Plasma Membrane Of Epithelial Cells Research Articles

Human rhinovirus-induced inflammatory responses are inhibited by phosphatidylserine containing liposomes.

Human rhinovirus (HRV) infections are major contributors to the healthcare burden associated with acute exacerbations of chronic airway disease, such as chronic obstructive pulmonary disease and asthma. Cellular responses to HRV are mediated through pattern recognition receptors that may in part signal from membrane microdomains. We previously found Toll-like receptor signaling is reduced, by targeting membrane microdomains with a specific liposomal phosphatidylserine species, 1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-L-serine (SAPS). Here we explored the ability of this approach to target a clinically important pathogen. We determined the biochemical and biophysical properties and stability of SAPS liposomes and studied their ability to modulate rhinovirus-induced inflammation, measured by cytokine production, and rhinovirus replication in both immortalized and normal primary bronchial epithelial cells. SAPS liposomes rapidly partitioned throughout the plasma membrane and internal cellular membranes of epithelial cells. Uptake of liposomes did not cause cell death, but was associated with markedly reduced inflammatory responses to rhinovirus, at the expense of only modest non-significant increases in viral replication, and without impairment of interferon receptor signaling. Thus using liposomes of phosphatidylserine to target membrane microdomains is a feasible mechanism for modulating rhinovirus-induced signaling, and potentially a prototypic new therapy for viral-mediated inflammation.Supplementary informationThe online version of this article (doi:10.1038/mi.2015.137) contains supplementary material, which is available to authorized users.

Mapping CFTR Clusters in 3D via Single Molecule Step Photobleaching Analysis in Epithelial Cells

Protein oligomerization is crucial for cellular regulation. Various methods have been used to study the oligomerization of membrane proteins and multimeric channels including single molecule step photobleaching subunit counting. However, the membrane proteins function in a quasi 2D environment within a larger 3D cellular context encompassing membrane delivery and recycling via exocytosis and endocytosis and scaffolding and functional interactions within multiprotein complexes. We present PISA (photobleaching intensity step analysis), a new single molecule approach to map oligomerization states of fluorescently tagged proteins in 3D in cells where the membrane interface is imaged via TIRF microscopy. PISA counts oligomer subunits via sequential photobleaching steps, and additionally maps these distributions with nm precision in z due to magnitude differences of the intensity steps within the TIRF evanescent field. We applied PISA to map distributions of clusters of cystic fibrosis transmembrane conductance regulator (CFTR) in primary human airway epithelial cells. CFTR is an anion channel which conducts chloride and bicarbonate ions through the apical plasma membrane of epithelial cells and is known to interact with other proteins which form a macromolecular complex that regulates its activity at the cell surface. We measured membrane distributions of CFTR-eGFP as well as intracellular clusters of CFTR that were distributed at distances over 100 nm from the membrane. Treatment of the cells with vasoactive intestinal peptide (VIP) and cholesterol oxidase (COase) both resulted in a shift of CFTR clusters towards the membrane. VIP treatment resulted in larger CFTR cluster size (mean 2.9 subunits/cluster) and population (mean 28 units/ROI) as compared to untreated cells (mean 2.1 subunits/cluster) and cluster population (mean 22 units/ROI). COase treatment resulted only in lower cluster population (mean 9 units/ROI). Results on untreated cells were corroborated by TEM.

Annexin A2 and S100A10 in the mammalian oviduct.

