Free Cell Surface Research Articles

4D Force Detection of Cell Adhesion and Contractility.

Mechanical signals establish two-way communication between mammalian cells and their environment. Cells contacting a surface exert forces via contractility and transmit them at the areas of focal adhesions. External stimuli, such as compressive and pulling forces, typically affect the adhesion-free cell surface. Here, we demonstrate the collaborative employment of Fluidic Force Microscopy and confocal Traction Force Microscopy supported by the Cellogram solver to enable a powerful integrated force probing approach, where controlled vertical forces are applied to the free surface of individual cells, while the concomitant deformations are used to map their transmission to the substrate. Force transmission across human cells is measured with unprecedented temporal and spatial resolution, enabling the investigation of the cellular mechanisms involved in the adaptation, or maladaptation, to external mechanical stimuli. Altogether, the system enables facile and precise force interrogation of individual cells, with the capacity to perform population-based analysis.

Interwoven MOF-Coated Janus Cells as a Novel Carrier of Toxic Proteins.

Two major issues in cell-mediated drug delivery systems (c-DDS) are the availability of free cell surfaces for the binding of the cells to the target or to their microenvironment and internalization of the cytotoxic drug. In this study, the Janus structure, MOF nanoparticles, and tannic acid (TA) are utilized to address these issues. Janus carrier cells coated with metal-organic frameworks (MOFs) are produced by asymmetrically immobilizing the nanoparticles of a MOF based on zinc with cytotoxic enzymes that are internally encapsulated on the surface of carrier cells. By maintaining the biological and structural features of regular living cells, the MOF-coated Janus cells developed in the present study preserve the intrinsic binding capacity of the cells to their microenvironment. Interconnected MOFs loaded onto the other face of the Janus cells cannot penetrate the cell. Therefore, the carrier cells are protected from the cytotoxic drug contained in MOFs. These MOF-Janus carrier cells are demonstrated to successfully eliminate three-dimensional (3D) tumor spheroids when a chemotherapeutic protein of proteinase K is released from the MOF nanoparticles in an acid environment. The ease with which the MOF-Janus carrier cells are prepared (in 15 min), and the ability to carry a variety of enzymes and even multiple ones should make the developed system attractive as a general platform for drug delivery in various applications, including combination therapy.

Gustatory ultrastructures of an amphihaline migratory fish hilsa Tenualosa ilisha.

This study was conducted with the tongue samples of different life stages of hilsa, that is, adult Marine hilsa, adult Riverine hilsa, and Riverine juvenile hilsa, respectively. Three types of taste buds (Types I, II, and III based on their elevation from the epithelium at different levels) of the tongue, which may be to ensure full utilization of the gustatory ability of the fish were rocorded. Presence of specific taste buds indicate that the fish hilsa dwells in turbid waters with a possible preference toward diatom like planktonic food source. Enhanced expression of taste receptors (T1R1 and T1R3) and associated stimulatory G-proteins subunits on tongue also indicate occurrence of amino acid like substances that guided sensory cues for feeding by this fish. A firm regularity or stringency of the free surface of the epithelial cells may be attributed to compactly arranged microridges. These structures protect against physical abrasions potentially caused during food manoeuvring and swallowing. In our present observations, the surface architectures of the tongue of hilsa are discussed within the background of migratory adaptation of the species in the context of feeding and habitat preferences.

Modifications in the gills of hill stream Moth catfish, Hara hara (Erethistidae, Siluriformes): A light and scanning electron microscope investigation

Present study reports significant modifications in surface ultrastructure, histological organization, and histochemical localization of glycoproteins (GPs) in the gills of a hill stream catfish, Hara hara. Punctate microridges on free surface of epithelial cells covering gill arches, gill rakers, gill filaments and secondary lamellae are considered to provide adaptive plasticity to gills in relation to the environment inhabited by fish. Short and stout gill rakers are considered to prevent food particles to pass in opercular chamber along with respiratory current that could damage delicate gill filaments. Mucous goblet cells show presence of different classes of glycoproteins. GPs with oxidizable vicinal diols are considered to control acidity of acidic GPs. GPs with carboxyl groups have been implicated with defensive mechanism against microorganisms. GPs with O-sulphate esters are associated to trap and to lubricate food particles for easy swallowing. Taste buds on gill arches and gill rakers function to select palatable food particles. Occurrence of taste buds on the gill filaments is regarded significant adaptation to analyse the chemical nature of water. This study could play a significant role to understand adjustment of gills in the hill stream fish.

