacts directly with multiple integrins of beta1 and beta3 subfamilies, but not with beta2 integrins. Complexes of transglutaminase with integrins are formed inside the cell during biosynthesis and accumulate on the surface and in focal adhesions. Together our results demonstrate that tissue transglutaminase mediates the interaction of integrins with fibronectin, thereby acting as an integrin-associated coreceptor to promote cell adhesion and spreading.

Differential onset of expression of alpha 7 and beta 1D integrins during mouse heart and skeletal muscle development.

beta 1D is a recently identified isoform of the beta 1 integrin subunit selectively expressed in skeletal and cardiac muscles. In the present study we determined the temporal expression of beta 1D and its association with alpha subunits during mouse development. By immunohistochemistry and western blot analysis we demonstrated that beta 1D begins to be expressed in skeletal muscles of 17 days embryo (stage E17). Its level progressively increases reaching maximal values few days after birth and remaining high in adult mice. At earlier stages of development (E11-E17) the beta 1A isoform is expressed in skeletal muscle cells. After E17 beta 1A is downregulated and disappears from muscle fibers few days after birth. In cardiac muscle the regulation of the beta 1D expression is different: beta 1D and beta 1A are coexpressed in the heart of E11 embryo. Subsequently expression of beta 1A declines, while beta 1D increases until it becomes the unique beta 1 isoform in cardiomyocytes few days after birth. Previous studies (Belkin et al J. Cell Biol. 132: 211-226, 1996) demonstrated that beta 1D in adult mouse cardiomyocytes is exclusively associated with alpha 7B. Western blot analysis shows that alpha 7B starts to be expressed in the heart only at stage E17, while beta 1D is expressed already at E11 embryo, indicating that alpha subunits other than alpha 7 should associate with beta 1D in early developmental stages. To investigate this aspect, beta 1 associated alpha subunits were identified by western blotting from cardiomyocytes integrin complexes immunoprecipitated with alpha subunit specific antibodies. We found that, during cardiomyocyte development, beta 1D associates with several alpha subunits namely with alpha 5, alpha 6A and alpha 7B. In conclusion these data show that the expression of the beta 1D muscle specific integrin during development occurs much earlier in heart than in skeletal muscle and it can dimerize with different alpha subunits.

Maturation of cell-substratum focal adhesions induced by depolymerization of microtubules is mediated by increased cortical tension.

Dynamics of alterations of focal adhesions (FA) induced by a microtubule-depolymerizing drug, colcemid, was examined in several types of fibroblastic cells. Evolution of individual FA in cultured cells was monitored by interference-reflection microscopy (IRM); at the end of the monitoring period (3 hours) the cells were fixed and immunofluorescence microscopy of the same FA was performed with an antibody against vinculin. Control and colcemid-treated cells remained non-motile and did not show lamellipodial activity at the edges. During the incubation, formation of new FA or disappearance of pre-existing FA did not occur in either colcemid-treated or control cultures. However, FA in colcemid-treated cells significantly increased in size in the course of a 3 hour incubation. The growth of FA was centripetal and sometimes was accompanied by the fusion of several adjacent FA. Immunofluorescence examination showed that colcemid-induced growth of FA was accompanied by accumulation of several proteins specific for these structures including vinculin, talin, paxillin and pp125FAK kinase. Immunoblotting with anti-vinculin antibody showed that incubation with colcemid considerably increased the amount of vinculin associated with the ventral membranes due to its partial redistribution from a soluble pool into the growing adhesions. A substantial increase in tyrosine phosphorylation of pp125FAK was also observed in colcemid-treated cells. In cells plated on elastic silicone rubber films, colcemid induced formation of wrinkles in the films and these wrinkles relaxed after treatment with cytochalasin D. These results confirm that microtubule depolymerization increases traction transmitted to the substratum by the actin cortex and shows that an increase in cortical tension accompanies maturation of FA. Taken together, these data show that short-term incubation with colcemid does not affect the formation of initial FA. In contrast, microtubule depolymerization considerably stimulates the maturation FA, manifested by their centripetal growth. Maturation is proposed to be mediated by increased cortical tension, which is caused by microtubule depolymerization.

beta1D integrin inhibits cell cycle progression in normal myoblasts and fibroblasts.