In many mammals, upon entry into the female reproductive tract, a subpopulation of sperm is stored in the oviduct forming a functional reservoir. In the oviducts of pig and cow, Annexin A2 (AnxA2) has been linked to the binding of sperm. This protein may exist as a monomer or bound to S100A10 and both forms are associated with different biological functions. S100A10 has not yet been reported in the oviduct. The objective of this work is to analyze for the presence of S100A10 in the oviduct and to advance the study of AnxA2 and S100A10 in this organ. This work shows the presence of both proteins, AnxA2 and S100A10, in the oviduct of human, pig, cow, cat, dog and rabbit. At least in pig, AnxA2 is found devoid of S100A10 in the outer surface of the apical plasma membrane of oviductal epithelial cells, indicating that it binds to sperm as a monomer or in association with proteins different from S100A10. In the apical cytoplasm of pig oviductal epithelial cells, AnxA2 is associated with S100A10. In primary culture of porcine oviductal cells, the expression of ANXA2 is increased by progesterone, while the expression of S100A10 is increased by progesterone and estradiol. The widespread detection of both proteins in the oviduct of mammals indicates a probable conserved function in this organ. In summary, S100A10 and AnxA2 are widespread in the mammalian oviduct but AnxA2 binds sperm in vivo devoid of S100A10 and may be related to reservoir formation.

Novel Approaches for Potential Therapy of Cystic Fibrosis.

Cystic fibrosis (CF) is a lethal autosomal recessive disease that causes severe damage to the respiratory and digestive systems. It results from a dysfunctional CF Transmembrane Conductance Regulator (CFTR) protein, which is a cAMP- regulated epithelial chloride channel. CFTR is also a subtype of the ABC-transporter superfamily, and is expressed primarily in the apical membrane of epithelial cells in the airways, pancreas, and intestines. A single amino acid deletion of phenylalanine (Phe) is the most common mutation in CF patients known as F508del-CFTR. Normally, wild-type CFTR is largely degraded before reaching the cell membrane and F508del-CFTR virtually never reaches the cell surface. Ultimately, our goal is to correct dysfunctional CFTR proteins in CF patients. Via high-throughput screening techniques, several novel compounds for potential drugs effective in reversing the molecular CF defect and prohibiting further progression of CF have recently been discovered. S-nitrosothiols (SNOs) are small, naturally occurring endogenous cell signaling compounds, which have potential relevance to human lung diseases, including CF. Remarkably, researchers have found that the level of SNOs are reduced in the CF airway. It was previously reported that different types of SNOs, such as GSNO and S-nitrosoglutathione diethyl ester will increase CFTR maturation and function at the plasma membrane in human airway epithelial cells. The mechanisms by which SNOs improve CFTR maturation remain elusive. Currently, clinical trials are still investigating the effectiveness and safety of novel corrector and potentiator drugs for F508del- CFTR. This review article offers a summary of our knowledge on the most up-to-date CF therapies.

Molecular machinery for vasotocin-dependent transepithelial water movement in amphibians: aquaporins and evolution.

Amphibians represent the first vertebrates to adapt to terrestrial environments, and are successfully distributed around the world. The ventral skin, kidney, and urinary bladder are important osmoregulatory organs for adult anuran amphibians. Water channel proteins, called aquaporins (AQPs), play key roles in transepithelial water absorption/reabsorption in these organs. At least 43 types of AQPs were identified in anurans; a recent phylogenetic analysis categorized anuran AQPs among 16 classes (AQP0-14, 16). Anuran-specific AQPa2 was assigned to AQP6, then was further subdivided into the ventral skin-type (AQP6vs; AQPa2S), whose expression is confined to the ventral skin, and the urinary bladder-type (AQP6ub; AQPa2U), which is basically expressed in the urinary bladder. For the osmoregulatory organs, AQP3 is constitutively located in the basolateral plasma membrane of tight-junctioned epithelial cells. AQP6vs, AQP2 and/or AQP6ub are also expressed in these epithelial cells and are translocated to the apical membrane in response to arginine vasotocin, thereby regulating water absorption/reabsorption. It was suggested recently that two subtypes of AQP6vs contribute to cutaneous water absorption in Ranid species. In addition, AQP5 (AQP5a) and AQP5L (AQP5b) were identified from Xenopus tropicalis Gray, 1864, and AQP5 was localized to the apical membrane of luminal epithelial cells of the urinary bladder in dehydrated Xenopus. This finding suggested that AQP5 may be involved in water reabsorption from this organ under dehydration. Based on the hitherto reported information, we propose models for the evolution of water-absorbing/reabsorbing mechanisms in anuran osmoregulatory organs in association with AQPs.

