Epithelial Cell Shape Research Articles

Histomorphometric Profile of the Corneal Response to Short-term Reverse-geometry Orthokeratology Lens Wear in Primate Corneas

To investigate the histological changes in primate cornea induced by short-term overnight orthokeratology (OK). Nine young adult primates were used. One animal served as negative control. The remaining 8 animals wore reverse-geometry OK lenses for periods of 4, 8, 16, and 24 hours on 1 eye with the other eye as control. Central and midperipheral corneal thickness, as well as ultrastructural changes in corneal epithelium, stroma and endothelium in response to OK lenses, were evaluated. OK significantly reduced the thickness of the central cornea in all treatment groups. The central corneal thinning was both stromal and epithelial in origin. Substantial midperipheral corneal thickening was seen in 16-hour and 24-hour lens-wear groups and this effect was both stromal and epithelial in origin as well. Histology evidence indicated the primary epithelial response in the central cornea was compression of cells that resulted in wing cells becoming shorter and basal cells being squatted rather than lost or migration of cell layers. These pronounced cell shape changes occurred without compromising the structural integrity of the desmosomes. The thickened corneal epithelium has normal cell layers. The squamous cells have larger surface sizes and are composed of oval instead of flattened nuclei. This implied delayed surface cell exfoliation at the thickened midperipheral epithelium. Physical presence of OK lens over the cornea did not influence the microstructures of microvilli and microplicae, endothelium, and collagen distribution. The primate cornea, particularly the corneal epithelium, responds rapidly to the application of reverse-geometry OK lenses with significant epithelial cell shape alterations with short-term OK lens wear. This finding suggests that the corneal epithelium is moldable in response to the physical forces generated by the OK lenses.

JAM‐A regulates epithelial integrin expression and cell shape by activation of Rap1a and Rap1b through a complex with Afadin and PDZ‐GEFs

Junctional adhesion molecule‐A (JAM‐A) is a tight junction protein implicated in the regulation of epithelial β1 integrin expression, cell shape and barrier function. We previously determined that this regulation requires both dimerization and PDZ binding motifs on different regions of JAM‐A. To gain further insight into JAM‐A mediated signaling in vitro, we downregulated expression of scaffolding proteins and found that loss of Afadin resulted in cellular changes similar to those observed with loss of JAM‐A. Immunofluorescence (IF) and immunoprecipitation (IP) studies revealed co‐association of Afadin with JAM‐A. Interestingly, the small GTPase Rap1 is known to interact with Afadin, regulate integrin function and have decreased activity after loss of JAM‐A. Downregulation of Rap1a resulted in decreased β1 integrin protein levels whereas loss of Rap1b resulted in altered cell shape. Since Rap1 activation is dependent on guanine nucleotide exchange factors (GEFs) we assessed expression of GEFs in vitro and identified PDZ‐GEF1 and PDZ‐GEF2 as major transcripts in colonic epithelia. IF and IP studies revealed co‐association of the PDZ‐GEFs with JAM‐A and loss of the PDZ‐GEFs resulted in diminished Rap1 activity. These results suggest that dimerized JAM‐A signals through a complex that may contain both Afadin and PDZ‐GEFs which, in turn, activate Rap1a and Rap1b to regulate β1 integrin levels and cell shape.Supported by NIH DK‐61379

Tmod3 regulates polarized epithelial cell morphology

Although the role of the actin cytoskeleton in morphogenesis of polarized epithelial sheets is generally accepted as centrally important, the regulation of actin dynamics in this process remains unclear. Here, we show that the pointed-end capping protein Tmod3 contributes to epithelial cell shape within confluent monolayers of polarized epithelial cells. Tmod3 localizes to lateral cell membranes in polarized epithelia of several cell types. Reduction of Tmod3 levels by shRNA leads to a loss of F-actin and tropomyosins from lateral cell membranes, and a decrease in epithelial cell height, without effects on localisation of tight junction or adherens junction proteins, or any apparent changes in cell-cell adhesion. Instead, distribution of alphaII-spectrin on lateral membranes is disrupted upon reduction of Tmod3 levels, suggesting that loss of Tmod3 function leads to destabilization and disassembly of tropomyosin-coated actin filaments followed by disorganization of the spectrin-based membrane skeleton on lateral membranes. These data demonstrate for the first time a role for pointed-end capping in morphology regulation of polarized epithelial cells through stabilization of F-actin on lateral membranes. We propose that Tmod3-capped tropomyosin-actin filaments provide crucial links in the spectrin membrane skeleton of polarized epithelial cells, enabling the membrane skeleton to maintain cell shape.