Integrins are alphabeta heterodimeric transmembrane receptors involved in the regulation of cell growth and differentiation. The beta1 integrin subunit is widely expressed in vivo and is represented by four alternatively spliced cytoplasmic domain isoforms. beta1D is a muscle-specific variant of beta1 integrin and a predominant beta1 isoform in striated muscles. In the present study we showed that expression of the exogenous beta1D integrin in C2C12 myoblasts and NIH 3T3 or REF 52 fibroblasts inhibited cell proliferation. Unlike the case of the common beta1A isoform, adhesion of beta1D-transfected C2C12 myoblasts specifically via the expressed integrin did not activate mitogen-activated protein kinases. The beta1D-induced growth inhibitory signal was shown to occur late in the G1 phase of the cell cycle, before the G1-S transition. Ha-(12R)Ras, but not (Delta22W)Raf-1 oncogene, was able to overcome completely the beta1D-triggered cell growth arrest in NIH 3T3 fibroblasts. Since perturbation of the beta1D amino acid sequence in beta1A/beta1D chimeric integrins decreased the growth inhibitory signal, the entire cytoplasmic domain of beta1D appeared to be important for this function. However, an interleukin-2 receptor-beta1D chimera containing the cytoplasmic domain of beta1D still efficiently inhibited cell growth, showing that the ectodomain and the ligand-binding site in beta1D were not required for the growth inhibitory signal. Together, our data showed a new specific function for the alternatively spliced beta1D integrin isoform. Since the onset of beta1D expression during myodifferentiation coincides with the timing of myoblast withdrawal from the cell cycle, the growth inhibitory properties of beta1D demonstrated in this study might reflect the major function for this integrin in commitment of differentiating skeletal muscle cells in vivo.

Rho-mediated contractility exposes a cryptic site in fibronectin and induces fibronectin matrix assembly.

Many factors influence the assembly of fibronectin into an insoluble fibrillar extracellular matrix. Previous work demonstrated that one component in serum that promotes the assembly of fibronectin is lysophosphatidic acid (Zhang, Q., W.J. Checovich, D.M. Peters, R.M. Albrecht, and D.F. Mosher. 1994. J. Cell Biol. 127:1447-1459). Here we show that C3 transferase, an inhibitor of the low molecular weight GTP-binding protein Rho, blocks the binding of fibronectin and the 70-kD NH2-terminal fibronectin fragment to cells and blocks the assembly of fibronectin into matrix induced by serum or lysophosphatidic acid. Microinjection of recombinant, constitutively active Rho into quiescent Swiss 3T3 cells promotes fibronectin matrix assembly by the injected cells. Investigating the mechanism by which Rho promotes fibronectin polymerization, we have used C3 to determine whether integrin activation is involved. Under conditions where C3 decreases fibronectin assembly we have only detected small changes in the state of integrin activation. However, several inhibitors of cellular contractility, that differ in their mode of action, inhibit cell binding of fibronectin and the 70-kD NH2-terminal fibronectin fragment, decrease fibronectin incorporation into the deoxycholate insoluble matrix, and prevent fibronectin's assembly into fibrils on the cell surface. Because Rho stimulates contractility, these results suggest that Rho-mediated contractility promotes assembly of fibronectin into a fibrillar matrix. One mechanism by which contractility could enhance fibronectin assembly is by tension exposing cryptic self-assembly sites within fibronectin that is being stretched. Exploring this possibility, we have found a monoclonal antibody, L8, that stains fibronectin matrices differentially depending on the state of cell contractility. L8 was previously shown to inhibit fibronectin matrix assembly (Chernousov, M.A., A.I. Faerman, M.G. Frid, O.Y. Printseva, and V.E. Koteliansky. 1987. FEBS (Fed. Eur. Biochem. Soc.) Lett. 217:124-128). When it is used to stain normal cultures that are developing tension, it reveals a matrix indistinguishable from that revealed by polyclonal anti-fibronectin antibodies. However, the staining of fibronectin matrices by L8 is reduced relative to the polyclonal antibody when the contractility of cells is inhibited by C3. We have investigated the consequences of mechanically stretching fibronectin in the absence of cells. Applying a 30-35% stretch to immobilized fibronectin induced binding of soluble fibronectin, 70-kD fibronectin fragment, and L8 monoclonal antibody. Together, these results provide evidence that self-assembly sites within fibronectin are exposed by tension.

