Laminin-5 and hemidesmosomes: role of the alpha 3 chain subunit in hemidesmosome stability and assembly.

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.

Optimization of electroporation for transfection of mammalian cell lines.

Electroporation can be a highly efficient method for introducing DNA molecules into cultured cells for transient expression of genes or for permanent genetic modification. However, effective transformation by electroporation requires careful optimization of electric field strength and pulse characteristics. We have used the transient expression of the firefly luciferase gene as a rapid and sensitive indicator of gene expression to describe the effects on transfection efficiency of altering electroporation field strength and shape. Using the luciferase assay, we investigated the correlation of cell viability with optimal transfection efficiency and determined the optimal parameters for a number of phenotypically distinct mammalian cell lines derived from the nervous and immune systems. The efficiency of electroporation under optimal conditions was compared with that obtained using DEAE-dextran or calcium phosphate-mediated transformation. Transfection by electroporation using square wave pulses, as opposed to exponentially decaying pulses, was found to be significantly increased by repetitive pulses. These methods improve the ability to obtain high efficiency gene transfer into many mammalian cell types.

Introduction and expression of DNA molecules in eukaryotic cells by electroporation.

Exposing eukaryotic cells to brief, high voltage electrical fields can induce the uptake of exogenous materials, presumably through the transient formation of micropores in the cell membrane. This phenomenon has been exploited for the introduction of cloned DNA molecules into cells for permanent transformation or for transient expression and analysis of gene products. The magnitude and characteristics of the generated electrical field are critical for successful electroporation and simple, transient expression assays using indicator genes allow the calibration and optimization of electroporation conditions for a wide variety of eukaryotic cell types. These techniques may allow the genetic modification of a variety of host cells which cannot be easily transformed by other methods.