Effects of microwave radiation on lens hydration and expression of PKC-α and transcription factors in lens epithelial cells

To observe the effects of low power microwave radiation on lens hydration and lens epithelial cells in vitro, and detect the expression of PKC-alpha, c-fos and c-jun in lens epithelial cells. Rabbit lens were exposed to microwave radiation with frequency of 2450 MHz and power density of 0.5, 2.0 and 5.0 mW/cm(2) in vitro. The hydration of lens was measured after 8 hours. Morphological changes of lens epithelial cells were observed using a phase-contrast microscope and Hoechst 33258 staining. Expression of PKC-alpha, c-fos and c-jun were analyzed using gel electrophoresis and western blot analysis. After 2.0 and 5.0 mW/cm(2) microwave radiation, the hydration of lens was increased compared to control groups (P<0.05), the shape of lens epithelial cells showed shrinking and disorder and cells nuclei appeared chromatin condensation. There was no change of lens and lens epithelial cells after 0.5 mW/cm(2) microwave radiation. The expression of PKC-alpha was significantly increased in cell membrane, however, decreased in cell cytoplasm after 2.0 mW/cm(2) microwave radiation for 2, 4, 6 and 8 hours. There was significantly increased expression of c-fos and c-jun protein compared with control groups (P<0.05, P<0.01). Low power microwave radiation higher than 2.0 mW/cm(2) can activate PKC-alpha by increasing its expression in cell membrane, then induce high expression of c-fos and c-jun, which may relate to cellular signaling pathway of microwave radiation injury to lens and lens epithelial cells.

Ras signaling modulates activity of the ecdysone receptor EcR during cell migration in the drosophila ovary

Ecdysone Receptor (EcR) mediates effects of the hormone ecdysone during larval molts, pupal metamorphosis, and adult female oogenesis. In the ovary, egg chamber formation requires interactions between the somatic follicle cell (FC) epithelium and the germ line nurse cell/oocyte cyst. Previous work has shown EcR is required in the germ line for egg chamber maturation, and here we examine EcR requirements in the FC at late stages of oogenesis. EcR protein is ubiquitous in the FC but its activity is restricted, visualized by activity of the "ligand sensor" hs-GAL4-EcR ligand binding domain fusion and EcRE-lacZ reporter gene expression. GAL4-EcR is activated in the FC by an ecdysone agonist and repressed by tissue-specific Ras GTPase signals. To determine the significance of restricted sites of EcR activity in the FC, we used targeted misexpression of the dominant negative EcR (EcR-DN) molecules EcR(F645A) and EcR(W650A). EcR-DN expression at stage 10 reduced EcRE-lacZ expression in the nurse cell FC and resulted in abnormal FC migrations, including aberrant centripetal migration and dorsal appendage tube formation, leading to the formation of cup-shaped eggs with shortened, branched dorsal appendages at stage 14. Clones of FC expressing EcR-DN displayed cell-autonomous increases in DE-cadherin expression and abnormal epithelial junction formation. EcR-DN expression caused thin eggshell phenotypes that correlated with both reduced levels of chorion gene expression and reduction in chorion gene amplification. Our results indicate that tissue-specific modulation of EcR activity by the Ras signaling pathway refines temporal ecdysone signals that regulate FC differentiation and cadherin-mediated epithelial cell shape changes.

Integrin-ECM interactions regulate the changes in cell shape driving the morphogenesis of theDrosophilawing epithelium

During development, morphogenesis involves migration and changes in the shape of epithelial sheets, both of which require coordination of cell adhesion. Thus, while modulation of integrin-mediated adhesion to the ECM regulates epithelial motility, cell-cell adhesion via cadherins controls the remodelling of epithelial sheets. We have used the Drosophila wing epithelium to demonstrate that cell-ECM interactions mediated by integrins also regulate the changes in cell shape that underly epithelial morphogenesis. We show that integrins control the transitions from columnar to cuboidal cell shape underlying wing formation, and we demonstrate that eliminating the ECM has the same effect on cell shape as inhibiting integrin function. Furthermore, lack of integrin activity also induces detachment of the basal lamina and failure to assemble the basal matrix. Hence, we propose that integrins control epithelial cell shape by mediating adherence of these cells to the ECM. Finally, we show that the ECM has an instructive rather than a structural role, because inhibition of Raf reverses the cell shape changes caused by perturbing integrins.