Keratins Regulate p38MAPK-Dependent Desmoglein Binding Properties in Pemphigus.

Keratins are crucial for the anchorage of desmosomes. Severe alterations of keratin organization and detachment of filaments from the desmosomal plaque occur in the autoimmune dermatoses pemphigus vulgaris and pemphigus foliaceus (PF), which are mainly caused by autoantibodies against desmoglein (Dsg) 1 and 3. Keratin alterations are a structural hallmark in pemphigus pathogenesis and correlate with loss of intercellular adhesion. However, the significance for autoantibody-induced loss of intercellular adhesion is largely unknown. In wild-type (wt) murine keratinocytes, pemphigus autoantibodies induced keratin filament retraction. Under the same conditions, we used murine keratinocytes lacking all keratin filaments (KtyII k.o.) as a model system to dissect the role of keratins in pemphigus. KtyII k.o. cells show compromised intercellular adhesion without antibody (Ab) treatment, which was not impaired further by pathogenic pemphigus autoantibodies. Nevertheless, direct activation of p38MAPK via anisomycin further decreased intercellular adhesion indicating that cell cohesion was not completely abrogated in the absence of keratins. Direct inhibition of Dsg3, but not of Dsg1, interaction via pathogenic autoantibodies as revealed by atomic force microscopy was detectable in both cell lines demonstrating that keratins are not required for this phenomenon. However, PF-IgG shifted Dsg1-binding events from cell borders toward the free cell surface in wt cells. This led to a distribution pattern of Dsg1-binding events similar to KtyII k.o. cells under resting conditions. In keratin-deficient keratinocytes, PF-IgG impaired Dsg1-binding strength, which was not different from wt cells under resting conditions. In addition, pathogenic autoantibodies were capable of activating p38MAPK in both KtyII wt and k.o. cells, the latter of which already displayed robust p38MAPK activation under resting conditions. Since inhibition of p38MAPK blocked autoantibody-induced loss of intercellular adhesion in wt cells and restored baseline cell cohesion in keratin-deficient cells, we conclude that p38MAPK signaling is (i) critical for regulation of cell adhesion, (ii) regulated by keratins, and (iii) targets both keratin-dependent and -independent mechanisms.

Mechanics of Fluid-Filled Interstitial Gaps. II. Gap Characteristics in Xenopus Embryonic Ectoderm

The ectoderm of the Xenopus embryo is permeated by a network of channels that appear in histological sections as interstitial gaps. We characterized this interstitial space by measuring gap sizes, angles formed between adjacent cells, and curvatures of cell surfaces at gaps. From these parameters, and from surface-tension values measured previously, we estimated the values of critical mechanical variables that determine gap sizes and shapes in the ectoderm, using a general model of interstitial gap mechanics. We concluded that gaps of 1–4 μm side length can be formed by the insertion of extracellular matrix fluid at three-cell junctions such that cell adhesion is locally disrupted and a tension difference between cell-cell contacts and the free cell surface at gaps of 0.003 mJ/m2 is generated. Furthermore, a cell hydrostatic pressure of 16.8 ± 1.7 Pa and an interstitial pressure of 3.9 ± 3.6 Pa, relative to the central blastocoel cavity of the embryo, was found to be consistent with the observed gap size and shape distribution. Reduction of cell adhesion by the knockdown of C-cadherin increased gap volume while leaving intracellular and interstitial pressures essentially unchanged. In both normal and adhesion-reduced ectoderm, cortical tension of the free cell surfaces at gaps does not return to the high values characteristic of the free surface of the whole tissue.

