Four-dimensional imaging of filter-grown polarized epithelial cells

Understanding how epithelial cells generate and maintain polarity and function requires live cell imaging. In order for cells to become fully polarized, it is necessary to grow them on a permeable membrane filter; however, the translucent filter obstructs the microscope light path required for quantitative live cell imaging. Alternatively, the membrane filter may be excised but this eliminates selective access to apical and basolateral surfaces. Conversely, epithelial cells cultured directly on glass exhibit different phenotypes and functions from filter grown cells. Here, we describe a new method for culturing polarized epithelial cells on a Transwell filter insert that allows superior live cell imaging with spatial and temporal image resolution previously unachievable using conventional methods. Cells were cultured on the underside of a filter support. Epithelial cells grown in this inverted configuration exhibit a fully polarized architecture, including the presence of functional tight junctions. This new culturing system permits four-dimensional (three spatial dimension over time) imaging of endosome and Golgi apparatus dynamics, and permits selective manipulation of the apical and basolateral surfaces. This new technique has wide applicability for visualization and manipulation of polarized epithelial cells.     

Involvement of NHERF1 in apical membrane localization of MRP4 in polarized kidney cells

Multidrug resistance protein 4 (MRP4/ABCC4), a member of the ATP-binding cassette protein superfamily, confers resistance to nucleoside and nucleotide analogs as well as camptothecin derivatives. MRP4 also mediates the efflux of certain cyclic nucleotides, eicosanoids, conjugated steroids, and uric acid. Depending on the cell type, MRP4 may localize to either apical or basolateral membranes in polarized cells. The adaptor protein NHERF1 has previously been implicated in MRP4 internalization in non-polarized cells. We have now found that NHERF1 levels are very low in polarized MDCKI cells which express MRP4 on basolateral membranes relative to polarized LLC-PK1 cells which express MRP4 on apical membranes. Furthermore, ectopic expression of FLAG-tagged NHERF1 in MDCKI cells and in MDCKI cells stably expressing eGFP-tagged MRP4 causes endogenous MRP4 and eGFP-MRP4, respectively, to traffic to the apical membranes. These data establish NHERF1 as a major determinant of MRP4 trafficking to apical membranes of mammalian kidney cells.     

Proliferative effects of apical, but not basal, matrix metalloproteinase-7 activity in polarized MDCK cells

Matrix metalloproteinase-7 (MMP-7) is primarily expressed in glandular epithelium. Therefore, its mechanism of action may be influenced by its regulated vectorial release to either the apical and/or basolateral compartments, where it would act on its various substrates. To gain a better understanding of where MMP-7 is released in polarized epithelium, we have analyzed its pattern of secretion in polarized MDCK cells expressing stably transfected human MMP-7 (MDCK-MMP-7), and HCA-7 and Caco2 human colon cancer cell lines. In all cell lines, latent MMP-7 was secreted to both cellular compartments, but was 1.5- to 3-fold more abundant in the basolateral compartment as compared to the apical. However, studies in the MDCK system demonstrated that MMP-7 activity was 2-fold greater in the apical compartment of MDCK-MMP-7(HIGH)-polarized monolayers, which suggests the apical co-release of an MMP-7 activator. In functional assays, MMP-7 over-expression increased cell saturation density as a result of increased cell proliferation with no effect on apoptosis. Apical MMP-7 activity was shown to be responsible for the proliferative effect, which occurred, as demonstrated by media transfer experiments, through cleavage of an apical substrate and not through the generation of a soluble factor. Taken together, our findings demonstrate the importance of MMP-7 secretion in relation to its mechanism of action when expressed in a polarized epithelium.     

A novel method for isolation of membrane proteins: a baculovirus surface display system

We have developed a novel baculovirus surface display (BVSD) system for the isolation of membrane proteins. We expressed a reporter gene that encoded hemagglutinin gene fused in frame with the signal peptide and transmembrane domain of the baculovirus gp64 protein, which is displayed on the surface of BmNPV virions. The expression of this fusion protein on the virion envelope allowed us to develop two methods for isolating membrane proteins. In the first method, we isolated proteins directly from the envelope of budding BmNPV virions. In the second method, we isolated proteins from cellular membranes that had disintegrated due to viral egress. We isolated 6756 proteins. Of these, 1883 have sequence similarities to membrane proteins and 1550 proteins are homologous to known membrane proteins. This study indicates that membrane proteins can be effectively isolated using our BVSD system. Using an analogous method, membrane proteins can be isolated from other eukaryotic organisms, including human beings, by employing a host cell-specific budding virus.     