Cholesterol Modulates CFTR Confinement in the Plasma Membrane of Primary Epithelial Cells

The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma-membrane anion channel that, when mutated, causes the disease cystic fibrosis. Although CFTR has been detected in a detergent-resistant membrane fraction prepared from airway epithelial cells, suggesting that it may partition into cholesterol-rich membrane microdomains (lipid rafts), its compartmentalization has not been demonstrated in intact cells and the influence of microdomains on CFTR lateral mobility is unknown. We used live-cell imaging, spatial image correlation spectroscopy, and k-space image correlation spectroscopy to examine the aggregation state of CFTR and its dynamics both within and outside microdomains in the plasma membrane of primary human bronchial epithelial cells. These studies were also performed during treatments that augment or deplete membrane cholesterol. We found two populations of CFTR molecules that were distinguishable based on their dynamics at the cell surface. One population showed confinement and had slow dynamics that were highly cholesterol dependent. The other, more abundant population was less confined and diffused more rapidly. Treatments that deplete the membrane of cholesterol caused the confined fraction and average number of CFTR molecules per cluster to decrease. Elevating cholesterol had the opposite effect, increasing channel aggregation and the fraction of channels displaying confinement, consistent with CFTR recruitment into cholesterol-rich microdomains with dimensions below the optical resolution limit. Viral infection caused the nanoscale microdomains to fuse into large platforms and reduced CFTR mobility. To our knowledge, these results provide the first biophysical evidence for multiple CFTR populations and have implications for regulation of their surface expression and channel function.

Sorting nexin 27 regulates basal and stimulated brush border trafficking of NHE3.

Sorting nexin 27 (SNX27) contains a PDZ domain that is phylogenetically related to the PDZ domains of the NHERF proteins. Studies on nonepithelial cells have shown that this protein is located in endosomes, where it regulates trafficking of cargo proteins in a PDZ domain-dependent manner. However, the role of SNX27 in trafficking of cargo proteins in epithelial cells has not been adequately explored. Here we show that SNX27 directly interacts with NHE3 (C-terminus) primarily through the SNX27 PDZ domain. A combination of knockdown and reconstitution experiments with wild type and a PDZ domain mutant (GYGF → GAGA) of SNX27 demonstrate that the PDZ domain of SNX27 is required to maintain basal NHE3 activity and surface expression of NHE3 in polarized epithelial cells. Biotinylation-based recycling and degradation studies in intestinal epithelial cells show that SNX27 is required for the exocytosis (not endocytosis) of NHE3 from early endosome to plasma membrane. SNX27 is also required to regulate the retention of NHE3 on the plasma membrane. The findings of the present study extend our understanding of PDZ-mediated recycling of cargo proteins from endosome to plasma membrane in epithelial cells.

Aquaporin‐1 retards renal cyst development in polycystic kidney disease by inhibition of Wnt signaling