Shroom family proteins regulate γ-tubulin distribution and microtubule architecture during epithelial cell shape change

Cell shape changes require the coordination of actin and microtubule cytoskeletons. The molecular mechanisms by which such coordination is achieved remain obscure, particularly in the context of epithelial cells within developing vertebrate embryos. We have identified a novel role for the actin-binding protein Shroom3 as a regulator of the microtubule cytoskeleton during epithelial morphogenesis. We show that Shroom3 is sufficient and also necessary to induce a redistribution of the microtubule regulator gamma-tubulin. Moreover, this change in gamma-tubulin distribution underlies the assembly of aligned arrays of microtubules that drive apicobasal cell elongation. Finally, experiments with the related protein, Shroom1, demonstrate that gamma-tubulin regulation is a conserved feature of this protein family. Together, the data demonstrate that Shroom family proteins govern epithelial cell behaviors by coordinating the assembly of both microtubule and actin cytoskeletons.

Mammary epithelial cell: Influence of extracellular matrix composition and organization during development and tumorigenesis

Stromal–epithelial interactions regulate mammary gland development and are critical for the maintenance of tissue homeostasis. The extracellular matrix, which is a proteinaceous component of the stroma, regulates mammary epithelial growth, survival, migration and differentiation through a repertoire of transmembrane receptors, of which integrins are the best characterized. Integrins modulate cell fate by reciprocally transducing biochemical and biophysical cues between the cell and the extracellular matrix, facilitating processes such as embryonic branching morphogenesis and lactation in the mammary gland. During breast development and cancer progression, the extracellular matrix is dynamically altered such that its composition, turnover, processing and orientation change dramatically. These modifications influence mammary epithelial cell shape, and modulate growth factor and hormonal responses to regulate processes including branching morphogenesis and alveolar differentiation. Malignant transformation of the breast is also associated with significant matrix remodeling and a progressive stiffening of the stroma that can enhance mammary epithelial cell growth, perturb breast tissue organization, and promote cell invasion and survival. In this review, we discuss the role of stromal–epithelial interactions in normal and malignant mammary epithelial cell behavior. We specifically focus on how dynamic modulation of the biochemical and biophysical properties of the extracellular matrix elicit a dialogue with the mammary epithelium through transmembrane integrin receptors to influence tissue morphogenesis, homeostasis and malignant transformation.

A UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferase Is Required for Epithelial Tube Formation

Epithelial tubes are essential for the proper function of a diverse array of eukaryotic organs. Here we present a novel class of genes required for maintaining epithelial cell shape, polarity, and paracellular barrier function in the Drosophila embryonic tracheal system. Mutations in one member of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family (pgant35A) are recessive lethal and result in tracheal tubes that are irregular in diameter and morphology. Further analysis of the pgant35A mutants reveals diminished levels of the apical determinant Crbs and the luminal marker 2A12, concomitant with increased staining in cytoplasmic vesicles within tracheal cells. GalNAc-containing glycoproteins are severely diminished along the apical region of the tracheal system as well. Tracheal cells become irregular in size and shape, and septate junction proteins are mislocalized to a more apical position. Most notably, paracellular barrier function is lost in the tracheal system of the mutants. Overexpression of wild type pgant35A under control of the trachea-specific breathless (btl) promoter results in partial rescue of the lethality. We propose a model where pgant35A is required to establish proper apical composition of tracheal cells by influencing apical delivery of proteins/glycoproteins. Disruption of the normal apical content results in altered cell morphology and loss of paracellular barrier function. These studies demonstrate a previously unrecognized requirement for mucin-type O-glycosylation in epithelial tube integrity and have obvious implications for epithelial morphogenesis in higher eukaryotes, since a unique ortholog to pgant35A exists in mammals.