Abstract 5066: Impact of Brk-inhibitors on nondirectional and directed migration of breast cancer cells

Abstract The non-receptor tyrosine kinase Brk (breast tumor kinase, PTK-6) has been linked to various effects in tumor cells including cell proliferation, differentiation and migration. Modulation of cellular signaling occurs due to protein scaffolding and substrate phosphorylation, while effects depend on cell type and molecular context. Recent findings suggest, that some hallmark effects of cancer may be linked to the active kinase whereas inactive Brk seems to be involved in different physiological processes of epithelial cells. Novel α-carboline derivatives were described previously and identified as Brk inhibitors in in-vitro testing. We examined the effect of four candidates MK5, MK136, MK138 and MK150 in breast cancer cell lines regarding Brk as a modulator of cell migration. Cell lines MDA-MB-231 and MDA-MB-468 were cultivated as indicated by the distributor. Inhibition of non-directional migration over a given period of time was determined by scratch assays barring proliferation by addition of 50 mM hydroxyurea. The gap was created using silicon inserts and TScratch software was used to evaluate cell free surface area. Directional migration was investigated by trans-well assay utilizing FCS as attractant and an 8 μm pore membrane in combination with xCellingence real time measurement. Statistical evaluation was conducted using 1-way ANOVA followed by Dunnett's test. In scratch assays samples incubated with 1 μM of each substance showed a larger cell free area after 24 hours for MDA-MB-231 cells and after 30 hours for MDA-MB-46 cells. The increase was significant in MDA-MB-231 cells for substance MK136 (32.07±17.21%, p<0.1) and highly significant for MK138 (52.37±7.00%, p<0.001) compared to DMSO treated control (7.73±3.22%). Furthermore, the effect of MK138 increased in a concentration-dependent manner yielding consistently highly significant differences at a concentration of 1 μM (36.77±14.53%, N = 8 vs. 9.870±1.12%, N = 4). MK138 displayed a significant difference in MDA-MB-468 cells as well (37.27±12.56% vs. 10.07±3.04%, p<0.01). In transwell experiments MDA-MB-231 cells showed significantly impaired migration when incubated with MK138 and MK150. For MK138 a concentration-dependent effect was found similar to the scratch assay. In cell line MDA-MB-468 the inhibitor MK138 accounted for a significant decrease in directional migration as well. Our findings indicate an influence of pharmacological Brk-inhibition on the migratory ability of breast cancer cells. These effects were evident for all tested substances and constantly significant and concentration-dependent for MK138 making unspecific effects unlikely. The mechanism of Brk activity in modulation of migratory processes needs to be further addressed to better understand and exploit its influence on tumor cell migration, dissemination and metastasis. This work was supported by the German Research Foundation (DFG, Grant No. RI1196/4-1). Citation Format: Markus Oelze, Franziska I. Kluwe, Tom Wersig, Wolfgang Sippl, Andreas Hilgeroth, Christoph A. Ritter. Impact of Brk-inhibitors on nondirectional and directed migration of breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5066.

Ultrastructural study on the foot and the shell of the oyster Pinctada radiata (leach, 1814),(Bivalvia Petridae).

The shape and microscopic structure of the foot of the oyster were described by light and electron microscopy. The foot is wedge-shaped with vertical furrows on the surface; it is composed of an epithelial layer, a connective tissue layer and a muscular layer. The epithelial layer was composed mostly of ciliated columnar epithelia and secretory cells. The cilia and microvilli were observed on the free surface of epithelial cells. The muscle fibers were composed of thin and thick microfilaments. Thick and thin filaments in muscle cells were reported in the foot as well as lateral projections of the cytoplasm contained granules. The outer surface of the periostracum of the shell is reported to be microridge and microfringe were present as well. The simplicity of periostracum of the shell layers was observed as thin simple layer whereas the prismatic layer was the opposite. Wedge-shaped and elongated prisms were reported near the outer surface. Fine lamellae were representing the nacreous layer and were divided by laminar inclusion. Micro-fringes were considered to be helping the direction of bivalve

Numerical simulation of deformation and fracture of space protective shell structures from concrete and fiber concrete under pulse loading

This paper presents results of numerical simulation of interaction between aircraft Boeing 747-400 and protective shell of nuclear power plant. The shell is presented as complex multilayered cellular structure comprising layers of concrete and fiber concrete bonded with steel trusses. Numerical simulation was held three-dimensionally using the author's algorithm and software taking into account algorithms for building grids of complex geometric objects and parallel computations. The dynamics of stress-strain state and fracture of structure were studied. Destruction is described using two-stage model that allows taking into account anisotropy of elastic and strength properties of concrete and fiber concrete. It is shown that wave processes initiate destruction of shell cellular structure—cells start to destruct in unloading wave, originating after output of compression wave to the free surfaces of cells.

