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.

Inhibition of Rho A activity causes pemphigus skin blistering.

The autoimmune blistering skin diseases pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are mainly caused by autoantibodies against desmosomal cadherins. In this study, we provide evidence that PV-immunoglobulin G (IgG) and PF-IgG induce skin blistering by interference with Rho A signaling. In vitro, pemphigus IgG caused typical hallmarks of pemphigus pathogenesis such as epidermal blistering in human skin, cell dissociation, and loss of desmoglein 1 (Dsg 1)-mediated binding probed by laser tweezers. These changes were accompanied by interference with Rho A activation and reduction of Rho A activity. Pemphigus IgG-triggered keratinocyte dissociation and Rho A inactivation were p38 mitogen-activated protein kinase dependent. Specific activation of Rho A by cytotoxic necrotizing factor-y abolished all pemphigus-triggered effects, including keratin retraction and release of Dsg 3 from the cytoskeleton. These data demonstrate that Rho A is involved in the regulation of desmosomal adhesion, at least in part by maintaining the cytoskeletal anchorage of desmosomal proteins. This may open the possibility of pemphigus treatment with the epidermal application of Rho A agonists.

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.

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.

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.

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.

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.

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.

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.

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 foliaceus IgG causes dissociation of desmoglein 1-containing junctions without blocking desmoglein 1 transinteraction.

Autoantibodies against the epidermal desmosomal cadherins desmoglein 1 (Dsg1) and Dsg3 have been shown to cause severe to lethal skin blistering clinically defined as pemphigus foliaceus (PF) and pemphigus vulgaris (PV). It is unknown whether antibody-induced dissociation of keratinocytes is caused by direct inhibition of Dsg1 transinteraction or by secondary cellular responses. Here we show in an in vitro system that IgGs purified from PF patient sera caused cellular dissociation of cultured human keratinocytes as well as significant release of Dsg1-coated microbeads attached to Dsg-containing sites on the keratinocyte cellular surface. However, cell dissociation and bead release induced by PF-IgGs was not caused by direct steric hindrance of Dsg1 transinteraction, as demonstrated by single molecule atomic force measurements and by laser trapping of surface-bound Dsg1-coated microbeads. Rather, our experiments strongly indicate that PF-IgG-mediated dissociation events must involve autoantibody-triggered cellular signaling pathways, resulting in destabilization of Dsg1-based adhesive sites and desmosomes.

Intestinal LI-cadherin acts as a Ca2+-dependent adhesion switch.

Cadherins are Ca(2+)-dependent transmembrane glycoproteins that mediate cell-cell adhesion and are important for the structural integrity of epithelia. LI-cadherin and the classical E-cadherin are the predominant two cadherins in the intestinal epithelium. LI-cadherin consists of seven extracellular cadherin repeats and a short cytoplasmic part that does not interact with catenins. In contrast, E-cadherin is composed of five cadherin repeats and a large cytoplasmic domain that is linked via catenins to the actin cytoskeleton. Whereas E-cadherin is concentrated in adherens junctions, LI-cadherin is evenly distributed along the lateral contact area of intestinal epithelial cells. To investigate if the particular structural properties of LI-cadherin result in a divergent homotypic adhesion mechanism, we analyzed the binding parameters of LI-cadherin on the single molecule and the cellular level using atomic force microscopy, affinity chromatography and laser tweezer experiments. Homotypic trans-interaction of LI-cadherin exhibits low affinity binding with a short lifetime of only 1.4 s. Interestingly, LI-cadherin binding responds to small changes in extracellular Ca(2+) below the physiological plasma concentration with a high degree of cooperativity. Thus, LI-cadherin might serve as a Ca(2+)-regulated switch for the adhesive system on basolateral membranes of the intestinal epithelium.

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).

Ca2+ dependency of N-cadherin function probed by laser tweezer and atomic force microscopy.

This study was undertaken to provide a biophysical basis for the hypothesis that activity-dependent modulation of cadherin-mediated adhesion by transient changes of extracellular calcium ([Ca2+]e) is causally involved in coordination of synaptic plasticity. Characterization of homophilic N-cadherin binding by atomic force microscopy and laser tweezer trapping of N-cadherin-coated microbeads attached to the cell surface of cultured neuronal cells showed that adhesive activity of N-cadherin is effectively regulated between 0.3 and 0.8 mm [Ca2+]e. Furthermore, we show that an increase of [Ca2+]i, which is known to be essential for induction of synaptic plasticity, causes significant reduction of cadherin-mediated bead adhesion that could be completely suppressed by inhibition of actin depolymerization. The results of this study show that N-cadherin has ideal biophysical properties to serve as a Ca2+-dependent sensor for synaptic activity and, at the same time, is strategically located to control synaptic adhesion. A drop of [Ca2+]e and a concomitant increase of [Ca2+]i may act in concert to modulate N-cadherin-based adhesive contacts at synaptic sites.

Transmembrane cooperative linkage in cellular adhesion.

