AFM functional imaging on vascular endothelial cells.

Vascular endothelial (VE)-cadherin is predominantly responsible for the mechanical linkage between endothelial cells, where VE-cadherin molecules are clustered and linked through their cytoplasmic domain to the actin-based cytoskeleton. Clustering and linkage of VE-cadherin to actin filaments is a dynamic process and changes according to the functional state of the cells. Here nano-mapping of VE-cadherin was performed using simultaneous topography and recognition imaging (TREC) technique onto microvascular endothelial cells from mouse myocardium (MyEnd). The recognition maps revealed prominent 'dark' spots (domains or clusters) with the sizes from 10 to 250 nm. These spots arose from a decrease of oscillation amplitude during specific binding between VE-cadherin cis-dimers. They were assigned to characteristic structures of the topography images. After treatment with nocodazole so as to depolymerize microtubules, VE-cadherin domains with a typical ellipsoidal form were still found to be collocalized with cytoskeletal filaments supporting the hypothesis that VE-cadherin is linked to actin filaments. Compared to other conventional techniques such as immunochemistry or single molecule optical microscopy, TREC represents an alternative method to quickly obtain the local distribution of receptors on cell surface with an unprecedented lateral resolution of several nanometers.

Glucocorticoid effects on endothelial barrier function in the murine brain endothelial cell line cEND incubated with sera from patients with multiple sclerosis.

Compromised blood-brain barrier integrity is a major hallmark of active multiple sclerosis (MS). Alterations in brain endothelial tight junction protein and gene expression occur early during neuroinflammation but there is little known about the underlying mechanisms. In this study, we analysed barrier compromising effects of sera from MS patients and barrier restoring effects of glucocorticoids on blood-brain barrier integrity in vitro. cEND murine brain microvascular endothelial cell monolayers were incubated with sera from patients in active phase of disease or in relapse. Data were compared with effects of the glucocorticoid dexamethasone alone or in combination with MS sera on barrier integrity. Tight junction protein levels and gene expression were evaluated concomitant with barrier integrity. We reveal downregulation of claudin-5 and occludin protein and mRNA and an accompanying upregulation in expression of matrix metalloproteinase MMP-9 after incubation with serum from active disease and remission and also a minor reconstitution of barrier functions related to dexamethasone treatment. Moreover, we for the first time describe downregulation of claudin-5 and occludin protein after incubation of cEND cells with sera from patients in remission phase of MS. Our findings reveal direct and differential effects of MS sera on blood-brain barrier integrity.

Extending the knowledge in histochemistry and cell biology.

Central to modern Histochemistry and Cell Biology stands the need for visualization of cellular and molecular processes. In the past several years, a variety of techniques has been achieved bridging traditional light microscopy, fluorescence microscopy and electron microscopy with powerful software-based post-processing and computer modeling. Researchers now have various tools available to investigate problems of interest from bird's- up to worm's-eye of view, focusing on tissues, cells, proteins or finally single molecules. Applications of new approaches in combination with well-established traditional techniques of mRNA, DNA or protein analysis have led to enlightening and prudent studies which have paved the way toward a better understanding of not only physiological but also pathological processes in the field of cell biology. This review is intended to summarize articles standing for the progress made in "histo-biochemical" techniques and their manifold applications.

Cell-cell contact formation governs Ca2+ signaling by TRPC4 in the vascular endothelium: evidence for a regulatory TRPC4-beta-catenin interaction.

TRPC4 is well recognized as a prominent cation channel in the vascular endothelium, but its contribution to agonist-induced endothelial Ca(2+) entry is still a matter of controversy. Here we report that the cellular targeting and Ca(2+) signaling function of TRPC4 is determined by the state of cell-cell adhesions during endothelial phenotype transitions. TRPC4 surface expression in human microvascular endothelial cells (HMEC-1) increased with the formation of cell-cell contacts. Epidermal growth factor recruited TRPC4 into the plasma membrane of proliferating cells but initiated retrieval of TRPC4 from the plasma membrane in quiescent, barrier-forming cells. Epidermal growth factor-induced Ca(2+) entry was strongly promoted by the formation of cell-cell contacts, and both siRNA and dominant negative knockdown experiments revealed that TRPC4 mediates stimulated Ca(2+) entry exclusively in proliferating clusters that form immature cell-cell contacts. TRPC4 co-precipitated with the junctional proteins beta-catenin and VE-cadherin. Analysis of cellular localization of fluorescent fusion proteins provided further evidence for recruitment of TRPC4 into junctional complexes. Analysis of TRPC4 function in the HEK293 expression system identified beta-catenin as a signaling molecule that enables cell-cell contact-dependent promotion of TRPC4 function. Our results place TRPC4 as a Ca(2+) entry channel that is regulated by cell-cell contact formation and interaction with beta-catenin. TRPC4 is suggested to serve stimulated Ca(2+) entry in a specific endothelial state during the transition from a proliferating to a quiescent phenotype. Thus, TRPC4 may adopt divergent, as yet unappreciated functions in endothelial Ca(2+) homeostasis and emerges as a potential key player in endothelial phenotype switching and tuning of cellular growth factor signaling.