ObjectiveAutosomal dominant polycystic kidney disease (ADPKD) is one of the most common human monogenic diseases. Aquaporin‐1 (AQP1) is expressed mainly at epithelial cell plasma membranes in renal proximal tubules, as well as in epithelial cells lining the cysts in ADPKD kidneys, postulating that AQP1 may be involved in cytogenesis. Here we investigated the role of AQP1 on ADPKD development.Methods: We used Aqp1/Pkd1 double knockout mice, embryonic kidney organ cultures, and matrix‐grown MDCK cells in this study to investigate the role of AQP1 in ADPKD. Expression of Wnt signaling‐related proteins were detected in MDCK and AQP1‐MDCK cells. Co‐immunoprecipitation was used to determine the interaction between AQP1 and β‐catenin,GSK3β,LRP6 and Axin1.Results: Kidney size and cyst number were significantly greater in AQP1‐null PKD mice than in AQP1‐expressing PKD mice. In embryonic kidney cultures, AQP1 deletion increased cyst development. In matrix‐grown MDCK cells, AQP1 inhibited cystogenesis and promoted tubulogenesis. AQP1 decreased β‐catenin and cyclinD1 expression, suggesting down‐regulation of Wnt signaling. Co‐immunoprecipitation showed AQP1 interaction with β‐catenin, GSK3β, LRP6 and Axin1. Biochemical analysis revealed decreased p‐β‐catenin and increased β‐catenin expression in Aqp1/Pkd1 double knockout mice, suggesting enhanced Wnt signaling.ConclusionsThese results suggest that AQP1 may be involved in maintaining the stability of the “destruction complex” to promote β‐catenin phosphorylation and suppress the Wnt signaling. This study reveals a previously unrecognized role of AQP1 in ADPKD and hence may provide new therapeutic targets.

Akt Substrate of 160 kD Regulates Na+,K+-ATPase Trafficking in Response to Energy Depletion and Renal Ischemia.

Renal ischemia and reperfusion injury causes loss of renal epithelial cell polarity and perturbations in tubular solute and fluid transport. Na(+),K(+)-ATPase, which is normally found at the basolateral plasma membrane of renal epithelial cells, is internalized and accumulates in intracellular compartments after renal ischemic injury. We previously reported that the subcellular distribution of Na(+),K(+)-ATPase is modulated by direct binding to Akt substrate of 160 kD (AS160), a Rab GTPase-activating protein that regulates the trafficking of glucose transporter 4 in response to insulin and muscle contraction. Here, we investigated the effect of AS160 on Na(+),K(+)-ATPase trafficking in response to energy depletion. We found that AS160 is required for the intracellular accumulation of Na(+),K(+)-ATPase that occurs in response to energy depletion in cultured epithelial cells. Energy depletion led to dephosphorylation of AS160 at S588, which was required for the energy depletion-induced accumulation of Na,K-ATPase in intracellular compartments. In AS160-knockout mice, the effects of renal ischemia on the distribution of Na(+),K(+)-ATPase were substantially reduced in the epithelial cells of distal segments of the renal tubules. These data demonstrate that AS160 has a direct role in linking the trafficking of Na(+),K(+)-ATPase to the energy state of renal epithelial cells.

Involvement of the Cdc42 pathway in CFTR post-translational turnover and in its plasma membrane stability in airway epithelial cells.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is expressed on the apical plasma membrane (PM) of epithelial cells. The most common deleterious allele encodes a trafficking-defective mutant protein undergoing endoplasmic reticulum-associated degradation (ERAD) and presenting lower PM stability. In this study, we investigated the involvement of the Cdc42 pathway in CFTR turnover and trafficking in a human bronchiolar epithelial cell line (CFBE41o-) expressing wild-type CFTR. Cdc42 is a small GTPase of the Rho family that fulfils numerous cell functions, one of which is endocytosis and recycling process via actin cytoskeleton remodelling. When we treated cells with chemical inhibitors such as ML141 against Cdc42 and wiskostatin against the downstream effector N-WASP, we observed that CFTR channel activity was inhibited, in correlation with a decrease in CFTR amount at the cell surface and an increase in dynamin-dependent CFTR endocytosis. Anchoring of CFTR to the cortical cytoskeleton was then presumably impaired by actin disorganization. When we performed siRNA-mediated depletion of Cdc42, actin polymerization was not impacted, but we observed actin-independent consequences upon CFTR. Total and PM CFTR amounts were increased, resulting in greater activation of CFTR. Pulse-chase experiments showed that while CFTR degradation was slowed, CFTR maturation through the Golgi apparatus remained unaffected. In addition, we observed increased stability of CFTR in PM and reduction of its endocytosis. This study highlights the involvement of the Cdc42 pathway at several levels of CFTR biogenesis and trafficking: (i) Cdc42 is implicated in the first steps of CFTR biosynthesis and processing; (ii) it contributes to the stability of CFTR in PM via its anchoring to cortical actin; (iii) it promotes CFTR endocytosis and presumably its sorting toward lysosomal degradation.