Sphingolipid transport from the trans-Golgi network to the apical surface in permeabilized MDCK cells

We have measured the transport of de novo synthesized fluorescent analogs of sphingomyelin and glucosylceramide from the trans-Golgi network (TGN) to the apical membrane in basolaterally permeabilized Madin-Darby canine kidney (MDCK) cells. Sphingolipid transport was temperature, ATP and cytosol dependent. Introduction of bovine serum albumin (BSA), which binds fluorescent sphingolipid monomer, into the permeabilized cells, did not affect lipid transport to the apical membrane. Both fluorescent sphingomyelin and glucosylceramide analogs were localized to the lumenal bilayer leaflet of isolated TGN-derived vesicles. These results strongly suggest that both sphingolipids are transported from the TGN to the apical membrane via vesicular traffic.     

A novel and efficient method for the isolation and purification of polysaccharides from lily bulbs by Saccharomyces cerevisiae fermentation

A water-soluble polysaccharide from lily bulbs was isolated and purified by Saccharomyces cerevisiae fermentation. Proteins present in lily bulb extract were removed by extracellular proteases secreted by S. cerevisiae during fermentation. This novel method differs from traditional protein removal methods. A suitable yeast strain was selected. Culture conditions were optimized. Response surface methodology (RSM) was utilized to evaluate the effects of variables on the lily polysaccharide (LP) yield and the protein removal ratio (PRR). The results of applying RSM revealed that the optimum fermentation conditions were 87.5 g L⁻¹ lily bulb powder, pH 5.6, and temperature 27.9 °C. When lily bulb extract was cultured with S. cerevisae under optimum conditions, the LP yield and the PRR were 6.56% and 91.46%, respectively. These values are in close agreement with the value predicted by the model. The resulting LP curding was further purified by DEAE Sepharose Fast Flow chromatography after isolation by alcohol precipitation post-fermentation. DEAE chromatography resulted in a fraction, LP-1 (yield: 4.46%) with a molecular weight of 65.0 kDa. LP-1 consisted of glucose and mannose in a molar ratio of 1:1.2.     

A novel rapid procedure for the isolation of outer membrane proteins from Campylobacter jejuni

A new molecular filtration based method for the recovery and fractionation of cell envelope fragments from Campylobacter jejuni has been developed. The process, which uses a novel combination of filtration and selective solubilization, offers major advantages over currently available methods. Inner and outer membranes associated with cell envelope fragments of Campylobacter jejuni, recovered onto a regenerated cellulose filter under 1 bar negative pressure, can be sequentially treated with Triton X-100 and Triton X-100/EDTA to yield a fraction principally composed of solubilised Outer Membrane Protein (OMP). The method is rapid, efficient and uses low cost easily available equipment to produce electrophoretic patterns and protein yields similar to the standard procedures used by previous workers.     

Maxi K+ channels from the apical membranes of rabbit oviduct epithelial cells

Large conductance (approximately 210 pS), K⁺-selective channels were identified in excised, insideout patches obtained from the apical membranes of both ciliated and nonciliated epithelial cells grown as monolayers from the primary culture of rabbit oviduct. The open probability of channels showing stable gating was increased at positive membrane potentials and was sensitive to the concentration of free calcium ions at the cytosolic surface of the patch ([Ca²⁺] _i ). In these respects, the channel resembled maxi K⁺ channels found in a number of other cell types. The distributions of dwell-times in the open state were most consistently described by two exponential components. Four exponential components were fitted to the distributions of dwelltimes in the closed state. Depolarizations and [Ca²⁺] _i increases had similar effects on the distribution of open dwell-times, causing increases in the two open time constants ( _o1 and _o2) and the fraction of events accounted for by the longer component of the distribution. In contrast, calcium ions and voltage had distinct effects on the distribution of closed dwelltimes. While the three shorter closed time constants ( _c1, _c2 and _c3) were reduced by depolarizing membrane potentials, increases in [Ca²⁺] _i caused decreases in the longer time constants ( _c3 and _c4). It is concluded that oviduct large conductance Ca²⁺-activated K⁺ channels can enter at least two major open states and four closed states.A.F.J. was supported by a research fellowship from the Japan Society for the Promotion of Science and received a grant for laboratory expenses from the Ministry of Education, Science and Culture, Japan. The authors wish to thank Dr. Shigetoshi Oiki for valuable discussion of the analysis of gating kinetics and Dr. Jeman Kim (Kyoto Pharmaceutical University) for making the transmission electron micrographs.     