beta1-integrin cytoplasmic subdomains involved in dominant negative function.

The beta1-integrin cytoplasmic domain consists of a membrane proximal subdomain common to the four known isoforms ("common" region) and a distal subdomain specific for each isoform ("variable" region). To investigate in detail the role of these subdomains in integrin-dependent cellular functions, we used beta1A and beta1B isoforms as well as four mutants lacking the entire cytoplasmic domain (beta1TR), the variable region (beta1COM), or the common region (beta1 deltaCOM-B and beta1 deltaCOM-A). By expressing these constructs in Chinese hamster ovary and beta1 integrin-deficient GD25 cells (Wennerberg et al., J Cell Biol 132, 227-238, 1996), we show that beta1B, beta1COM, beta1 deltaCOM-B, and beta1 deltaCOM-A molecules are unable to support efficient cell adhesion to matrix proteins. On exposure to Mn++ ions, however, beta1B, but none of the mutants, can mediate cell adhesion, indicating specific functional properties of this isoform. Analysis of adhesive functions of transfected cells shows that beta1B interferes in a dominant negative manner with beta1A and beta3/beta5 integrins in cell spreading, focal adhesion formation, focal adhesion kinase tyrosine phosphorylation, and fibronectin matrix assembly. None of the beta1 mutants tested shows this property, indicating that the dominant negative effect depends on the specific combination of common and B subdomains, rather than from the absence of the A subdomain in the beta1B isoform.

Muscle beta1D integrin reinforces the cytoskeleton-matrix link: modulation of integrin adhesive function by alternative splicing.

Expression of muscle-specific beta1D integrin with an alternatively spliced cytoplasmic domain in CHO and GD25, beta1 integrin-minus cells leads to their phenotypic conversion. beta1D-transfected nonmuscle cells display rounded morphology, lack of pseudopodial activity, retarded spreading, reduced migration, and significantly enhanced contractility compared with their beta1A-expressing counterparts. The transfected beta1D is targeted to focal adhesions and efficiently displaces the endogenous beta1A and alphavbeta3 integrins from the sites of cell-matrix contact. This displacement is observed on several types of extracellular matrix substrata and leads to elevated stability of focal adhesions in beta1D transfectants. Whereas a significant part of cellular beta1A integrin is extractable in digitonin, the majority of the transfected beta1D is digitonin-insoluble and is strongly associated with the detergent-insoluble cytoskeleton. Increased interaction of beta1D integrin with the actin cytoskeleton is consistent with and might be mediated by its enhanced binding to talin. In contrast, beta1A interacts more strongly with alpha-actinin, than beta1D. Inside-out driven activation of the beta1D ectodomain increases ligand binding and fibronectin matrix assembly by beta1D transfectants. Phenotypic effects of beta1D integrin expression in nonmuscle cells are due to its enhanced interactions with both cytoskeletal and extracellular ligands. They parallel the transitions that muscle cells undergo during differentiation. Modulation of beta1 integrin adhesive function by alternative splicing serves as a physiological mechanism reinforcing the cytoskeleton- matrix link in muscle cells. This reflects the major role for beta1D integrin in muscle, where extremely stable association is required for contraction.

