1. ¹Skirball Institute for Biomolecular Medicine, New York University School of Medicine, New York, New York, USA
2. ²Department of Cell Biology, New York University School of Medicine, New York, New York, USA
3. ³Vetsuisse Faculty, Institute of Animal Pathology, University of Bern, Bern, Switzerland
4. ⁴Department Dermatology, New York University School of Medicine, New York, New York, USA
5. ⁵New York Structural Biology Center, New York, New York, USA

Correspondence: Dr David L. Stokes, Skirball Institute 3-13, New York University School of Medicine, 540 First Avenue, New York, New York 10012, USA. E-mail: stokes@saturn.med.nyu.edu

Received 4 January 2008; Revised 17 March 2008; Accepted 4 April 2008; Published online 22 May 2008.

Abstract

Desmosomes are adhesive junctions that provide mechanical coupling between cells. Plakoglobin (PG) is a major component of the intracellular plaque that serves to connect transmembrane elements to the cytoskeleton. We have used electron tomography and immunolabeling to investigate the consequences of PG knockout on the molecular architecture of the intracellular plaque in cultured keratinocytes. Although knockout keratinocytes form substantial numbers of desmosome-like junctions and have a relatively normal intercellular distribution of desmosomal cadherins, their cytoplasmic plaques are sparse and anchoring of intermediate filaments is defective. In the knockout, -catenin appears to substitute for PG in the clustering of cadherins, but is unable to recruit normal levels of plakophilin-1 and desmoplakin to the plaque. By comparing tomograms of wild type and knockout desmosomes, we have assigned particular densities to desmoplakin and described their interaction with intermediate filaments. Desmoplakin molecules are more extended in wild type than knockout desmosomes, as if intermediate filament connections produced tension within the plaque. On the basis of our observations, we propose a particular assembly sequence, beginning with cadherin clustering within the plasma membrane, followed by recruitment of plakophilin and desmoplakin to the plaque, and ending with anchoring of intermediate filaments, which represents the key to adhesive strength.