Human cellular prion protein hPrPC is sorted to the apical membrane of epithelial cells

Propagation of the scrapie isoform of the prion protein (PrP(Sc)) depends on the expression of endogenous cellular prion (PrP(C)). During oral infection, PrP(Sc) propagates, by conversion of the PrP(C) to PrP(Sc), from the gastrointestinal tract to the nervous system. Intestinal epithelium could serve as the primary site for PrP(C) conversion. To investigate PrP(C) sorting in epithelia cells, we have generated both a green fluorescent protein (EGFP) or hemagglutinin (HA) tagged human PrP(C) (hPrP(C)). Combined molecular, biochemical, and single living polarized cell imaging characterizations suggest that hPrP(C) is selectively targeted to the apical side of Madin-Darby canine kidney (MDCKII) and of intestinal epithelia (Caco2) cells.     

A novel lectin from the sponge Haliclona cratera: isolation,characterization and biological activity

A lectin from the Adriatic sponge Haliclona cratera was purified by ion-exchange and gel chromatography. The molecular mass of the lectin is approximately 29 kDa. Purified lectin is rich in hydrophobic and basic amino acids and has an isoelectric point at pH 8.6. H. cratera lectin is relatively heat- and pH-stable. It agglutinates native and trypsinized, papainized and neuraminidase-treated human A, B, O, AB and sheep erythrocytes, and the hemagglutinating activity is independent of Ca(2+), Mn(2+) and Mg(2+) ions; D-galactose and N-acetyl-D-galactosamine are found to be moderate inhibitors of the activity. H. cratera lectin displays cytotoxic effect on HeLa and FemX cells and weak mitogenic effect on human T-lymphocytes pretreated with phytohemagglutinin (PHA).     

A simple and biosafe method for isolation of human umbilical vein endothelial cells

Human umbilical vein endothelial cells (HUVEC_S) are used as an irreplaceable tool for the study of vascular diseases. However, the technicians who isolate HUVECs are largely exposed to potential infectious threats. Here we report the development of a specialized instrument to protect researchers from known or unknown infectious agents when they operate on human umbilical cords. This instrument can be assembled by common laboratory supplies and adapted to accommodate umbilical cords of different lengths. When the cord is enclosed within the instrument, the risk of sample contamination and operator infection is greatly reduced. Using our instrument, endothelial cells were successfully isolated from human umbilical veins without contamination. The cells were verified by their cobblestone-like morphology and by immunofluorescence staining (Factor VIII and CD31 positivity and α-SMA negativity). Our instrument simplifies and optimizes the cell extraction process, and most importantly elevates the biosafety to a higher level during the isolation of human umbilical vein endothelial cells.     

Prominin-2 is a cholesterol-binding protein associated with apical and basolateral plasmalemmal protrusions in polarized epithelial cells and released into urine

Prominin-2 is a pentaspan membrane glycoprotein structurally related to the cholesterol-binding protein prominin-1, which is expressed in epithelial and non-epithelial cells. Although prominin-1 expression is widespread throughout the organism, the loss of its function solely causes retinal degeneration. The finding that prominin-2 appears to be restricted to epithelial cells, such as those found in kidney tubules, raises the possibility that prominin-2 functionally substitutes prominin-1 in tissues other than the retina and provokes a search for a definition of its morphological and biochemical characteristics. Here, we have investigated, by using MDCK cells as an epithelial cell model, whether prominin-2 shares the biochemical and morphological properties of prominin-1. Interestingly, we have found that, whereas prominin-2 is not restricted to the apical domain like prominin-1 but is distributed in a non-polarized fashion between the apical and basolateral plasma membranes, it retains the main feature of prominin-1, i.e. its selective concentration in plasmalemmal protrusions; prominin-2 is confined to microvilli, cilia and other acetylated tubulin-positive protruding structures. Similar to prominin-1, prominin-2 is partly associated with detergent-resistant membranes in a cholesterol-dependent manner, suggesting its incorporation into membrane microdomains, and binds directly to plasma membrane cholesterol. Finally, prominin-2 is also associated with small membrane particles that are released into the culture media and found in a physiological fluid, i.e. urine. Together, these data show that all the characteristics of prominin-1 are shared by prominin-2, which is in agreement with a possible redundancy in their role as potential organizers of plasma membrane protrusions.     