Localization of cranin (dystroglycan) at sites of cell-matrix and cell-cell contact: recruitment to focal adhesions is dependent upon extracellular ligands.

We report that cranin (dystroglycan) can become recruited to focal adhesions of cultured rat REF 52 fibroblasts and human aortic smooth muscle cells. Within mature focal adhesions, cranin was present within the plaque region defined by beta 1 integrin, vinculin and phosphotyrosine staining, but occupied a larger domain corresponding to the terminal segments of stress fibers that was more precisely co-extensive with the cytoskeletal proteins alpha-actinin, utrophin and aciculin. When REF 52 fibroblasts were plated on different substrata in the absence of protein synthesis and secretion in serum-free medium, focal clusters of cranin readily formed within 2 hours on matrix proteins that bind cranin directly (laminin or agrin) which were maintained as the focal adhesions became mature. In contrast, cranin failed to become targeted to cell-substratum attachment sites, either at early or later times, when cells were plated on a variety of other substrata that elicit formation of focal adhesions but do not bind cranin directly (fibronectin, vitronectin, collagen type IV, or anti-beta 1 integrin antibody TS2/16). These data strongly suggest that targeting of cranin to focal adhesions was dependent upon the presence of an extracellular ligand capable of binding cranin directly. However, some cultured nonmuscle cell lines (e.g., human umbilical vein endothelial cells, NIH 3T3 and CHO cells) failed to localize cranin to focal adhesions, even when plated on laminin. Cranin was also enriched at cell-cell adherens-type junctions of human normal breast MCF-10 epithelial cells, and at growth cones of E17 rat hippocampal axons. That cranin can become targeted to sites of cell-cell and cell-substratum contact in diverse cell types supports the hypothesis that cranin may be involved in mediating or regulating cell adhesion. The absence of muscle-specific and synapse-specific proteins within fibroblasts and epithelial cells provides a different context for thinking about cranin (dystroglycan) that may aid in discerning general principles of its structure and function.

Beta 1D integrin displaces the beta 1A isoform in striated muscles: localization at junctional structures and signaling potential in nonmuscle cells.

The cytoplasmic domains of integrins provide attachment of these extracellular matrix receptors to the cytoskeleton and play a critical role in integrin-mediated signal transduction. In this report we describe the identification, expression, localization, and initial functional characterization of a novel form of beta 1 integrin, termed beta 1D. This isoform contains a unique alternatively spliced cytoplasmic domain of 50 amino acids, with the last 24 amino acids encoded by an additional exon. Of these 24 amino acids, 11 are conserved when compared to the beta 1A isoform, but 13 are unique (Zhidkova, N. I., A. M. Belkin, and R. Mayne. 1995. Biochem. Biophys. Res. Commun. 214:279-285; van der Flier, A., I. Kuikman, C. Baudoin, R, van der Neuf, and A. Sonnenberg. 1995. FEBS Lett. 369:340-344). Using an anti-peptide antibody against the beta 1D integrin subunit, we demonstrated that the beta 1D isoform is synthesized only in skeletal and cardiac muscles, while very low amounts of beta 1A were detected by immunoblot in striated muscles. Whereas beta 1A could not be detected in adult skeletal muscle fibers and cardiomyocytes by immunofluorescence, beta 1D was localized to the sarcolemma of both cell types. In skeletal muscle, beta 1D was concentrated in costameres, myotendinous, and neuromuscular junctions. In cardiac muscle this beta 1 isoform was found in costamers and intercalated discs. beta 1D was associated with alpha 7A and alpha 7B in adult skeletal muscle. In cardiomyocytes of adult heart, alpha 7B was the major partner for the beta 1D isoform. beta 1D could not be detected in proliferating C2C12 myoblasts, but it appeared immediately after myoblast fusion and its amount continued to rise during myotube growth and maturation. In contrast, expression of the beta 1A isoform was downregulated during myodifferentiation in culture and it was completely displaced by beta 1D in mature differentiated myotubes. We also analyzed some functional properties of the beta 1D integrin subunit. Expression of human beta 1D in CHO cells led to its localization at focal adhesions. Clustering of this integrin isoform on the cell surface stimulated tyrosine phosphorylation of pp125FAK (focal adhesion kinase) and caused transient activation of mitogen-activated protein (MAP) kinases. These data indicate that beta 1D and beta 1A integrin isoforms are functionally similar with regard to integrin-mediated signaling.