TRIM27/MRTF-B-Dependent Integrin β1 Expression Defines Leading Cells in Cancer Cell Collectives

For collective invasion, cancer cells form cohesive groups comprised of leading cells (LCs) at the forefront and following cells (FCs) at the rear. However, the molecular mechanisms that define LCs and FCs remain elusive. Here, we demonstrated that LCs, but not FCs, upregulated the expression of integrin β1 after the loss of intercellular adhesion. The LC-specific expression of integrin β1 was posttranscriptionally regulated by the TRIM27/MRTF-B complex in response to the loss of intercellular adhesion, thereby regulating the stability and translation of integrin β1 mRNA via microRNA-124 in LCs. Accordingly, depletion of TRIM27 and MRTF-B abrogated the upregulation of integrin β1 in LCs and blocked the invasion of cancer cell groups in vitro and in vivo. Therefore, our findings revealed that the specific function of LCs was defined by intrinsic mechanisms related to the presence of the cell's free surface, providing insights into the regulation of intratumor heterogeneity.

Investigation of Hydrogen Sulfide Gas as a Treatment against P. falciparum, Murine Cerebral Malaria, and the Importance of Thiolation State in the Development of Cerebral Malaria

IntroductionCerebral malaria (CM) is a potentially fatal cerebrovascular disease of complex pathogenesis caused by Plasmodium falciparum. Hydrogen sulfide (HS) is a physiological gas, similar to nitric oxide and carbon monoxide, involved in cellular metabolism, vascular tension, inflammation, and cell death. HS treatment has shown promising results as a therapy for cardio- and neuro- pathology. This study investigates the effects of fast (NaHS) and slow (GYY4137) HS-releasing drugs on the growth and metabolism of P. falciparum and the development of P. berghei ANKA CM. Moreover, we investigate the role of free plasma thiols and cell surface thiols in the pathogenesis of CM.Methods P. falciparum was cultured in vitro with varying doses of HS releasing drugs compared with artesunate. Growth and metabolism were quantified. C57Bl/6 mice were infected with P. berghei ANKA and were treated with varying doses and regimes of HS-releasing drugs. Free plasma thiols and cell surface thiols were quantified in CM mice and age-matched healthy controls.ResultsHS-releasing drugs significantly and dose-dependently inhibited P. falciparum growth and metabolism. Treatment of CM did not affect P. berghei growth, or development of CM. Interestingly, CM was associated with lower free plasma thiols, reduced leukocyte+erythrocyte cell surface thiols (infection day 3), and markedly (5-fold) increased platelet cell surface thiols (infection day 7).ConclusionsHS inhibits P. falciparum growth and metabolism in vitro. Reduction in free plasma thiols, cell surface thiols and a marked increase in platelet cell surface thiols are associated with development of CM. HS drugs were not effective in vivo against murine CM.

Morphological adaptation of the buccal cavity in relation to feeding habits of the omnivorous fish Clarias gariepinus: A scanning electron microscopic study

The surface architecture of the buccal cavity of the omnivorous fish Clarias gariepinus was studied in relation to its food and feeding habits. The buccal cavity of the present fish was investigated by means of a scanning electron microscope. This cavity may be distinguished into the roof and the floor. Papilliform and molariform teeth which are located in the buccal cavity are associated with seizing, grasping, holding of the prey, crushing and grinding of various food items. Three types of taste buds (Types I, II & III) were found at different levels in the buccal cavity. Type I taste buds were found in relatively high epidermal papillae. Type II taste buds were mostly found in low epidermal papillae. Type III taste buds never raise above the normal level of the epithelium. These types may be useful for ensuring full utilization of the gustatory ability of the fish. A firm consistency or rigidity of the free surface of the epithelial cells may be attributed to compactly arranged microridges. These structures protect against physical abrasions potentially caused during food maneuvering and swallowing. Furthermore, protection of the epithelium from abrasion is enhanced with mucous cell secretions which lubricate ingested food items.