Definition of distinct compartments in polarized Madin-Darby canine kidney (MDCK) cells for membrane-volume sorting, polarized sorting and apical recycling

Previous studies of fibroblasts have demonstrated that recycling of endocytic receptors occurs through a default mechanism of membrane-volume sorting. Epithelial cells require an additional level of polar membrane sorting, but there are conflicting models of polar sorting, some suggesting that it occurs in early endosomes, others suggesting it occurs in a specialized apical recycling endosome (ARE). The relationship between endocytic sorting to the lysosomal, recycling and transcytotic pathways in polarized cells was addressed by characterizing the endocytic itineraries of LDL, transferrin (Tf) and IgA, respectively, in polarized Madin-Darby canine kidney (MDCK) cells. Quantitative analyses of 3-dimensional images of living and fixed polarized cells demonstrate that endocytic sorting occurs sequentially. Initially internalized into lateral sorting endosomes, Tf and IgA are jointly sorted from LDL into apical and medical recycling endosomes, in a manner consistent with default sorting of membrane from volume. While Tf is recycled to the basolateral membrane from recycling endosomes, IgA is sorted to the ARE prior to apical delivery. Quantifications of the efficiency of sorting of IgA from Tf between the recycling endosomes and the ARE match biochemical measurements of transepithelial protein transport, indicating that all polar sorting occurs in this step. Unlike fibroblasts, rab11 is not associated with Tf recycling compartments in either polarized or glass-grown MDCK cells, rather it is associated with the compartments to which IgA is directed after sorting from Tf. These results complicate a suggested homology between the ARE and the fibroblast perinuclear recycling compartment and provide a framework that justifies previous conflicting models of polarized sorting.     

Establishment of a simple and effective isolation method for cyanophycin from recombinant Saccharomyces cerevisiae

An efficient, time-saving, and cost-effective method for isolation of the polyamide cyanophycin from recombinant Saccharomyces cerevisiae was established. Due to its simple procedure, this isolation method may be also applicable at industrial scale and also to other intracellular compounds in this yeast. Production of cyanophycin gained preferential interest in the past, as degradation products thereof are of pharmaceutical and technical interest. Recently, it was shown that Saccharomyces cerevisiae represents a putative candidate for cyanophycin synthesis at industrial scale. For identification of optimal isolation procedures, several parameters such as heat stress, freeze drying, and freeze/thaw cycles of transgenic yeast cells were compared for their effectiveness of cyanophycin isolation. Additionally, optimal resuspension solutions for the applied cells and minimal required materials or chemicals were determined to make the process most environmentally and economically friendly. Maximal cyanophycin granule polypeptide yields of 21% (w/w) were obtained after incubation of dry cells at 70 °C or 80 °C and precipitation of the polymer with two volumes of ethanol.     

A simple process for the isolation of epithelial cells from bacteria-contaminated samples using anchoring molecules

A simple and efficient tool to isolate epithelial cells from bacteria-contaminated samples has been developed using two different microparticles functionalized with chemical molecules. The epithelial cells could be captured simply by biocompatible anchors for membranes (BAM), consisting of poly(ethylene glycol) functionalized with oleyl-chain-conjugated NHS (N-hydroxysuccinimide) on glass microparticles, whereas bacteria were adsorbed on 3-aminopropyltrimethoxysilane (ATPS)-functionalized magnetic microparticles. In the case of samples highly contaminated with bacteria, epithelial cells were not isolated successfully by both of the single BAM- and antibody-functionalized microparticles. Therefore, serial isolation steps of these two different chemical functionalized microparticles were introduced. The concentration of bacteria was decreased dramatically by using APTS-functionalized magnetic particles prior to the isolation of epithelial cells by BAM microparticles. With these serial processes, successful isolation of epithelial cells was achieved from bacteria-contaminated epithelial samples. The applicability of this method was verified with bacteria-contaminated intestinal samples biopsied from a BALB/C mouse for primary cell cultivation.     