Nano- and Microscale Holes Modulate Cell-Substrate Adhesion, Cytoskeletal Organization, and $-\beta 1$ Integrin Localization in Sv40 Human Corneal Epithelial Cells

Human corneal epithelial cells (HCECs) interface with a basement membrane in vivo that possesses complex nanoscale topographic features. We report that synthetic substrates patterned with nano- and microscale holes differentially modulate the proliferation, shape and adhesion of SV40 human corneal epithelial cells (SV40-HCECs) as a function of feature size: 1) Cell proliferation was inhibited on nanoscale features (features size less than 800 nm in pitch) compared to microscale features or planar substrates in identical culture conditions. 2) Cells on nanoscale holes had a stellate morphology compared to those on microscale features that were more evenly spread. 3) Cells adhered more to nanoscale features than to microscale features when exposed to shear stress in a laminar flow chamber. Transmission electron microscopy showed that cells cultured on the 400 nm pitch patterns had longer and more numerous filopodia and retraction fibers than cells cultured on the 1600 nm pitch patterns. Immunogold labeling of -beta1 integrins revealed that these receptors were localized at the cell periphery and in the aforementioned cytoskeletal elements. Our findings indicate that surface discontinuities and the activation of mechanochemical cell signaling mechanisms may contribute to the observed responses exhibited by SV40-HCECs cultured on nano- and microscale topography.

Deletion of JAM-A causes morphological defects in the corneal epithelium

Junctional adhesion molecule-A (JAM-A, JAM-1, F11R) is an Ig domain containing transmembrane protein that has been proposed to function in diverse processes including platelet activation and adhesion, leukocyte transmigration, angiogenesis, epithelial cell shape and endothelial cell migration although its function in vivo is less well established. In the mouse eye, JAM-A protein expression is first detected at 12.5dpc in the blood vessels of the tunica vasculosa, while it is first detected in both the corneal epithelium and lens between 13.5 and 14.5dpc. In the corneal epithelium, JAM-A levels remain appreciable throughout life, while JAM-A immunostaining becomes stronger in the lens as the animals age. Both the cornea and lens of mice lacking an intact JAM-A gene are transparent until at least a year of age, although the cells of the JAM-A null corneal epithelium are irregularly shaped. In wild-type mice, JAM-A protein is found at the leading edge of repairing corneal epithelial wounds, however, corneal epithelial wound repair was qualitatively normal in JAM-A null animals. In summary, JAM-A is expressed in the corneal epithelium where it appears to regulate cell shape.

Essential Roles of 3′-Phosphoadenosine 5′-Phoshosulfate Synthase in Embryonic and Larval Development of the Nematode Caenorhabditis elegans

Sulfation of biomolecules, which is widely observed from bacteria to humans, plays critical roles in many biological processes. All sulfation reactions in all organisms require activated sulfate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), as a universal donor. In animals, PAPS is synthesized from ATP and inorganic sulfate by the bifunctional enzyme, PAPS synthase. In mammals, genetic defects in PAPS synthase 2, one of two PAPS synthase isozymes, cause dwarfism disorder, but little is known about the consequences of the complete loss of PAPS synthesis. To define the developmental role of sulfation, we cloned a Caenorhabditis elegans PAPS synthase-homologous gene, pps-1, and depleted expression of its product by isolating the deletion mutant and by RNA-mediated interference. PPS-1 protein exhibits specific activity to form PAPS in vitro, and disruption of the pps-1 gene by RNAi causes pleiotropic developmental defects in muscle patterning and epithelial cell shape changes with a decrease in glycosaminoglycan sulfation. Additionally, the pps-1 null mutant exhibits larval lethality. These data suggest that sulfation is essential for normal growth and integrity of epidermis in C. elegans. Furthermore, reporter analysis showed that pps-1 is expressed in the epidermis and several gland cells but not in neurons and muscles, indicating that PAPS in the neurons and muscles is provided by other cells.