An anatomical assessment of the supracerebellar midline and paramedian approaches to the inferior colliculus for auditory midbrain implants using a neuronavigation model on cadaveric specimens.

The inferior colliculus (IC) is an alternative site for electrode placement in neural deafness due to its surgical accessibility and its well-known tonotopic stratification. In patients where tumor surgery has already occurred and the cerebellopontine angle contains scar tissue or tumor-remnants, midline and paramedian supracerebellar approaches are alternative routes. They are often avoided due to concerns regarding the venous drainage of the cerebellum, the electrode trajectory and the course of the electrode cable. We studied these surgical routes in five neuronavigated fixed cadaveric specimens. For paramedian and midline approaches, the transverse sinus was exposed 5.8mm on average. A mean of 1.6 cerebellar veins, with an average diameter of 2.0mm, draining to the tentorium were transected to reach the tentorial notch. Only 0.4 arterial branches were met. We conclude that the supracerebellar midline and paramedian approaches provide a good exposure of the IC and offer safe and viable alternative routes to the IC. Additionally, they provide a wider angle of action for optimal electrode placement.

Desmocollin 3-mediated binding is crucial for keratinocyte cohesion and is impaired in pemphigus.

Desmocollin (Dsc) 1-3 and desmoglein (Dsg) 1-4, transmembrane proteins of the cadherin family, form the adhesive core of desmosomes. Here we provide evidence that Dsc3 homo- and heterophilic trans-interaction is crucial for epidermal integrity. Single molecule atomic force microscopy (AFM) revealed homophilic trans-interaction of Dsc3. Dsc3 displayed heterophilic interaction with Dsg1 but not with Dsg3. A monoclonal antibody targeted against the extracellular domain reduced homophilic and heterophilic binding as measured by AFM, caused intraepidermal blistering in a model of human skin, and a loss of intercellular adhesion in cultured keratinocytes. Because autoantibodies against Dsg1 are associated with skin blistering in pemphigus, we characterized the role of Dsc3 binding for pemphigus pathogenesis. In contrast to AFM experiments, laser tweezer trapping revealed that pemphigus autoantibodies reduced binding of Dsc3-coated beads to the keratinocyte cell surface. These data indicate that loss of heterophilic Dsc3/Dsg1 binding may contribute to pemphigus skin blistering.

Endothelial barrier stabilization by a cyclic tandem peptide targeting VE-cadherin transinteraction in vitro and in vivo.

Inflammatory stimuli result in vascular leakage with potentially life threatening consequences. As a key barrier component, loss of vascular endothelial (VE-) cadherin-mediated adhesion often precedes endothelial breakdown. This study aimed to stabilize VE-cadherin transinteraction and endothelial barrier function using peptides targeting the VE-cadherin adhesive interface. After modelling the transinteracting VE-cadherin structure, an inhibiting single peptide (SP) against a VE-cadherin binding pocket was selected, which specifically blocked VE-cadherin transinteraction as analyzed by single molecule atomic force microscopy (AFM). The tandem peptide (TP) consisting of two SP sequences in tandem was designed to strengthen VE-cadherin adhesion by simultaneously binding and cross-bridging two interacting cadherin molecules. Indeed, in AFM experiments TP specifically rendered VE-cadherin transinteraction resistant against an inhibitory monoclonal antibody. Moreover, TP reduced VE-cadherin lateral mobility and enhanced binding of VE-cadherin-coated microbeads to cultured endothelial cells, but acted independently of the actin cytoskeleton. TP also stabilized endothelial barrier properties against the Ca(2+) ionophore A23187 and the inhibitory antibody. Finally, TP abolished endothelial permeability increase induced by tumour necrosis factor-alpha in microperfused venules in vivo. Stabilization of VE-cadherin adhesion by cross-bridging peptides may therefore be a novel therapeutic approach for the treatment of vascular hyperpermeability.

