The formation of a biological seal around the transmucosal portion of dental implants may be crucial for the long-term success of these therapies. Data to date suggest that the gingival epithelium attaches to dental implants through the formation of hemidesmosomes. Biochemical and genetic data indicate that the laminin isoform, laminin-5, a component of basement membranes, plays a crucial role in the assembly and maintenance of hemidesmosomes. We report the use of soluble laminin-5 as a biological coating of titanium-alloy to promote cell attachment of the gingival epithelial cell line, IHGK. Monoclonal antibodies reactive with laminin-5 depleted the coating solution of all cell attachment activity and blocked cell attachment to laminin-5-coated disks. Immunodepletion with antibodies to fibronectin had no effect. Finally, we demonstrate that IHGK cells assembled hemidesmosomes within 24 h of attachment to laminin-5-coated titanium alloy but not to the titanium alloy alone. These results suggest that soluble laminin-5 may have clinical applications as a dental implant coating to promote the formation of a biological seal.

Laminin-5r is a basement membrane component that promotes rapid adhesion and hemidesmosome formation in epithelial cells. We raised monoclonal antibodies and identified their corresponding epitopes on the constituent chains of laminin-5r by western blotting, Using a combination of immunoprecipitation and ELISA assays, we determined that these epitopes are differentially exposed on two forms of the laminin-5r heterotrimer: soluble (passively adsorbed onto plastic) and cell-associated. Antibody 5C5 epitope is exposed on the cell-associated form, but not the soluble/passively adsorbed form of laminin-5r, Epitopes reactive with antibodies CM6, FM3, and TR1 are also preferentially exposed on cell-associated laminin-5r, such that reactivity of these antibodies with the cell-associated form is fourfold higher than with the soluble/passively adsorbed form in ELISA assays, Incubation of passively adsorbed laminin-5r with the human epithelial cell line SCC12 induced exposure of 5C5 and CM6, FM3, or TR1 epitopes, These data suggest that cells actively modify laminin-5r, perhaps during matrix assembly, and that the 5C5 epitope may serve as a marker for assembled laminin-5r matrix.

Hemidesmosomes are complex macromolecular structures which integrate elements of the extracellular matrix and the cytoskeleton of epithelial cells. To characterize cell-matrix interactions in the hemidesmosome, we have made use of 804G cells which possess the unusual ability to assemble hemidesmosomes in vitro. During the course of our studies, we have raised a set of monoclonal antibodies against rat laminin-5, the major structural element comprising 804G matrix. One of these, termed CM6, recognizes the 150 kDa alpha chain of rat laminin-5 and binds the globular (G) domain of intact laminin-5 molecules as determined by rotary shadowing. CM6 antibodies perturb formed hemidesmosomes in 804G cells. In particular, within 1 hour of incubation of 804G cells with CM6 antibodies, colocalization of laminin-5 and alpha 6 beta 4 integrin is lost and by 2 hours, staining generated by hemidesmosomal antibodies appears primarily cytoplasmic in the perinuclear zone. Ultrastructurally, CM6 antibodies first appear to induce detachment of hemidesmosomes from the underlying matrix. Next, portions of the basal cell surface invaginate to form vesicles whose cytoplasmic-facing surface is coated with hemidesmosomes still associated with keratin intermediate filaments. Anchoring filaments extend into the inside compartment of the vesicles. We have also studied the impact of CM6 antibodies on a model system in which the matrix of 804G cells induces de novo assembly of hemidesmosomes in human keratinocytes. This process involves the plasma membrane reorganization of the hemidesmosome associated integrin alpha 6 beta 4 as well as a redistribution of other hemidesmosome components such as the 230 kDa bullous pemphigoid antigen. Pretreatment of 804G matrix with CM6 antibodies blocks such plasma membrane reorganization of hemidesmosome components and inhibits hemidesmosome formation. Our studies indicate a crucial role for the G domain of the alpha chain of laminin-5 in both nucleation of hemidesmosome assembly as well as maintenance of hemidesmosome structural integrity.

HaCaT cells, an immortalized keratinocyte line, incubated in plastic wells in the presence of conditioned medium from 804G cells adhered and spread rapidly in less than 30 min. In contrast, cells plated in fibroblast or keratinocyte conditioned medium adhered poorly and remained rounded at 30 min. Immunodepletion of 804G conditioned medium with polyclonal antisera to laminin-5r, but not control antisera, abolished rapid cell spreading. Electron microscopy of HaCaT cells spread by incubation in 804G conditioned medium, but not control medium, revealed mature hemidesmosomes after 24 h. Rapid spreading was also observed in wells precoated with 804G conditioned medium or 804G cell-deposited matrix, but not with fibronectin, vitronectin, or laminin-1. Immunoblotting of 804G conditioned medium with anti-laminin-5r antibodies unveiled polypeptides of 150, 140, 135, and 100 kDa, identical by electrophoretic mobility to immunoreactive polypeptides in 804G deposited matrix. Our results suggest that addition of laminin-5r in a soluble form is sufficient to promote rapid spreading and hemidesmosome assembly in keratinocytes. The mechanism of soluble laminin-5r action may include efficient surface "priming" for cell adhesion. Soluble laminin-5r may have a physiologic role in morphogenesis and repair of the epidermis and may be of use for therapeutic applications.

