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.

The intermediate filament (IF)–binding protein desmoplakin (DP) is essential for desmosome function and tissue integrity, but its role in junction assembly is poorly understood. Using time-lapse imaging, we show that cell–cell contact triggers three temporally overlapping phases of DP-GFP dynamics: (1) the de novo appearance of punctate fluorescence at new contact zones after as little as 3 min; (2) the coalescence of DP and the armadillo protein plakophilin 2 into discrete cytoplasmic particles after as little as 15 min; and (3) the cytochalasin-sensitive translocation of cytoplasmic particles to maturing borders, with kinetics ranging from 0.002 to 0.04 μm/s. DP mutants that abrogate or enhance association with IFs exhibit delayed incorporation into junctions, altering particle trajectory or increasing particle pause times, respectively. Our data are consistent with the idea that DP assembles into nascent junctions from both diffusible and particulate pools in a temporally overlapping series of events triggered by cell–cell contact and regulated by actin and DP–IF interactions.

Although a number of cell adhesion proteins have been identified as caspase substrates, the potential role of differentiation-specific desmosomal cadherins during apoptosis has not been examined. Here, we demonstrate that UV-induced caspase cleavage of the human desmoglein 1 cytoplasmic tail results in distinct 17-and 140-kDa products, whereas metalloproteinase-dependent shedding of the extracellular adhesion domain generates a 75-kDa product. In vitro studies identify caspase-3 as the preferred enzyme that cleaves desmoglein 1 within its unique repeating unit domain at aspartic acid 888, part of a consensus sequence not conserved among the other desmosomal cadherins. Apoptotic processing leads to decreased cell surface expression of desmoglein 1 and re-localization of its C terminus diffusely throughout the cytoplasm over a time course comparable with the processing of other desmosomal proteins and cytoplasmic keratins. Importantly, whereas classic cadherins have been reported to promote cell survival, short hairpin RNA-mediated suppression of desmoglein 1 in differentiated keratinocytes protected cells from UV-induced apoptosis. Collectively, our results identify desmoglein 1 as a novel caspase and metalloproteinase substrate whose cleavage likely contributes to the dismantling of desmosomes during keratinocyte apoptosis and also reveal desmoglein 1 as a previously unrecognized regulator of apoptosis in keratinocytes.Desmosomes are vertebrate cell junctions that anchor the intermediate filament cytoskeleton to the plasma membrane at sites of cell-cell contact and in so doing form a supracellular scaffolding that is essential for maintaining tissue integrity (for review, see Ref. 1). The molecular components of the desmosome fall into three main families: desmosomal cadherins, armadillo family proteins, and plakins. The two types of desmosomal cadherins, desmogleins (Dsgs) 4 and desmocollins (Dscs), are thought to mediate calcium dependent cell-cell adhesion at the membrane, which is strengthened through indirect interactions with the intermediate filament cytoskeleton (for review, see Ref.2). The armadillo family protein, plakoglobin (Pg), interacts directly with the cytoplasmic tail of the desmosomal cadherins (3-6), thereby connecting the transmembrane glycoproteins to the obligate desmosomal protein, desmoplakin (DP) (7). DP links the cell surface to the cytoskeleton by associating with Pg at its N terminus and intermediate filaments through its C terminus (8 -10). Plakophilins, additional members of the armadillo family, can interact with desmosomal cadherin cytoplasmic domains (11, 12), enhance recruitment of DP to the membrane, and likely facilitate lateral clustering of the plaque components to enhance the mechanical strength of the junction (13,14).Four isoforms of Dsgs and three isoforms of Dscs have been identified in humans (15-17). The cytoplasmic domains of all of the Dsg isoforms contain regions of unknown function, including a unique repeating unit domain (RUD). Each Dsg isoform includes a RUD c...

The long-term effects of interferon treatment on cell lines that maintain human papillomavirus type 31 (HPV-31) episomes have been examined. High doses and prolonged interferon treatment resulted in growth arrest of HPV-positive cells, with a high percentage of cells undergoing apoptosis. These effects were not seen with interferon treatment of either normal human keratinocytes or cells derived from HPV-negative squamous carcinomas, which exhibited only slight decreases in their rates of growth. Within 2 weeks of the initiation of treatment, a population of HPV-31-positive cells that were resistant to interferon appeared consistently and reproducibly. The resistant cells had growth and morphological characteristics similar to those of untreated cells. Long-term interferon treatment of HPV-positive cells also resulted in a reduction in HPV episome levels but did not significantly decrease the number of integrated copies of HPV. Cells that maintained HPV genomes lacking E5 were sensitive to interferon, while cells expressing only the E6/E7 genes were resistant. In contrast, cells that expressed E2 from a tetracycline-inducible promoter were found to be significantly more sensitive to interferon treatment than parental cells. This suggests that at least a portion of the sensitivity to interferon could be mediated through the E2 protein. These studies indicate that cells maintaining HPV episomes are highly sensitive to interferon treatment but that resistant populations arise quickly.Human papillomaviruses (HPV) are small double-stranded DNA viruses that infect epithelial tissues. More than 85 subtypes have been identified, and each of these types exhibits strict tissue specificity (13). About one-third of HPV types infect the anogenital epithelia and induce the most common form of sexually transmitted disease (71). HPV infect cells in the basal layer of epithelia and establish a latent infection in these cells. Production of HPV virions, however, requires infected cells to migrate away from the basal layer and undergo differentiation (28,31,71). The HPV that infect the anogenital region can be divided into high-risk and low-risk HPV types depending on their association with malignancy. The low-risk HPVs, such as HPV type 6 (HPV-6) and HPV-11, cause hyperproliferative lesions of external genitalia and are rarely associated with malignancies. In contrast, the high-risk HPVs, , are the etiological agents of cervical cancer (28,31,35,71). The difference in clinical outcome between low-and high-risk HPV infections has been an area of major research interest.The genomes of all genital HPV types encode 8 to 10 proteins. In the high-risk HPVs, E6 and E7 function as oncogenes. E6 binds to the cellular ubiquitin ligase, E6AP, which then targets p53 for degradation (29,53,54,67). In addition, E6 activates the expression of htert, the catalytic subunit of telomerase. E7 binds to and inhibits the activity of retinoblastoma protein, pRB, and promotes the constitutive activation of E2F family members (9,16,38,44). The E1 and E2 g...

The diagnosis of Hodgkin's disease is based upon the finding of characteristic Sternberg‐Reed cells in appropriate cellular and architectural environments. The demonstration of cells with the nuclear and cytoplasmic features of Sternberg‐Reed cells is necessary, but not sufficient for the diagnosis of this disease. Many investigators, however, have erroneously regarded these cells as pathognomonic. This report emphasizes that cells indistinguishable from, or closely resembling, Sternberg‐Reed cells may be found in conditions other than Hodgkin's disease. Their presence, therefore, should not be considered a diagnostic mandate. Thirteen cases are presented in which biopsy sections of both benign and malignant lesions revealed cells closely resembling or indistinguishable from Sternberg‐Reed cells.