Lipopolysaccharide-induced endothelial barrier breakdown is cyclic adenosine monophosphate dependent in vivo and in vitro.

OBJECTIVES: To determine whether cyclic adenosine monophosphate (cAMP) is critically involved in lipopolysaccharide (LPS)-induced breakdown of endothelial barrier functions in vivo and in vitro. DESIGN: Experimental laboratory research. SETTING: Research laboratory. SUBJECTS: Wistar rats and cultured human microvascular endothelial cells. INTERVENTION: Permeability measurements in single postcapillary venules in vivo and permeability measurements and cell biology techniques in vitro. MEASUREMENTS AND RESULTS: We demonstrate that within 120 minutes LPS increased endothelial permeability in rat mesenteric postcapillary venules in vivo and caused a barrier breakdown in human dermal microvascular endothelial cells in vitro. This was associated with the formation of large intercellular gaps and fragmentation of vascular endothelial cadherin immunostaining. Furthermore, claudin 5 immunostaining at cell borders was drastically reduced after LPS treatment. Interestingly, activity of the small GTPase Rho A, which has previously been suggested to mediate the LPS-induced endothelial barrier breakdown, was not increased after 2 hours. However, activity of Rac 1, which is known to be important for maintenance of endothelial barrier functions, was significantly reduced to 64 +/- 8% after 2 hours. All LPS-induced changes of endothelial cells were blocked by a forskolin-mediated or rolipram-mediated increase of cAMP. Consistently, enzyme-linked immunosorbent assay-based measurements demonstrated that LPS significantly decreased intracellular cAMP. CONCLUSION: In summary, our data demonstrate that LPS disrupts endothelial barrier properties by decreasing intracellular cAMP. This mechanism may involve inactivation of Rac 1 rather than activation of Rho A.

Peptides Targeting the Desmoglein 3 Adhesive Interface Prevent Autoantibody-induced Acantholysis in Pemphigus.

Pemphigus vulgaris (PV) autoantibodies directly inhibit desmoglein (Dsg) 3-mediated transinteraction. Because cellular signaling also seems to be required for PV pathogenesis, it is important to characterize the role of direct inhibition in pemphigus acantholysis to allow establishment of new therapeutic approaches. Therefore, we modeled the Dsg1 and Dsg3 sequences into resolved cadherin structures and predicted peptides targeting the adhesive interface of both Dsg3 and Dsg1. In atomic force microscopy single molecule experiments, the self-designed cyclic single peptide specifically blocked homophilic Dsg3 and Dsg1 transinteraction, whereas a tandem peptide (TP) consisting of two combined single peptides did not. TP did not directly block binding of pemphigus IgG to their target Dsg antigens but prevented PV-IgG-induced inhibition of Dsg3 transinteraction in cell-free (atomic force microscopy) and cell-based (laser tweezer) experiments, indicating stabilization of Dsg3 bonds. Similarly, PV-IgG-mediated acantholysis and disruption of Dsg3 localization in HaCaT keratinocytes was partially blocked by TP. This is the first evidence that direct inhibition of Dsg3 binding is important for PV pathogenesis and that peptidomimetics stabilizing Dsg transinteraction may provide a novel approach for PV treatment.

State-of-the-art technologies, current opinions and developments, and novel findings: news from the field of histochemistry and cell biology.

Investigations of cell and tissue structure and function using innovative methods and approaches have again yielded numerous exciting findings in recent months and have added important data to current knowledge, inspiring new ideas and hypotheses in various fields of modern life sciences. Topics and contents of comprehensive expert reviews covering different aspects in methodological advances, cell biology, tissue function and morphology, and novel findings reported in original papers are summarized in the present review.

Glucocorticoids regulate the human occludin gene through a single imperfect palindromic glucocorticoid response element.