Micromorphology and Ultrastructure of the Foot of the Equilateral Venus <i>Gomphina veneriformis</i> (Bivalvia: Veneridae)

The shape and microscopic structure of the foot of the equilateral Venus, Gomphina veneriformis are described by light and electron microscopy along with the substrate conditions of their habitat. The habitat sediment of G. veneriformis is composed of sand (2 - 0.063 mm in diameter), mainly. The foot is wedge-shaped with multiple vertical furrows on the surface. Although the foot is composed of an epithelial layer, a connective tissue layer and a muscular layer, the boundary between the connective tissue and muscular layer is not clear. The epithelial layer was composed mostly of ciliated columnar epithelia and secretory cells. Epithelial cells forming the apical region of the fold were long columnar, while cells of the interfold were mostly short columnar. The cilia and microvilli were commonly observed on the free surface of epithelial cells, while tight junctions of apico-lateral aspect and membrane interdigitations were found between the epithelial cells. Secretory cells were found to contain acidic mucopolysaccharide, and were classified into two types in accordance with the shapes and ultrastructures of secretory granules. The muscle fibers were composed of thin and thick microfilaments, the proportions of which were 81.3% and 18.7%, respectively. It was determined that such morphology and structural characteristics of the foot of G. veneriformis would present advantageous conditions for borrowing into substrate and mobility.

Modifications in the surface organisation of the epidermis on the outer surface of the operculum and the epithelium lining the inner surface of the operculum in certain fresh water teleosts

The outer surface of the opercular epidermis (OE) and the epithelium lining the inner surface of the operculum (EISO) ofMacrognathus aculeatum,Colisa fasciataandGlossogobius giuriswas investigated using scanning electron microscopy to unravel their surface ultrastructure. In the fish species investigated, the OE is thick, compared to the EISO. The OE and the EISO remain covered with a mosaic pavement of irregularly polygonal epithelial cells. The free surface of the epithelial cells is thrown into a series of microridges. Modifications in the pattern of microridges could be caused by various intrinsic or extrinsic factors. Interspersed between the epithelial cells in the OE mucous cell pores, mitochondria-rich cells, taste buds and superficial neuromasts could be located. In the EISO, in contrast, taste buds and neuromasts are absent. The modifications in the surface organisations at the OE and EISO in different fish species have been associated to perform a variety of functions and have been discussed in the light of the varied conditions experienced by them.

A Combined Confocal-Total Internal Reflection Fluorescence (TIRF) Single-Cell Microscopy Investigation of CEACAM1 Dynamics

The carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs) represent a subset of the immunoglobulin superfamily of cell adhesion molecules that mediate intercellular adhesion. One member of this subset, CEACAM1, is particularly interesting since it is down-regulated in tumourigenic cell lines. CEACAM1 exists as an equilibrium between monomers and dimers; however, the spatial and temporal distribution upon cell-substrate contact is not known.In order to determine CEACAM1 dynamics, a coupled confocal-total internal reflection fluorescence (TIRF)-atomic force microscopy (AFM) platform was used. Live YFP-CEACAM1 labelled cells were isolated on AFM tips for controlled positioning of cells. Confocal microscopy was used to map CEACAM1 receptors over the entire cell surface. Coupled TIRF microscopy monitored the free cell surface as it was brought into contact with a substrate (i.e. another cell, a model membrane, modified glass). Confocal and TIRF microscopy homoFRET measurements were used to determine the distribution of monomeric and dimeric CEACAM1 receptors prior to and upon cell-substrate contact. Through this understanding of how molecular organization affects intercellular binding and signal transduction, it may be possible to identify peptide or pharmacological drug strategies to create CEACAM-focused therapies for cancer.View Large Image | View Hi-Res Image | Download PowerPoint Slide

HgCdTe monolithic infrared detector

AbstractWe report the processes of fabricating monolithic 32×32 infrared detector based on (310) HgCdTe/CdTe/ZnTe/ Si photosensitive heterostructure which was grown by a molecular‐beam epitaxy technique in the free surface of ROIC cells. Optimum parameters of the technological processes were determined. The minimum temperature of preepitaxial thermal annealing in vacuum of ROIC was determined as 450 °C. The dark and photo current–voltage characteristics were measured and analyzed. A good sensitivity of diodes was observed. The product R0×A ∼ 105 Ohm×cm2. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Ovipositor ultrastructure of the striped bitterling Acheilognathus yamatsutae (Teleostei: Acheilognathinae) during spawning season