Evidence of a Structural and Functional Ammonium Transporter RhBG·Anion Exchanger 1·Ankyrin-G Complex in Kidney Epithelial Cells

The renal ammonium transporter RhBG and anion exchanger 1 kAE1 colocalize in the basolateral domain of α-intercalated cells in the distal nephron. Although we have previously shown that RhBG is linked to the spectrin-based skeleton through ankyrin-G and that its NH3 transport activity is dependent on this association, there is no evidence for an interaction of kAE1 with this adaptor protein. We report here that the kAE1 cytoplasmic N terminus actually binds to ankyrin-G, both in yeast two-hybrid analysis and by coimmunoprecipitation in situ in HEK293 cells expressing recombinant kAE1. A site-directed mutagenesis study allowed the identification of three dispersed regions on kAE1 molecule linking the third and fourth repeat domains of ankyrin-G. One secondary docking site corresponds to a major interacting loop of the erythroid anion exchanger 1 (eAE1) with ankyrin-R, whereas the main binding region of kAE1 does not encompass any eAE1 determinant. Stopped flow spectrofluorometry analysis of recombinant HEK293 cells revealed that the Cl(-)/HCO3 (-) exchange activity of a kAE1 protein mutated on the ankyrin-G binding site was abolished. This disruption impaired plasma membrane expression of kAE1 leading to total retention on cytoplasmic structures in polarized epithelial Madin-Darby canine kidney cell transfectants. kAE1 also directly interacts with RhBG without affecting its surface expression and NH3 transport function. This is the first description of a structural and functional RhBG·kAE1·ankyrin-G complex at the plasma membrane of kidney epithelial cells, comparable with the well known Rh·eAE1·ankyrin-R complex in the red blood cell membrane. This renal complex could participate in the regulation of acid-base homeostasis.

Modulation of TRIM72 Alters the Repair Capacity of Lung Epithelial Cells

The molecular mechanisms for alveolar epithelial cell plasma membrane wounding and repair are largely unknown. Previous studies identified TRIM72 (also known as mitsugumin 53) from striated muscle tissues and linked its physiological functions in these tissues to membrane repair, cell proliferation, and glucose metabolism. Here, using biochemical and immunochemical assays, we have characterized TRIM72 expression in lung tissue and cells, and investigated the physiological function of TRIM72 in the repair of alveolar epithelial cells. In vivo injury of alveolus resident cells was introduced by high tidal volume ventilation, and repair-defective cells were labeled by postinjury administration of propidium iodide and quantified in optical slices of subpleural alveoli. Primary alveolar epithelial cells were isolated to study plasma membrane wounding and repair in vitro, using a dual-dye incorporation assay following glass bead–induced damage to separately quantify injured versus repaired cells. Our results show that TRIM72 protein was expressed in alveolar epithelia of mouse, rat, and human lungs. High tidal volume ventilation of trim72 knockout mice caused significantly more fatal damage of alveolus cells as compared with the wild-type mice. Strikingly, injury estimates in trim72 overexpressor lungs were significantly lower than in the wild-type. The in vitro cell wounding assay further revealed that absence of TRIM72 led to significant repair defects in alveolar epithelial cells, whereas the total number of wounded cells (repaired plus nonrepaired) was similar in the wild-type and trim72 knockout groups. The cellular function of TRIM72 in the lung may be further linked to caveolin 1, as physical and physiological interactions were identified between these two proteins. These data suggest an important role for TRIM72 in the repair of alveolar epithelial cells under plasma membrane stress failure and potential reversal of cell repair defects by TRIM72 overexpression.