The 65kDa protein occludin is an essential element of the blood-brain barrier. This integral membrane protein represents an important part of the tight junctions, which seal and protect the blood brain barrier against paracellular diffusion of solutes to the brain parenchyme and are therefore responsible for the high resistance and low permeability between cerebral capillary endothelial cells. However, the molecular basis for the regulation of occludin gene expression is only incompletely understood. In former projects we showed that treatment of a brain microvascular cell line, cEND, with glucocorticoids resulted in increased occludin expression in cell-cell-contacts [Förster, C., Silwedel, C., Golenhofen, N., Burek, M., Kietz, S., Mankertz, J., Drenckhahn, D., 2005. Occludin as direct target for glucocorticoid-induced improvement of blood-brain barrier properties in a murine in vitro system. J. Physiol. 565, Pt 2, 475-486]. Induction of occludin expression by glucocorticoids was shown to be dependent on the glucocorticoid receptor. This study aims to identify the underlying molecular mechanism of gene expression and to identify potential glucocorticoid receptor binding sites within the occludin promoter, the glucocorticoid response elements. We identified one candidate glucocorticoid response element within the distal part of the occludin promoter that differs from the consensus glucocorticoid response element by the presence of a 4-basepair instead of a 3-basepair spacer between two highly degenerate halfsites (5'-ACATGTGTTTACAAAT-3'). Chromatin immunoprecipitation assay and site-directed mutagenesis confirmed binding of the glucocorticoid receptor to this site. The need for glucocorticoid receptor dimerization to induce gene expression was further confirmed by transfection studies using wild type and glucocorticoid receptor dimerization-deficient expression vectors, indicating that transactivation of occludin occurs through the glucocorticoid response element (GRE).

The Drosophila p21-activated kinase Mbt modulates DE-cadherin-mediated cell adhesion by phosphorylation of Armadillo.

Phosphorylation by tyrosine and serine/threonine kinases regulate the interactions between components of the cadherin-catenin cell-adhesion complex and thus can influence the dynamic modulation of cell adhesion under normal and disease conditions. Previous mutational analysis and localization experiments suggested an involvement of single members of the family of PAKs (p21-activated kinases) in the regulation of cadherin-mediated cell adhesion, but the molecular mechanism remained elusive. In the present study, we address this question using the Drosophila PAK protein Mbt, which is most similar to vertebrate PAK4. Previous phenotypic analysis showed that Mbt has a function to maintain adherens junctions during eye development and indicated a requirement of the protein in regulation of the actin cytoskeleton and the cadherin-catenin complex. Here we show that activation of Mbt leads to destabilization of the interaction of the Drosophila beta-catenin homologue Armadillo with DE-cadherin resulting in a decrease in DE-cadherin-mediated adhesion. Two conserved phosphorylation sites in Armadillo were identified that mediate this effect. The findings of the present study support the previous observation that activation of the human Mbt homologue PAK4 leads to anchorage-independent growth and provide a functional link between a PAK protein and the cadherin-catenin complex.

Pemphigus vulgaris IgG directly inhibit desmoglein 3-mediated transinteraction.

The autoimmune blistering skin disease pemphigus is caused by autoantibodies against keratinocyte surface Ags. In pemphigus vulgaris (PV), autoantibodies are primarily directed against desmosomal cadherins desmoglein (Dsg) 3 and Dsg 1, whereas pemphigus foliaceus (PF) patients only have Abs against Dsg 1. At present, it is unclear whether Dsg autoantibodies contribute to pemphigus pathogenesis by direct inhibition of Dsg transinteraction. Using atomic force microscopy, we provide evidence that PV-IgG directly interfere with homophilic Dsg 3 but, similar to PF-IgG, not with homophilic Dsg 1 transinteraction, indicating that the molecular mechanisms in PV and PF pathogenesis substantially differ. PV-IgG (containing Dsg 3 or Dsg 1 and Dsg 3 autoantibodies) as well as PV-IgG Fab reduced binding activity of Dsg 3 by approximately 60%, comparable to Ca(2+) depletion. Similarly, the mouse monoclonal PV Ab AK 23 targeting the N-terminal Dsg 3 domain and AK 23 Fab reduced Dsg 3 transinteraction. In contrast, neither PV-IgG nor PF-IgG blocked Dsg 1 transinteraction. In HaCaT monolayers, however, both PV- and PF-IgG caused keratinocyte dissociation as well as loss of Dsg 1 and Dsg 3 transinteraction as revealed by laser tweezer assay. These data demonstrate that PV-IgG and PF-IgG reduce Dsg transinteraction by cell-dependent mechanisms and suggest that in addition, Abs to Dsg 3 contribute to PV by direct inhibition of Dsg transinteraction.

Molecular composition of tight and adherens junctions in the rat olfactory epithelium and fila.