Junctional Adhesion Molecule A (JAM‐A) signals through Afadin and Rap1

JAM-A is a transmembrane tight junction protein involved in regulation of barrier function and leukocyte transmigration, yet the mechanisms governing these functions are incompletely understood. The extracellular domain of JAM-A is known to participate in protein-protein interactions while the cytoplasmic tail of JAM-A has a PDZ motif that interacts with scaffolding proteins such as Afadin and ZO-1 that are linked to signaling molecules such as small GTPases. We have previously shown that JAM-A siRNA-mediated knockdown causes a decrease in the levels of β1 integrin in an epithelial cell line (SKCO15). We now provide evidence that this decrease in β1 integrin is due to loss of Rap1a activity and that loss of β1 integrin is due to aberrant recycling. Furthermore we show that siRNA-mediated knockdown of JAM-A, Afadin, and Rap1b, but not Rap1a, results in dramatic alterations in epithelial cell shape characterized by loss of cell-cell interactions, flattening and abundant spikes. Such alterations in cell shape appeared to be dependent upon dimerization of JAM-A. Additionally, decreases in JAM-A, Rap1b, and Afadin expression were observed to be associated with an increase in expression of the actin monomer shuttle, Twinfillin. Knockdown of both Twinfillin and JAM-A resulted in partial reversal of the morphologic alterations observed with knockdown of JAM-A alone. Together, these findings suggest that JAM-A dimerization promotes activation of Rap1, presumably mediated by Afadin. We also predict that Rap1b activation is associated with increased twinfillin expression, which alters actin cytoskeleton dynamics and cell morphology. Supported by NIH R01 DK61379

Essential requirement for Rho family GTPase signaling in Pax3 induced mesenchymal–epithelial transition

We previously showed that the transcription factor Pax3 regulates mesenchymal-to-epithelial transition (MET) in cultured osteogenic Saos-2 cells. Herein we demonstrate that Pax3 induced MET in these cells requires intact Pax3 DNA binding motifs and is associated with the altered expression and activity of numerous proteins involved in signal transduction pathways that regulate cytoskeleton remodeling, the majority of which were not previously detected by mRNA expression array analysis. Proper levels of active Rho GTPases are essential for Pax3 induced MET. Rac activity and actomyosin contractility via Rho/ROCK signaling are required for the formation of circumferential actin bundles, epithelial discoid cell shape and the regulation of membrane protrusions. Precise spatial activation of Rho GTPase signaling components is also paramount for MET. Endogenous PAK2, Rac1 and PIX, a Rac/Cdc42-GEF, localize to focal adhesions. Dynamic localization of PAK and PIX to focal adhesions is required for Pax3 induced MET and is dependent on full PAK activity because kinase dead or GTPase-binding deficient mutants of PAK sequester PIX at focal adhesions and disrupt Pax3 induced phenotypic MET. All together, our results define roles for Rho GTPases and their effectors in MET and newly identify proteins and signal transduction cascades regulated by Pax3.

Tubulogenesis: An Inside Job

New work shows that a dynamic and highly patterned apical extracellular matrix regulates epithelial cell shape and tube size from within the lumen of the Drosophila tracheal system.

Quantitative analysis of keratin filament networks in scanning electron microscopy images of cancer cells

The keratin filament network is an important part of the cytoskeleton. It is involved in the regulation of shape and viscoelasticity of epithelial cells. The morphology of keratin networks depends on post-translational modifications of keratin monomers. In-vitro studies indicated that network characteristics, such as filament crosslink density, determines the biophysical properties of the filament network. This report presents a quantitative method for the morphological analysis of keratin filament networks. Visualization of filaments was based on prefixation extraction of epithelial cells and scanning electron microscopy (SEM). SEM images were processed by a skeletonization algorithm to obtain a graph structure that represents individual filaments as well as their connections. This method was applied to investigate the effects of transforming growth factor alpha (TGFalpha) on the morphology of keratin networks in pancreatic cancer cells. TGFalpha contributes to pancreatic cancer progression and activates signalling pathways phosphorylating keratin monomers. Using this new method, a significant alteration to the keratin network morphology could be detected in response to TGFalpha.