A novel dystrophin/utrophin-associated protein is an enzymatically inactive member of the phosphoglucomutase superfamily.

A 60-kDa protein localised in adherens-type cellular junctions, and previously called aciculin, has been found to interact with the cytoskeletal proteins dystrophin and utrophin [Belkin, A. M. & Burridge, K. (1995) J. Biol. Chem. 270, 6328-6337]. In this study, we report the complete sequence of this protein, and show that it is a novel member of the phosphoglucomutase (PGM) family of proteins. The PGM-related protein (PGM-RP), which contains 506 amino acids (55.6 kDa), is smaller than PGM1 (566 amino acids, 61 kDa). The active site consensus sequences of prokaryotic and eukaryotic mutases are not conserved in PGM-RP, a finding consistent with the lack of enzymatic activity of PGM-RP in vitro, and the absence of a phosphorylated intermediate in vivo. The organisation of the PGM-RP gene is essentially identical to that of PGM1. We propose that the PGM-RP gene, which we have mapped to human chromosome 9qcen-q13, evolved from the PGM1 gene, and encodes a protein with a structural rather than an enzymatic role. PGM-RP is expressed predominantly in muscle with the highest levels in smooth muscle. The significance of the interaction between dystrophin/utrophin and an increasing number of cytoplasmic proteins including PGM-RP remains to be explored.

Localization of utrophin and aciculin at sites of cell-matrix and cell-cell adhesion in cultured cells.

Aciculin is a phosphoglucomutase-related cytoskeletal protein associated with dystrophin and/or utrophin in various tissues and cell types. Comparison of expression patterns for aciculin, dystrophin, and utrophin in cultured cells demonstrated that aciculin is coexpressed with utrophin, but not with dystrophin, in cultures of A7r5 smooth muscle cells and REF52 fibroblasts. Some other nonmuscle cells synthesized only trace levels of or no aciculin and utrophin. Aciculin was detected by immunoblotting in antiutrophin immunoprecipitates from A7r5 and REF52 cultured cells, indicating an association between these two proteins. The aciculin-utrophin complex in fibroblasts and smooth muscle cells was mostly resistant to Triton X-100 extraction and was detected predominantly in the Triton-insoluble fraction, enriched in actin and actin-associated proteins. By immunofluorescence both aciculin and utrophin were identified in a similar dot-like or streak-like pattern in A7r5 and REF52 cultured cells. Immunolocalization of utrophin in cultured fibroblasts and smooth muscle cells in combination with interference reflection microscopy demonstrated that utrophin staining was mostly codistributed, but not exclusively confined to the areas of focal adhesions, sites of closest cell attachment to the substrate. Double immunostaining of A7r5 and REF52 cells for aciculin and utrophin revealed a precise colocalization of both cytoskeletal proteins at focal adhesions and along microfilaments. Costaining of cultured fibroblasts and smooth muscle cells with antibodies against utrophin and major focal adhesion components, vinculin and talin, showed that utrophin is concentrated in focal adhesions both at initial stages of cell spreading and in well spread cells of nearly confluent monolayers. In MCF10 breast epithelial cells both utrophin and aciculin were localized at cell-cell adherens-type junctions. Our data show that utrophin is a cytoskeletal component of cell-matrix and cell-cell adhesions in various cultured cells. In certain cell types the aciculin-utrophin complexes may contribute to the linking actin filaments to the plasma membrane.