Tight and adherens junctions (TJs, AJs) between neurons, epithelial and glial cells provide barrier and adhesion properties in the olfactory epithelium (OE), and subserve functions such as compartmentalization and axon growth in the fila olfactoria (FO). Immunofluorescence and immunoelectronmicroscopy were combined in sections of rat OE and FO to document the cellular and subcellular localization of TJ proteins occludin(Occl), claudins(Cl) 1-5 and zonula occludens(ZO) proteins 1-3, and of AJ proteins N-cadherin(cad), E-cad, and alpha-, beta- and p120-catenin(cat). With the exception of Cl2, all TJ proteins were colocalized in OE junctions. Differences in relative immunolabeling intensities were noted between neuronal and epithelial TJs. In the FO, Cl5-reactivity was localized in olfactory ensheathing cell (OEC) junctions, Cl1-reactivity in the FO periphery, with differential colocalization with ZOs. Supporting cells formed N-cad-immunoreactive (ir) AJs with olfactory sensory neurons, E-cad-ir junctions with microvillar and gland duct cells, and both N-cad and E-cad-ir junctions in homotypic contacts. Alpha, beta- and p120-cat were localized in all AJs of the OE. AJs were scarce in the globose basal cell layer. Immature and mature neurons formed numerous contacts. In the FO, AJs were documented between OECs, between OECs and axons, and between axons. Most AJs colocalized N-cad with catenins, occasionally E-cad-ir AJs were found in the FO periphery. Characteristics of molecular composition suggest differential properties of TJs formed by neuronal, epithelial and glial cells in the OE and FO. The presence and molecular composition of AJs are consistent with a role of AJ proteins in neuroplastic processes in the peripheral olfactory pathway.

Heterotypic trans-interaction of LI- and E-cadherin and their localization in plasmalemmal microdomains.

Cadherins are calcium-dependent adhesion molecules important for tissue morphogenesis and integrity. LI-cadherin and E-cadherin are the two prominent cadherins in intestinal epithelial cells. Whereas LI-cadherin belongs to the subfamily of 7D (seven-domain)-cadherins defined by their seven extracellular cadherin repeats and short intracellular domain, E-cadherin is the prototype of classical cadherins with five extracellular domains and a highly conserved cytoplasmic part that interacts with catenins and thereby modulates the organization of the cytoskeleton. Here, we report a specific heterotypic trans-interaction of LI- with E-cadherin, two cadherins of distinct subfamilies. Using atomic force microscopy and laser tweezer experiments, the trans-interaction of LI- and E-cadherin was characterized on the single-molecule level and on the cellular level, respectively. This heterotypic interaction showed similar binding strength (20-52 pN at 200-4000 nm/s) and lifetime (0.8 s) as the respective homotypic interactions of LI- and E-cadherin. VE-cadherin, another classical cadherin, did not bind to LI-cadherin. In enterocytes, LI-cadherin and E-cadherin are located in different membrane regions. LI-cadherin is distributed along the basolateral membrane, whereas the majority of E-cadherin is concentrated in adherens junctions. This difference in membrane distribution was also reflected in Chinese hamster ovary cells stably expressing either LI- or E-cadherin. We found that LI-cadherin is localized almost exclusively in cholesterol-rich fractions, whereas E-cadherin is excluded from these membrane fractions. Given their different membrane localization in enterocytes, the heterotypic trans-interaction of LI- and E-cadherin might play a role during development of the intestinal epithelium when the cells do not yet have elaborate membrane specializations.

Differential effects of hydrocortisone and TNFalpha on tight junction proteins in an in vitro model of the human blood-brain barrier.

Homeostasis of the central nervous system (CNS) microenvironment is maintained by the blood-brain barrier (BBB) which regulates the transport of molecules from blood into brain and back. Many disorders change the functionality and integrity of the BBB. Glucocorticoids are being used sucessfully in the treatment of some disorders while their effects on others are questionable. In addition, conflicting results between clinical and experimental experience using animal models has arisen, so that the results of molecular studies in animal models need to be revisited in an appropriate in vitro model of the human BBB for more effective treatment strategies. Using the human brain microvascular endothelial cell line hCMEC/D3, the influence of glucocorticoids on the expression of barrier constituting adherens junction and tight junction transmembrane proteins (VE-cadherin, occludin, claudins) was investigated and compared to other established BBB models. In hCMEC/D3 cells the administration of glucocorticoids induced expression of the targets occludin 2.75 +/- 0.04-fold and claudin-5 up to 2.32 +/- 0.11-fold, which is likely to contribute to the more than threefold enhancement of transendothelial electrical resistance reflecting barrier tightness. Our analyses further provide direct evidence that the GC hydrocortisone prevents endothelial barrier breakdown in response to pro-inflammatory stimuli (TNFalpha administration), which could be demonstrated to be partly based on maintenance of occludin levels. Our studies strongly suggest stabilization of BBB function as a mode of GC action on a molecular level in the human brain vasculature.