Saccharomyces cerevisiae CNCM I-3856 prevents colitis induced by AIEC bacteria in the transgenic mouse model mimicking Crohn's disease.

Adherent-invasive Escherichia coli (AIEC), which colonize the ileal mucosa of patients with Crohn's disease (CD), are able to adhere to and invade intestinal epithelial cells. Overexpression of the glycoprotein CEACAM6 on host cells favors AIEC attachment and inflammation. We investigated the ability of Saccharomyces cerevisiae CNCM I-3856 to inhibit AIEC adhesion and to reduce colitis. Adhesion experiments were performed on T84 cells and on enterocytes from patients with CD with AIEC LF82 in the presence of S. cerevisiae. Colonization and symptoms of colitis were assessed in LF82-infected transgenic CEABAC10 mice treated with live S. cerevisiae or S. cerevisiae derivatives. Proinflammatory cytokines were quantified by enzyme linked immunosorbent assay. Intestinal permeability was assessed by measuring the 4 kDa dextran-FITC flux in the serum. S. cerevisiae strongly inhibited LF82 adhesion to T84 cells and to the brush border of CD enterocytes. Yeasts decreased LF82 colonization and colitis in CEABAC10 mice and restored barrier function through prevention of the LF82-induced expression of pore-forming tight junction claudin-2 at the plasma membrane of intestinal epithelial cells. These effects were accompanied by a decrease in proinflammatory cytokines IL-6, IL-1β, and KC release by the gut mucosa. Yeast derivatives exerted similar effects on LF82 colonization and colitis demonstrating that yeast viability was not essential to exert beneficial effects. S. cerevisiae yeasts reduce colitis induced by AIEC bacteria in CEACAM6-expressing mice. Such a probiotic strategy could be envisaged in a subgroup of patients with CD abnormally expressing CEACAM6 at the ileal mucosa and therefore susceptible to being colonized by AIEC bacteria.

PTRF is associated with caveolin 1 at the time of receptivity: but SDPR is absent at the same time.

The plasma membrane of uterine epithelial cells undergoes a number of changes during early pregnancy. The changes in the basolateral membrane at the time of implantation in particular change from being smooth to highly tortuous in morphology, along with a dramatic increase in the number of morphological caveolae at this time. The major protein of caveolar membranes is caveolin, and previous studies have shown that RNA pol I transcription factor (PTRF) and serum deprivation protein response (SDPR) are the two members of the cavin protein family. These proteins are known to be involved in caveolae biogenesis, where they directly bind to cholesterol and lipids and have been reported to promote membrane curvature. As there is an increase in membrane tortuosity and caveolae at the time of implantation, this study investigated PTRF and SDPR to explore the possible roles that they play in the morphology of the uterine epithelium during early pregnancy. PTRF protein abundance did not change in uterine epithelial cells during early pregnancy or in response to ovarian hormones. At the time of implantation in uterine epithelial cells, PTRF co-immunoprecipitated with caveolin 1, thereby demonstrating an association with caveolin-1 at the basal plasma membrane in caveolae. SDPR protein was observed to be present only at the time of fertilisation, and also under the influence of oestrogen alone, where a cytoplasmic localisation in uterine epithelial cells was observed. The localisation and expression PTRF and SDPR in uterine epithelial cells during early pregnancy suggest that they have roles in the maintenance of lipids and cholesterol in the plasma membrane. PTRF and lack of SDPR may contribute not only to the morphology of the basal plasma membrane as observed at the time of implantation, but also to the maintenance of epithelial polarity during early pregnancy.

Aquaporin-1 retards renal cyst development in polycystic kidney disease by inhibition of Wnt signaling.