Novel isoform of beta 1 integrin expressed in skeletal and cardiac muscle.

We describe a novel isoform of the beta 1 integrin subunit called beta 1D, which contains a unique cytoplasmic domain, and is expressed specifically in skeletal and cardiac muscle. The beta 1D isoform arises from splicing into the final transcript of an additional exon located between exons 6 and 7. The nucleotide sequence of beta 1D predicts a cytoplasmic domain of 50 amino acids in which the last 21 amino acids of the beta 1A integrin isoform are replaced by a related sequence of 24 amino acids. A beta 1D-specific anti-peptide polyclonal antibody was prepared and immunoprecipitation of tissue extracts with subsequent immunoblotting showed expression of beta 1D isoform only in striated muscle cells.

Association of aciculin with dystrophin and utrophin.

Aciculin is a recently identified 60-kDa cytoskeletal protein, highly homologous to the glycolytic enzyme phosphoglucomutase type 1, (Belkin, A. M., Klimanskaya, I. V., Lukashev, M. E., Lilley, K., Critchley, D., and Koteliansky, V. E. (1994) J. Cell Sci. 107, 159-173). Aciculin expression in skeletal muscle is developmentally regulated, and this protein is particularly enriched at cell-matrix adherens junctions of muscle cells (Belkin, A. M., and Burridge, K. (1994) J. Cell Sci. 107, 1993-2003). The purpose of our study was to identify cytoskeletal protein(s) interacting with aciculin in various cell types. Using immunoprecipitation from cell lysates of metabolically labeled differentiating C2C12 muscle cells with anti-aciculin-specific antibodies, we detected a high molecular weight band (M(r) approximately 400,000), consistently coprecipitating with aciculin. We showed that this 400 kDa band comigrated with dystrophin and immunoblotted with anti-dystrophin antibodies. The association between aciculin and dystrophin in C2C12 cells was shown to resist Triton X-100 extraction and the majority of the complex could be extracted only in the presence of ionic detergents. In the reverse immunoprecipitation experiments, aciculin was detected in the precipitates with different anti-dystrophin antibodies. Immunodepletion experiments with lysates of metabolically labeled C2C12 myotubes showed that aciculin is a major dystrophin-associated protein in cultured skeletal muscle cells. Double immunostaining of differentiating and mature C2C12 myotubes with antibodies against aciculin and dystrophin revealed precise colocalization of these two cytoskeletal proteins throughout the process of myodifferentiation in culture. In skeletal muscle tissue, both proteins are concentrated at the sarcolemma and at myotendinous junctions. In contrast, utrophin, an autosomal homologue of dystrophin, was not codistributed with aciculin in muscle cell cultures and in skeletal muscle tissues. Analytical gel filtration experiments with purified aciculin and dystrophin showed interaction of these proteins in vitro, indicating that their association in skeletal muscle is due to direct binding. Whereas dystrophin was shown to be a major aciculin-associated protein in skeletal muscle, immunoblotting of anti-aciculin immunoprecipitates with antibodies against utrophin showed that aciculin is associated with utrophin in cultured A7r5 smooth muscle cells and REF52 fibroblasts. Immunodepletion experiments performed with lysates of metabolically labeled A7r5 cells demonstrated that aciculin is a major utrophin-binding protein in this cell type. Taken together, our data show that aciculin is a novel dystrophin- and utrophin-binding protein. Association of aciculin with dystrophin (utrophin) in various cell types might provide an additional cytoskeletal-matrix transmembrane link at sites where actin filaments terminate at the plasma membrane.

Expression and localization of the phosphoglucomutase-related cytoskeletal protein, aciculin, in skeletal muscle.