The transmembrane integrins have been shown to interact with the cytoskeleton via noncovalent binding between cytoplasmic domains (CDs) of integrin β chains and various actin binding proteins within the focal adhesion complex. Direct or indirect integrin α chain CD binding to the actin cytoskeleton has not been reported. We show here that actin, as an abundant constituent of focal adhesion complex proteins isolated from fibroblasts, binds strongly and specifically to α2 CD, but not to α1 CD peptide. Similar specific binding to α2 CD peptide was seen for highly purified F actin, free of putative actin-binding proteins. The bound complex of actin and peptide was visualized directly by coprecipitation, and actin binding was abrogated by removal of a five amino acid sequence from the α2 CD peptide. Our findings may explain the earlier observation that, while integrins α2β1 and α1β1 both bind to collagen, only α2β1 can mediate contraction of extracellular collagen matrices.

This chapter focuses on the role of the intracellular cytoskeleton (CSK) in cell shape determination and tissue morphogenesis. The role of mechanical changes in the CSK during embryological development is reviewed. The chapter focuses on the mechanism by which mechanical forces are transmitted across the cell surface and through the CSK, as well as how they regulate cell shape. An analysis of the biomechanical basis of cell shape control addresses two central questions: (1) how do changes in mechanical forces alter CSK organization, and (2) how do changes in CSK structure regulate cell growth and function. The results from recent studies showing that the CSK can respond directly to mechanical stress are also reviewed. The particular type of mechanical response that living cells exhibit is consistent with a theory of CSK architecture that is based on tensional integrity and is known as “tensegrity”. Inherent to the tensegrity model is an efficient mechanism for integrating changes in structure and function at the tissue, cell, nuclear, and molecular levels. The chapter explores the possibility that CSK tensegrity may also provide a mechanical basis for cell locomotion as well as a structural mechanism for coupling mechanical and chemical signaling pathways inside the cell.

Focal adhesion complexes (FACs) containing integrin beta 1, talin, vinculin, talin, alpha-actinin, and paxillin formed within 15 min when round cells bound magnetic microbeads coated with integrin ligands, such as fibronectin or RGD-containing peptide, but not when coated with acetylated-low density lipoprotein. Newly formed FACs were isolated and collected for biochemical analysis using a combination of detergent extraction, sonication, dounce homogenization, and magnetic pelleting. Isolated bead complexes were greatly enriched for all FAC proteins when compared with either the whole cytoskeleton or basal cell membranes whereas actin (a general cytoskeletal marker) was relatively depleted. This method which permits isolation of intact FACs within minutes following integrin ligation should facilitate analysis of both FAC assembly and the molecular basis of integrin signaling.

A mAb AD7, raised against canine liver Golgi membranes, recognizes a novel, 200-kD protein (p200) which is found in a wide variety of cultured cell lines. Immunofluorescence staining of cultured cells with the AD7 antibody produced intense staining of p200 in the juxtanuclear Golgi complex and more diffuse staining of p200 in the cytoplasm. The p200 protein in the Golgi complex was colocalized with other Golgi proteins, including mannosidase II and beta-COP, a coatomer protein. Localization of p200 by immunoperoxidase staining at the electron microscopic level revealed concentrations of p200 at the dilated rims of Golgi cisternae. Biochemical studies showed that p200 is a peripheral membrane protein which partitions to the aqueous phase of Triton X-114 solutions and is phosphorylated. The p200 protein is located on the cytoplasmic face of membranes, since it was accessible to trypsin digestion in microsomal preparations, and is recovered in approximately equal amounts in membrane pellets and in the cytosol of homogenized cells. Immunofluorescence staining of normal rat kidney cells exposed to the toxin brefeldin A (BFA), showed that there was very rapid redistribution of p200, which was dissociated from Golgi membranes in the presence of this drug. The effect of BFA was reversible, since upon removal of the toxin, AD7 rapidly reassociated with the Golgi complex. In the BFA-resistant cell line PtK1, BFA failed to cause redistribution of p200 from Golgi membranes. Taken together, these results indicate that the p200 Golgi membrane-associated protein has many properties in common with the coatomer protein, beta-COP.

By means of a monoclonal antibody (BH3), we have identified a 57-kD protein (p57) that in interphase is restricted largely to the perinuclear region of the cell. Double label immunofluorescence microscopy suggests localization of p57 to the Golgi complex and associated membranous structures. Protease protection experiments and chemical extractability indicate that p57 is a peripheral membrane protein exposed to the cytoplasm. p57 displays unique behavior during mitosis. At the end of G2 or in early prophase, p57 leaves the perinuclear region and accumulates very rapidly within the nucleus, at a time when the nuclear envelope is still intact and before nuclear lamina disassembly. This relocation of p57 coincides with its hyperphosphorylation on serine and threonine residues. After nuclear envelope breakdown p57 becomes uniformly distributed throughout the mitotic cytoplasm until in late telophase when it returns to its perinuclear location and is once again excluded from the nucleus. The behavior of p57 during mitosis suggests that it may play a role in the cellular reorganization evident during mitotic prophase.