Water channel aquaporin-1 (AQP1) is expressed at epithelial cell plasma membranes in renal proximal tubules and thin descending limb of Henle. Recently, AQP1 was reported to interact with β-catenin. Here we investigated the relationship between AQP1 and Wnt signaling in in vitro and in vivo models of autosomal dominant polycystic kidney disease (PKD). AQP1 overexpression decreased β-catenin and cyclinD1 expression, suggesting down-regulation of Wnt signaling, and coimmunoprecipitation showed AQP1 interaction with β-catenin, glycogen synthase kinase 3β, LRP6, and Axin1. AQP1 inhibited cyst development and promoted branching in matrix-grown MDCK cells. In embryonic kidney cultures, AQP1 deletion increased cyst development by up to ∼ 40%. Kidney size and cyst number were significantly greater in AQP1-null PKD mice than in AQP1-expressing PKD mice, with the difference mainly attributed to a greater number of proximal tubule cysts. Biochemical analysis revealed decreased β-catenin phosphorylation and increased β-catenin expression in AQP1-null PKD mice, suggesting enhanced Wnt signaling. These results implicate AQP1 as a novel determinant in renal cyst development that may involve inhibition of Wnt signaling by an AQP1-macromolecular signaling complex.

Actin crosslinking protein filamin A during early pregnancy in the rat uterus.

During early pregnancy the endometrium undergoes a major transformation in order for it to become receptive to blastocyst implantation. The actin cytoskeleton and plasma membrane of luminal uterine epithelial cells (UECs) and the underlying stromal cells undergo dramatic remodelling to facilitate these changes. Filamin A (FLNA), a protein that crosslinks actin filaments and also mediates the anchorage of membrane proteins to the actin cytoskeleton, was investigated in the rat uterus at fertilisation (Day 1) and implantation (Day 6) to determine the role of FLNA in actin cytoskeletal remodelling of UECs and decidua during early pregnancy. Localisation of FLNA in UECs at the time of fertilisation was cytoplasmic, whilst at implantation it was distributed apically; its localisation is under the influence of progesterone. FLNA was also concentrated to the first two to three stromal cell layers at the time of fertilisation and shifted to the primary decidualisation zone at the time of implantation. This shift in localisation was found to be dependent on the decidualisation reaction. Protein abundance of the FLNA 280-kDa monomer and calpain-cleaved fragment (240kDa) did not change during early pregnancy in UECs. Since major actin cytoskeletal remodelling occurs during early pregnancy in UECs and in decidual cells, the changing localisation of FLNA suggests that it may be an important regulator of cytoskeletal remodelling of these cells to allow uterine receptivity and decidualisation necessary for implantation in the rat.

Z-Profiling of CFTR Oligomerization State Distributions via Single Molecule Step Photobleaching Analysis in Epithelial Cells

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel which conducts chloride and bicarbonate ions through the apical plasma membrane of epithelial cells. CFTR interacts with other proteins which form a macromolecular complex that regulates its activity at the cell surface. The number of CFTR molecules within these complexes remains uncertain due to cell variability and the difficulty of measuring this parameter in situ. We show a single molecule step photobleaching method which measures the oligomerization state of resolved clusters and then maps the z-position profile of individual CFTR molecules within the cluster based on imaging data collected by TIRF microscopy. DNA nanorods with defined fluorescent label spacing were used as controls to validate the analysis and to recover the oligomerization distribution that would be expected for a 24.5 nm building block length (24.6±2.5 nm, measured), and for the 13.5 nm height of the base layer of streptavidin on the coverslip (12.9±1.7 nm). Studies of primary airway epithelial cells expressing GFP-CFTR indicate that CFTR clustering state increases linearly with the z-position from the inside to the outside of the cell. Plots of z position versus oligomer subunit count yielded the same slope under control conditions (6.0±1.4 nm/count) and during protein kinase C stimulation (PKC; 6.0±3.7 nm/count). However, the number of counts per cluster was larger in PKC stimulated cells. Our results demonstrate that CFTR aggregation state varies with depth from the cell surface, and may be related to the mechanism of cluster assembly inside the cell and/or its function at the plasma membrane. We anticipate that our method can be generalized to monitor the z-position profile or oligomerization states of other membrane receptors and channel to determine structure/function relationships for such membrane complexes.