Recently, a 60/63 kDa cytoskeletal protein, highly homologous to the glycolytic enzyme phosphoglucomutase (PGM 1), was isolated from smooth muscle tissue and shown to localize in various adherens-type junctions of muscle and some nonmuscle cells. Since this protein, tentatively named 'aciculin', was enriched in muscle tissues and cells, we have attempted to study its expression and localization during myodifferentiation. C2C12 mouse myoblasts did not express any aciculin before cell fusion in culture. Immediately after cell fusion aciculin became detectable and its content continued to rise during myotube maturation. In early myotubes aciculin appeared first at cell tips and was predominantly localized to focal adhesions of immature myotubes. As myotubes matured in culture, aciculin became associated with growing myofibrils, and finally was found redistributed in striations, corresponding to sarcomere Z-discs. Immunoblotting showed that aciculin content in chicken breast skeletal muscle remained very low until day 11 of embryogenesis, but significantly increased in late prenatal and early postnatal development. By immunofluorescence, aciculin was not revealed in thigh skeletal muscle of day 11 chicken embryos, but was prominently localized at myotendinous junctions in thigh muscle of day 16 embryos. Myotendinous junctions appeared to be major sites of aciculin accumulation in developing and mature skeletal muscle fibers in vivo, suggesting some role for this protein in thin filament-membrane interactions and, potentially, in force transmission at these cell-matrix contacts. In adult skeletal muscle faint aciculin staining appeared at the sarcolemma and as striations in register with Z-discs. Since the protein was not identified in glycerinated myofibrils but was localized to striations in C2C12 myotubes and within the limited areas on skeletal muscle tissue sections, we conclude that aciculin is a component of skeletal muscle costameres. In cultured C2C12 myotubes we found some codistribution of aciculin with clusters of acetylcholine receptors, suggesting its presence at neuromuscular junctions. However, we did not detect any significant concentration of aciculin at neuromuscular junctions in both embryonic and adult skeletal muscle. Taken together, our data show that aciculin expression in skeletal muscle is differentiation-dependent and upregulated during muscle development, and that this novel cytoskeletal protein is a component of various cell-matrix adherens junctions in muscle cells.

A novel phosphoglucomutase-related protein is concentrated in adherens junctions of muscle and nonmuscle cells.

Using five monoclonal antibodies raised against a human uterine smooth muscle extract, we have identified a novel antigen which runs as a closely spaced doublet in SDS-gels. The proteins (60/63 kDa) co-purify, are present in a 1:1 ratio as judged by Coomassie Blue staining, and are immunologically closely related, if not identical. No N-terminal sequence could be obtained from a mixture of the 60/63 kDa proteins, but the sequence of four polypeptides liberated by V8 protease or cyanogen bromide cleavage showed that the proteins are closely related to the glycolytic enzyme phosphoglucomutase type 1. Affinity-purified polyclonal antibodies and three different monoclonal antibodies to the 60/63 kDa proteins cross-reacted with rabbit skeletal muscle phosphoglucomutase type 1, whilst two additional monoclonal antibodies were specific for the 60/63 kDa proteins. Peptide maps of the 60/63 kDa proteins and phosphoglucomutase 1 are markedly different, and the purified proteins have no detectable phosphoglucomutase activity. Staining of cultured smooth muscle cells and fibroblasts with antibodies to 60/63 kDa proteins showed that the antigen is concentrated in focal contacts at the ends of actin bundles and is also associated with actin filaments. About 60% of the cellular 60/63 kDa proteins were found in the detergent-insoluble fraction, suggesting a physical association with the cytoskeleton. The highest levels of protein immunoreactivity were found in muscles. The antigen is concentrated in muscle adherens junctions, including smooth muscle dense plaques, cardiomyocyte intercalated disks, and striated muscle myotendinous junctions. Among epithelial cells, the 63 kDa isoform of the protein was found only in cultured keratinocytes where immunofluorescent staining was localized in cell-to-cell adherens junctions. Expression of the 60/63 kDa proteins in vascular smooth muscle cells is developmentally regulated and correlates with the differentiated contractile phenotype of these cells.