No entry for TAT(44-57) into liposomes and intact MDCK cells: novel approach to study membrane permeation of cell-penetrating peptides

Cell penetrating peptides (CPPs) have been postulated to carry macromolecules across cell plasma membranes without the need of receptors, transporters, endocytosis or any energy-consuming mechanism.We developed an assay to study lipid bilayer permeation of CPPs. HIV-1 TAT peptides were conjugated to N-(4-carboxy-3-hydroxyphenyl)maleimide (SAM) and incubated with Tb³⁺-containing liposomes. Upon chelation of Tb³⁺ by an aromatic carboxylic acid, the fluorescence of Tb³⁺ increases many fold. The CPP TAT(44-57)-SAM and TAT(37-53)-SAM, as a negative control, were unable to enter liposomes consisting of phosphatidylcholine (PC) or a mix of PC, negatively charged lipids and cholesterol.In parallel, cell entry of fluorescein-labeled TAT peptides was studied using confocal laser scanning microscopy (CLSM). TAT(44-57)-fluorescein did not enter Madin Darby canine kidney (MDCK) cells with intact plasma membranes but accumulated at their basal side. Only cells with impaired plasma membranes, as identified by nuclear staining with ethidium homodimer-1 (EthD-1), showed accumulation of TAT(44-57).Our findings change the perspectives of the potential use of TAT peptides as carriers for intracellular targeting. SAM- and fluorescein-labeled TAT(44-57) cannot penetrate lipid bilayers and intact plasma membranes of MDCK cells, respectively.     

Distinct roles of Rab3B and Rab13 in the polarized transport of apical,basolateral, and tight junctional membrane proteins to the plasma membrane

Regulated transport of proteins to distinct plasma membrane domains is essential for the establishment and maintenance of cell polarity in all eukaryotic cells. The Rab family small G proteins play a crucial role in determining the specificity of vesicular transport pathways. Rab3B and Rab13 localize to tight junction in polarized epithelial cells and cytoplasmic vesicular structures in non-polarized fibroblasts, but their functions are poorly understood. Here we examined their roles in regulating the cell-surface transport of apical p75 neurotrophin receptor (p75NTR), basolateral low-density lipoprotein receptor (LDLR), and tight junctional Claudin-1 using transport assay in non-polarized fibroblasts. Overexpression of Rab3B mutants inhibited the cell-surface transport of LDLR, but not p75NTR and Claudin-1. In contrast, overexpression of Rab13 mutants impaired the transport of Claudin-1, but not LDLR and p75NTR. These results suggest that Rab3B and Rab13 direct the cell-surface transport of LDLR and Claudin-1, respectively, and may contribute to epithelial polarization.     

GM1 and GM3 gangliosides highlight distinct lipid microdomains within the apical domain of epithelial cells

The apical domain of epithelial cells is composed of distinct subdomains such as microvilli, primary cilia and a non-protruding region. Using the cholesterol-binding protein prominin-1 as a specific marker of plasma membrane protrusions we have previously proposed the co-existence of different cholesterol-based lipid microdomains (lipid rafts) within the apical domain [R?per, K., Corbeil, D. and Huttner, W.B. (2000), Retention of prominin in microvilli reveals distinct cholesterol-based lipid microdomains in the apical plasma membrane. Nat. Cell Biol. 2, 582-592]. To substantiate the hypothesis that the microvillar plasma membrane subdomains contain a distinct set of lipids compared to the planar portion we have investigated the distribution of prominin-1 and two raft-associated gangliosides GM(1) and GM(3) by fluorescence microscopy. GM(1) was found to co-localize with prominin-1 on microvilli whereas GM(3) was segregated from there suggesting its localization in the planar region. Regarding the primary cilium, overlapping fluorescent signals of GM(1) or GM(3) and prominin-1 were observed. Thus, our data demonstrate that specific ganglioside-enriched rafts are found in different apical subdomains and reveal that two plasma membrane protrusions with different structural bases (actin for the microvillus and tubulin for the cilium) are composed of distinct types of lipid.