LAMININ-5 AND HEMIDESMOSOMES - ROLE OF THE ALPHA-3 CHAIN SUBUNIT IN HEMIDESMOSOME STABILITY AND ASSEMBLY

Hemidesmosomes are complex macromolecular structures which integrate e lements 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 a ssemble 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 a chain of rat laminin-5 and binds the glo bular (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 an tibodies, colocalization of laminin-5 and alpha 6 beta 4 integrin is l ost and by 2 hours, staining generated by hemidesmosomal antibodies ap pears primarily cytoplasmic in the perinuclear zone, Ultrastructurally , CM6 antibodies first appear to induce detachment of hemidesmosomes f rom the underlying matrix, Next, portions of the basal cell surface in vaginate to form vesicles whose cytoplasmic-facing surface is coated w ith hemidesmosomes still associated with keratin intermediate filament s, Anchoring filaments extend into the inside compartment of the vesic les, We have also studied the impact of CM6 antibodies on a model syst em in which the matrix of 804G cells induces de novo assembly of hemid esmosomes in human keratinocytes, This process involves the plasma mem brane reorganization of the hemidesmosome associated integrin alpha 6 beta 4 as well as a redistribution of other hemidesmosome components s uch as the 230 kDa bullous pemphigoid antigen, Pretreatment of 804G ma trix with CM6 antibodies blocks such plasma membrane reorganization of hemidesmosome components and inhibits hemidesmosome formation, Our st udies indicate a crucial role for the G domain of the alpha chain of l aminin-5 in both nucleation of hemidesmosome assembly as well as maint enance of hemidesmosome structural integrity.