Shroom regulates epithelial cell shape via the apical positioning of an actomyosin network

The actin-binding protein Shroom is essential for neural tube morphogenesis in multiple vertebrate organisms, indicating its function is evolutionarily conserved. Shroom facilitates neurulation by regulating the morphology of neurepithelial cells. Shroom localizes to the apical tip of adherens junctions of neural ectoderm cells in vivo and to the apical junctional complex (AJC) in MDCK cells. Induced expression of Shroom in polarized epithelia elicits apical constriction and dramatic reorganization of the apical arrangement and packing of cells without altering apical-basal polarity. These events likely mimic the cell shape changes and cellular movements required for neurulation in vivo. The observed phenotypes depend on the ability of Shroom to alter F-actin distribution and regulate the formation of a previously uncharacterized contractile actomyosin network associated with the AJC. Targeting the C-terminal domain of Shroom to the apical plasma membrane elicits constriction and reorganization of the actomyosin network, indicting that this domain mediates Shroom's activity. In vivo, Shroom-mutant neural epithelia show a marked reduction in apically positioned myosin. Thus, Shroom likely facilitates neural tube closure by regulating cell shape changes via the apical positioning of an actomyosin network in the neurepithelium.

Analysis of Cell Type-Specific Responses Mediated by the Type IV Secretion System of Helicobacter pylori

Helicobacter pylori persistently infects the human stomach and can cause gastritis, gastric ulceration, and gastric cancer. The type IV secretion system (TFSS) of virulent H. pylori strains translocates the CagA protein, inducing the dephosphorylation of host cell proteins and leading to changes in the morphology or shape of AGS gastric epithelial cells. Furthermore, the TFSS is involved in the induction of proinflammatory cytokines. While the H. pylori genes required for TFSS function have been investigated systematically, little is known about possible host cell factors involved. We infected 19 different mammalian cell lines individually with H. pylori and analyzed CagA translocation, dephosphorylation of host cell proteins, chemokine secretion (interleukin-8 and macrophage inflammatory protein 2), and changes in cellular phenotypes. Our results demonstrate that not only bacterial but also host cell factors determine the cellular response to infection. The identification of such unknown host cell factors will add to our understanding of host-pathogen interactions and might help in the development of new therapeutic strategies.

Three-dimensional polarization sensitizes hepatocytes to Fas/CD95 apoptotic signalling

Maintenance of epithelial cell shape and polarity determines many vital cell functions, including the appropriate response to external stimuli. Murine hepatocytes cultured in a three-dimensional Matrigel matrix formed highly polarized organoids characterized by specific localization of an ERM (ezrin/radixin/moesin) protein, radixin, at microvillus-lined membrane domains. These apical domains surrounded a lumen and were bordered by tight junctions. The hepatocyte organoids were functional as judged by the high level of albumin secretion and accumulation of bilirubin. Stimulation of the Fas/CD95 death receptor, which is highly hepatotoxic in vivo, was a strong inducer of apoptosis in the polarized organoids. This was in sharp contrast to the monolayer hepatocyte cultures, which were protected from death by exacerbated NF-kappaB signalling following engagement of the death receptors. Thus, hepatocytes in polarized, functional organoids modulate an intracellular signal transduction pathway, allowing the recapitulation of their physiological response to an apoptotic stimulus.

SMA-1 spectrin has essential roles in epithelial cell sheet morphogenesis in C. elegans

During Caenorhabditis elegans development, the embryo acquires its vermiform shape due to changes in the shape of epithelial cells, a process that requires an apically localized actin cytoskeleton. We show that SMA-1, an ortholog of β H-spectrin required for normal morphogenesis, localizes to the apical membrane of epithelial cells when these cells are rapidly elongating. In spc-1 α-spectrin mutants, SMA-1 localizes to the apical membrane but its organization is altered, consistent with the hypothesis these proteins act together to form an apically localized spectrin-based membrane skeleton (SBMS). SMA-1 is required to maintain the association between actin and the apical membrane; sma-1 mutant embryos fail to elongate because actin, which provides the driving force for cell shape change, dissociates from the apical membrane skeleton during morphogenesis. Analysis of sma-1 expression constructs and mutant strains indicates SMA-1 maintains the association between actin and the apical membrane via interactions at its N-terminus and this activity is independent of α-spectrin. SMA-1 also preserves dynamic changes in the organization of the apical membrane skeleton. Taken together, our results show the SMA-1 SBMS plays a dynamic role in converting changes in actin organization into changes in epithelial cell shape during C. elegans embryogenesis.