Intermediate filament–membrane attachments function synergistically with actin-dependent contacts to regulate intercellular adhesive strength

Abstract

By tethering intermediate filaments (IFs) to sites of intercellular adhesion, desmosomes facilitate formation of a supercellular scaffold that imparts mechanical strength to a tissue. However, the role IF–membrane attachments play in strengthening adhesion has not been directly examined. To address this question, we generated Tet-On A431 cells inducibly expressing a desmoplakin (DP) mutant lacking the rod and IF-binding domains (DPNTP). DPNTP localized to the plasma membrane and led to dissociation of IFs from the junctional plaque, without altering total or cell surface distribution of adherens junction or desmosomal proteins. However, a specific decrease in the detergent-insoluble pool of desmoglein suggested a reduced association with the IF cytoskeleton. DPNTP-expressing cell aggregates in suspension or substrate-released cell sheets readily dissociated when subjected to mechanical stress whereas controls remained largely intact. Dissociation occurred without lactate dehydrogenase release, suggesting that loss of tissue integrity was due to reduced adhesion rather than increased cytolysis. JD-1 cells from a patient with a DP COOH-terminal truncation were also more weakly adherent compared with normal keratinocytes. When used in combination with DPNTP, latrunculin A, which disassembles actin filaments and disrupts adherens junctions, led to dissociation up to an order of magnitude greater than either treatment alone. These data provide direct in vitro evidence that IF–membrane attachments regulate adhesive strength and suggest furthermore that actin- and IF-based junctions act synergistically to strengthen adhesion.

Plakoglobin suppresses keratinocyte motility through both cell–cell adhesion-dependent and -independent mechanisms

“…Likewise, a reduction in desmosomes or desmosomal proteins has been observed in certain tumors (10)(11)(12)(13). Uncoupling desmosome attachment to intermediate filaments through expression of a dominant-negative (DN) desmoplakin (14) or loss of the desmosomal cadherin associated protein plakophilin 1 results in increased cell migration (15) correlated with a decrease in adhesive strength (16). These observations raise the question as to whether PG can also regulate cell motility through its regulation of intercellular adhesive strength.…”

“…Keratinocyte monolayers were lifted from culture dishes by using dispase, a recombinant protease that cleaves cell-matrix, but not cellcell, adhesion molecules. The resulting cell sheets were then subjected to mechanical stress and the number of fragments was used as an indicator of intercellular adhesive strength as described (16,23). At 0.05 mM calcium, neither adherens junction nor desmosome components accumulated at cell-cell borders, based on immunofluorescence analysis (data not shown).…”

“…Western blot and immunofluorescence were performed as described (16). Primary antibodies used were: PG, 1407 (19); ␤-catenin, c-2206 (Sigma); rabbit anti-myc (20); GFP, JL-8 (Becton Dickinson); and rabbit anti-GAPDH (Novus Biologicals, Littleton, CO), mouse anti-vSrc (Oncogene Science, Cambridge, MA).…”

“…The dispase-based dissociation assay was performed as described (16). The transwell migration assay was modified from a published protocol (21).…”

Kallikrein-5 Promotes Cleavage of Desmoglein-1 and Loss of Cell-Cell Cohesion in Oral Squamous Cell Carcinoma

“…Harvested cell monolayers were pulsed with dispase and subjected to controlled mechanical agitation followed by semi-quantitative analysis of sheet fragmentation (26,39). SCC25 monolayers dissociated into numerous smaller fragments consistent with reduced epithelial cohesion (Fig.…”