Various dynamic cellular activities require precise regulation of extracellular adhesion. Here we propose a simple thermodynamic model that does not depend on affinity regulation of transmembrane adhesion molecules but, rather, is based on the principles of collision-limited reactions. We show that the number of transmembrane adhesion molecules forming extracellular bonds depends on the degree of cytoskeletal damping of their lateral mobility (translational entropy) within the plane of the plasma membrane. This type of transmembrane cooperativity between cytoskeletal linkage and the number of extracellular bonds does not require high affinities to the cytoskeleton (micromolar range) and will be particularly effective at low extracellular affinities of adhesion molecules (millimolar range).

Mitochondrial and nuclear localization of kanadaptin.

Kanadaptin has originally been isolated as a kidney Cl-/HCO3- anion exchanger 1 (kAE1)-binding protein. Initial studies suggested, that in the kidney of the rabbit kanadaptin is expressed exclusively in all epithelial cells of the collecting duct. Transcripts of kanadaptin were also found in tissues not expressing kAE1, indicating additional roles for kanadaptin. With respect to this, we could recently demonstrate translocation of kanadaptin into the nucleus of mammalian cells in a nuclear localization sequence- and importin-dependent manner (Hübner et al., Biochem. J. 361, 287-296, 2002). In this study, we provide evidence, that kanadaptin is widely expressed in many tissues and that expression of kanadaptin in the mouse occurs early in embryonic development. In rat kidney we found the most intense immunofluorescence for kanadaptin in cells of the proximal tubule, consistent with the detection by in situ hybridization of high amounts of kanadaptin messenger RNA in proximal tubule cells. Immunostaining revealed localization of kanadaptin in two subcellular locations, nuclei and mitochondria. Whereas nuclear localization was demonstrated in virtually all cells, mitochondrial staining was restricted to certain cell types. Nuclear staining was only seen in cryosections, whereas mitochondrial staining was observed in both cryosections and semithin sections of freeze-dried plastic-embedded tissue. In the kidney mitochondrial staining was particularly prominent in proximal tubular epithelium. Most surprisingly, in the collecting duct epithelium (including acid-secreting intercalated cells) only negligible immunostaining, if at all, could be observed. Immunoelectron microscopy showed immunolabelling of the entire cross-sectional profile of mitochondria (matrix/inner membrane). Mitochondrial localization of kanadaptin was further documented by immunoblotting of mitochondria-enriched cellular fractions. Utilizing an interspecies heterokaryon assay, we could further demonstrate that kanadaptin has nuclear export activity. Thus, kanadaptin can be regarded to be a highly mobile nucleocytoplasmic shuttling and multilocalizing protein, but its role in mammalian cells remains still obscure.

Kidney Na+,K(+)-ATPase is associated with moesin.

Na+,K(+)-ATPase is a ubiquitous plasmalemmal membrane protein essential for generation and maintenance of transmembrane Na+ and K+ gradients in virtually all animal cell types. Activity and polarized distribution of renal Na+,(+)-ATPase appears to depend on connection of ankyrin to the spectrin-based membrane cytoskeleton as well as on association with actin filaments. In a previous study we showed copurification and codistribution of renal Na+,K(+)-ATPase not only with ankyrin, spectrin and actin, but also with two further peripheral membrane proteins, pasin 1 and pasin 2. In this paper we show by sequence analysis through mass spectrometry as well as by immunoblotting that pasin 2 is identical to moesin, a member of the FERM (protein 4.1, ezrin, radixin, moesin) protein family, all members of which have been shown to serve as cytoskeletal adaptor molecules. Moreover, we show that recombinant full-length moesin as well as its FERM domain bind to Na+,K(+)-ATPase and that this binding can be inhibited by an antibody specific for the ATPase activity-containing cytoplasmic loop (domain 3) of the Na+,K(+)-ATPase alpha-subunit. This loop has been previously shown to be a site essential for ankyrin binding. These observations indicate that moesin might not only serve as direct linker molecule of Na+,K(+)-ATPase to actin filaments but also modify ankyrin binding at domain 3 of Na+,K(+)-ATPase in a way similar to protein 4.1 modifying the binding of ankyrin to the cytoplasmic domain of the erythrocyte anion exchanger (AE1).

Dynamic force microscopy imaging of native membranes.

We employed magnetic ACmode atomic force microscopy (MACmode AFM) as a novel dynamic force microscopy method to image surfaces of biological membranes in their native environments. The lateral resolution achieved under optimized imaging conditions was in the nanometer range, even when the sample was only weakly attached to the support. Purple membranes (PM) from Halobacterium salinarum were used as a test standard for topographical imaging. The hexagonal arrangement of the bacteriorhodopsin trimers on the cytoplasmic side of PM was resolved with 1.5nm lateral accuracy, a resolution similar to images obtained in contact and tapping-mode AFM. Human rhinovirus 2 (HRV2) particles were attached to mica surfaces via nonspecific interactions. The capsid structure and 2nm sized protein loops of HRV2 were routinely obtained without any displacement of the virus. Globular and filamentous structures on living and fixed endothelial cells were observed with a resolution of 5-20nm. These examples show that MACmode AFM is a favorable method in studying the topography of soft and weakly attached biological samples with high resolution under physiological conditions.