The ovipositor of striped bitterling Acheilognathus yamatsutae was subjected to ultrastructure and histochemical analysis during spawning season using light and electron microscopy. Although the ovipositor of A. yamatsutae is a long cylindrical tube with smooth external surface, it was possible to confirm the presence of well-developed fingerprint structure using scanning electron microscopy. Internal aspect analysis of ovipositor revealed formation of 5–8 longitudinal folds. Cross section analysis revealed that the ovipositor is composed of an outer epithelial layer, a mid connective tissue layer, and an inner epithelial layer. The outer epithelial layer contains 7–9 cell layers composed mainly of epithelial and mucous cells. Result of AB–PAS (pH 2.5) and AF–AB reaction showed that mucous cells contained mainly acidic carboxylated mucosubstances. The connective tissue layer was loose and made mainly of collagen fibers and some muscle fibers, along with blood vessels and a small number of chromatophores. The inner epithelial layer, which is a single layer, is composed of columnar epithelia. Observation under transmission electron microscope enabled distinction of the outer epithelial layer into superficial, intermediate and basal layers. Although the types of cells in the superficial tissue layer were diverse, they all shared the development of glycocalyx covered microridges. The majority of epithelial cells in the intermediate layer were cuboidal shaped, while those in the basal layer were columnar. Two types (A and B) of secretory cells were observed in the outer epithelial layer. The connective tissue layer had two types of chromatophores including xantophore and melanophore, in addition to a well-developed nerve fiber bundles. Columnar epithelial cells, mitochondria-rich cells and rodlet cells were observed in the inner epithelial layer. Microvilli were well developed on the free surface of columnar epithelial cells.

Human Immunodeficiency Virus Type 1 and Influenza Virus Exit via Different Membrane Microdomains

Directed release of human immunodeficiency virus type 1 (HIV-1) into the cleft of the virological synapse that can form between infected and uninfected T cells, for example, in lymph nodes, is thought to contribute to the systemic spread of this virus. In contrast, influenza virus, which causes local infections, is shed into the airways of the respiratory tract from free surfaces of epithelial cells. We now demonstrate that such differential release of HIV-1 and influenza virus is paralleled, at the subcellular level, by viral assembly at different microsegments of the plasma membrane of HeLa cells. HIV-1, but not influenza virus, buds through microdomains containing the tetraspanins CD9 and CD63. Consequently, the anti-CD9 antibody K41, which redistributes its antigen and also other tetraspanins to cell-cell adhesion sites, interferes with HIV-1 but not with influenza virus release. Altogether, these data strongly suggest that the bimodal egress of these two pathogenic viruses, like their entry into target cells, is guided by specific sets of host cell proteins.

Expression of complement system regulatory molecules in the endometrium of normal ovulatory and hyperstimulated women correlate with menstrual cycle phase

The present study describes the temporal expression of complement regulatory molecules membrane cofactor protein (MCP), decay accelerating factor (DAF), CR1, and CD59 in the human endometrium throughout the normal menstrual cycle and in patients submitted to ovarian hyperstimulation. During its proliferative phase, the endometrium expresses MCP, with increased expression during the secretory phase. Phase-dependent expression also was observed for DAF and CD59, mainly in the secretory phase. Expression of CR1 was not detected. These results suggest the presence of complement system activity during the menstrual cycle, with greater expression of regulatory molecules during the secretory phase to protect the epithelial integrity of human endometrium.

Gp135/podocalyxin and NHERF-2 participate in the formation of a preapical domain during polarization of MDCK cells

Epithelial polarization involves the segregation of apical and basolateral membrane domains, which are stabilized and maintained by tight junctions and membrane traffic. We report that unlike most apical and basolateral proteins in MDCK cells, which separate only after junctions have formed, the apical marker gp135 signifies an early level of polarized membrane organization established already in single cells. We identified gp135 as the dog orthologue of podocalyxin. With a series of domain mutants we show that the COOH-terminal PSD-95/Dlg/ZO-1 (PDZ)–binding motif is targeting podocalyxin to the free surface of single cells as well as to a subdomain of the terminally polarized apical membrane. This special localization of podocalyxin is shared by the cytoplasmic PDZ-protein Na+/H+ exchanger regulatory factor (NHERF)-2. Depleting podocalyxin by RNA interference caused defects in epithelial polarization. Together, our data suggest that podocalyxin and NHERF-2 function in epithelial polarization by contributing to an early apical scaffold based on PDZ domain-mediated interactions.