Glucocorticoids increase VE-cadherin expression and cause cytoskeletal rearrangements in murine brain endothelial cEND cells.

Recent studies have shown the influence of glucocorticoids on the expression of the tight junction protein occludin in the brain capillary endothelial cell line cEND, contributing to improvement in endothelial barrier functions. In this study, we investigated glucocorticoid effects on the expression of the adherens junction proteins VE- (vascular-endothelial) cadherin, alpha-catenin and beta-catenin as well as that of ZO-1, the plaque protein shared by both adherens and tight junctions on stimulation with dexamethasone. We were able to show a positive influence of dexamethasone administration on VE-cadherin protein levels as well as a rearrangement of VE-cadherin protein to the cytoskeleton after dexamethasone treatment. Investigation of transcriptional activation of the VE-cadherin promoter by dexamethasone, however, did not point to direct glucocorticoid-mediated VE-cadherin gene induction but rather suggested indirect steroid effects leading to increased VE-cadherin protein synthesis. Dexamethasone was further shown to induce cellular differentiation into a cobblestone cellular morphology and reinforcement of adherens junctions concomitant with the increased anchorage of VE-cadherin to the actin cytoskeleton. We thus propose that glucocorticoid effects on VE-cadherin protein synthesis and organization are important for the formation of both adherens and tight junction, and for improved barrier properties in microvascular brain endothelial cells.

Pemphigus IgG causes skin splitting in the presence of both desmoglein 1 and desmoglein 3.

According to the desmoglein (Dsg) compensation concept, different epidermal cleavage planes observed in pemphigus vulgaris and pemphigus foliaceus have been proposed to be caused by different autoantibody profiles against the desmosomal proteins Dsg 1 and Dsg 3. According to this model, Dsg 1 autoantibodies would only lead to epidermal splitting in those epidermal layers in which no Dsg 3 is present to compensate for the functional loss of Dsg 1. We provide evidence that both pemphigus foliaceus-IgG containing Dsg 1- but not Dsg 3-specific antibodies and pemphigus vulgaris-IgG with antibodies to Dsg 1 and Dsg 3 were equally effective in causing epidermal splitting in human skin and keratinocyte dissociation in vitro. These effects were present where keratinocytes expressed both Dsg 1 and Dsg 3, demonstrating that Dsg 3 does not compensate for Dsg 1 inactivation. Rather, the cleavage plane in intact human skin caused by pemphigus autoantibodies was similar to the plane of keratinocyte dissociation in response to toxin B-mediated inactivation of Rho GTPases. Because we recently demonstrated that pemphigus-IgG causes epidermal splitting by inhibition of Rho A, we propose that Rho GTPase inactivation contributes to the mechanisms accounting for the cleavage plane in pemphigus skin splitting.

Imaging and force spectroscopy on desmoglein 1 using atomic force microscopy reveal multivalent Ca(2+)-dependent, low-affinity trans-interaction.

Desmoglein 1 is a desmosomal member of the cadherin family expressed in stratified epithelia. Desmoglein 1 is the target adhesion molecule of severe blistering skin diseases such as pemphigus or bullous impetigo. However, despite this enormous pathological relevance, the molecular binding properties of desmoglein 1 are largely unknown. Using atomic force microscopic imaging, we found that desmoglein 1 molecules displayed Ca(2+)-dependent conformational changes of the extracellular domains. By single-molecule force-distance cycles, we provide evidence that desmoglein 1 undergoes Ca(2+)-dependent (K (d) = 0.8 mM Ca(2+)) homophilic trans-interaction, which is highly relevant for the contribution of desmoglein 1 homophilic binding to keratinocyte cohesion in distinct epidermal layers. Moreover, while the single-unit unbinding force is comparable to other cadherins (approximately 40 pN at retrace velocity of 300 nm/s), apparent differences with respect to multivalency of interaction and lifetime of single bonds (0.17 s) were observed. Thus, besides the biophysical characterization of desmoglein 1, a main outcome of the study is that desmoglein 1 differs from other members of the cadherin family in terms of some molecular binding properties.