Transepithelial resistance in human bestrophin-1 stably transfected Madin–Darby canine kidney cells

Bestrophin-1 (Best1) is a transmembrane protein, found in the basolateral plasma membrane of retinal pigmented epithelial cells. The exact structure and functions of Best1 protein are still unclear. The protein is thought to be a regulator of ion channels, or an ion channel itself: it was shown to be permeable for chloride, thiocyanate, bicarbonate, glutamate and γ-aminobutyric acid (GABA). Mutations in the gene for Best1 are leading to best vitelliform macular dystrophy (BVMD) and are found in several other types of maculopathy. In order to obtain additional information about Best1 protein, we determined cell polarization of a stably transfected Madin–Darby canine kidney cell line II (MDCK II) cell line, expressing human Best1. We measured the transepithelial resistance of transfected and non-transfected MDCK cells by voltmeter EVOM, over 10 days at 24 hour intervals. The first few days (first–fourth day) both cell lines showed the same or similar values ​​of transmembrane resistance. As expected, on the fifth day the non-transfected cells showed maximum value of epithelial resistance, corresponding to the forming of monolayer. The transfected cells showed maximum value of transepithelial resistance on the ninth day of their cultivation. Phalloidin staining of actin demonstrated the difference in actin arrangements between transfected and non-transfected cells due to Best1. As a consequence of actin rearrangement, Best1 strongly affects the transepithelial resistance of polarizing stably transfected MDCK cells. Our results suggest that Best1 protein has an effect on transepithelial resistance and actin rearrangements of polarized stably transfected MDCK cells.

Calcium release-activated calcium (CRAC) channels mediate the β2-adrenergic regulation of Na,K-ATPase

β2-Adrenergic agonists have been shown to regulate Na,K-ATPase in the alveolar epithelium by recruiting Na,K-ATPase-containing vesicles to the plasma membrane of alveolar epithelial cells (AEC). Here, we provide evidence that β2-agonists induce store-operated calcium entry (SOCE) in AECs. This calcium entry is necessary for β2-agonist-induced recruitment of Na,K-ATPase to the plasma membrane of AECs. Specifically, we show that β2-agonists induce SOCE via stromal interaction molecule 1 (STIM1)-associated calcium release-activated calcium (CRAC) channels. We also demonstrate that the magnitude of SOCE affects the abundance of Na,K-ATPase at the plasma membrane of AECs.

Novel functional complexity of polycystin-1 by GPS cleavage in vivo: role in polycystic kidney disease.

Polycystin-1 (Pc1) cleavage at the G protein-coupled receptor (GPCR) proteolytic site (GPS) is required for normal kidney morphology in humans and mice. We found a complex pattern of endogenous Pc1 forms by GPS cleavage. GPS cleavage generates not only the heterodimeric cleaved full-length Pc1 (Pc1(cFL)) in which the N-terminal fragment (NTF) remains noncovalently associated with the C-terminal fragment (CTF) but also a novel (Pc1) form (Pc1(deN)) in which NTF becomes detached from CTF. Uncleaved Pc1 (Pc1(U)) resides primarily in the endoplasmic reticulum (ER), whereas both Pc1(cFL) and Pc1(deN) traffic through the secretory pathway in vivo. GPS cleavage is not a prerequisite, however, for Pc1 trafficking in vivo. Importantly, Pc1(deN) is predominantly found at the plasma membrane of renal epithelial cells. By functional genetic complementation with five Pkd1 mouse models, we discovered that CTF plays a crucial role in Pc1(deN) trafficking. Our studies support GPS cleavage as a critical regulatory mechanism of Pc1 biogenesis and trafficking for proper kidney development and homeostasis.