scholarly journals Viscoelasticity of basal plasma membranes and cortices derived from MDCK II cells

2021 ◽  
Author(s):  
Andreas Janshoff
Keyword(s):  
Mechanical Properties ◽  
Epithelial Cells ◽  
Plasma Membranes ◽  
Basolateral Membrane ◽  
Scaling Law ◽  
Focal Adhesions ◽  
Living Cells ◽  
The Body ◽  
Basolateral Membranes ◽  
Apical Side

In mature epithelial cells, however, cells adhere to one another through tight junctions, adherens junctions and desmosomes thereby displaying a pronounced apical-basal polarity. In vivo, the apical membrane has a larger surface area and faces the outer surface of the body or the lumen of internal cavities, whereas the basolateral membrane is oriented on the side away from the lumen and forms focal adhesions with the extracellular matrix. The mechanical properties of cells are largely determined by the architecture and dynamics of their viscoelastic cortex, which consists of a contractile, cross-linked actin mesh attached to the plasma membrane via linker proteins. Measuring the mechanical properties of adherent, polarized epithelial cells is usually limited to the upper, i.e., apical side of the cells due to their accessibility on culture dishes. Moreover, contributions from the cell interior comprising various filament types, organelles, and the crowded cytoplasm usually impede examination of the cortex alone. Here, we investigate the viscoelastic properties of basolateral membranes derived from polarized MDCK II epithelia in response to external deformation and compare them to living cells probed at the apical side. Therefore, we grew MDCK II cells on porous surfaces to confluency and removed the upper cell body by sandwich cleavage. The free-standing, defoliated cortices were subject to force indentation and relaxation experiments permitting a precise assessment of cortical viscoelasticity. A new theoretical framework to describe the force cycles is developed and applied to obtain the time-dependent area compressibility modulus of cell cortices from adherent cells. Compared to the viscoelastic response of living cells the basolateral membranes are substantially less fluid and stiffer but obey to the same universal scaling law if excess area is taken into account.

scholarly journals Putative linear motifs mediate the trafficking to apical and basolateral membranes

2020 ◽  
Author(s):  
Laszlo Dobson ◽  
András Zeke ◽  
Levente Szekeres ◽  
Tamás Langó ◽  
Gábor Tusnády
Keyword(s):  
Epithelial Cells ◽  
Drug Transport ◽  
Basolateral Membrane ◽  
Transmembrane Proteins ◽  
Transport Processes ◽  
Membrane Domains ◽  
Protein Distribution ◽  
Basolateral Membranes ◽  
Linear Motifs ◽  
Cellular Components

AbstractCell polarity refers to the asymmetric organisation of cellular components in various cells. Epithelial cells are the best known examples of polarized cells, featuring apical and basolateral membrane domains. Despite huge efforts, the exact rules governing the protein distribution in such domains are still elusive. In this study we examined linear motifs accumulating in these parts and based on the results we prepared ‘Classical’ and Convolutional Neural Networks to classify human transmembrane proteins localizing into apical/basolateral membranes. Asymmetric expression of drug transporters results in vectorial drug transport, governing the pharmacokinetics of numerous substances, yet the data on how proteins are sorted in epithelial cells is very scattered. The provided dataset may offer help to experimentalists to characterize novel molecular targets to regulate transport processes more precisely.

scholarly journals Water and solute permeabilities of medullary thick ascending limb apical and basolateral membranes

1998 ◽  
Vol 274 (3) ◽  
pp. F453-F462 ◽  
Author(s):  
Rickey Rivers ◽  
Anne Blanchard ◽  
Dominique Eladari ◽  
Francois Leviel ◽  
Michel Paillard ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Basolateral Membrane ◽  
Thick Ascending Limb ◽  
Membrane Structures ◽  
Membrane Unit ◽  
Small Surface ◽  
Basolateral Membranes ◽  
Medullary Thick Ascending Limb ◽  
Osmotic Water

The medullary thick ascending limb (MTAL) reabsorbs solute without water and concentrates [Formula: see text] in the interstitium without a favorable pH gradient, activities which require low water and NH3 permeabilities. The contributions of different apical and basolateral membrane structures to these low permeabilities are unclear. We isolated highly purified apical and basolateral MTAL plasma membranes and measured, by stopped-flow fluorometry, their permeabilities to water, urea, glycerol, protons, and NH3. Osmotic water permeability at 20°C averaged 9.4 ± 0.8 × 10−4 cm/s for apical and 11.9 ± 0.5 × 10−4cm/s for basolateral membranes. NH3 permeabilities at 20°C averaged 0.0023 ± 0.00035 and 0.0035 ± 0.00080 cm/s for apical and basolateral membranes, respectively. These values are consistent with those obtained in isolated perfused tubules and can account for known aspects of MTAL function in vivo. Because the apical and basolateral membrane unit permeabilities are similar, the ability of the apical membrane to function as the site of barrier function arises from its very small surface area when compared with the highly redundant basolateral membrane.

Selective permeability barrier to urea in shark rectal gland

2005 ◽  
Vol 289 (1) ◽  
pp. F83-F89 ◽  
Author(s):  
Joshua D. Zeidel ◽  
John C. Mathai ◽  
John D. Campbell ◽  
Wily G. Ruiz ◽  
Gerard L. Apodaca ◽  
...  
Keyword(s):  
Activation Energy ◽  
Epithelial Cells ◽  
Water Flow ◽  
Water Permeability ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Water Flux ◽  
Rectal Gland ◽  
Basolateral Membranes ◽  
Urea Permeability

Elasmobranchs such as the dogfish shark Squalus acanthius achieve osmotic homeostasis by maintaining urea concentrations in the 300- to 400-mM range, thus offsetting to some degree ambient marine osmolalities of 900–1,000 mosmol/kgH2O. These creatures also maintain salt balance without losing urea by secreting a NaCl-rich (500 mM) and urea-poor (18 mM) fluid from the rectal gland that is isotonic with the plasma. The composition of the rectal gland fluid suggests that its epithelial cells are permeable to water and not to urea. Because previous work showed that lipid bilayers that permit water flux do not block flux of urea, we reasoned that the plasma membranes of rectal gland epithelial cells must either have aquaporin water channels or must have some selective barrier to urea flux. We therefore isolated apical and basolateral membranes from shark rectal glands and determined their permeabilities to water and urea. Apical membrane fractions were markedly enriched for Na-K-2Cl cotransporter, whereas basolateral membrane fractions were enriched for Na-K-ATPase. Basolateral membrane osmotic water permeability (Pf) averaged 4.3 ± 1.3 × 10−3 cm/s, whereas urea permeability averaged 4.2 ± 0.8 × 10−7 cm/s. The activation energy for water flow averaged 16.4 kcal/mol. Apical membrane Pf averaged 7.5 ± 1.6 × 10−4 cm/s, and urea permeability averaged 2.2 ± 0.4 × 10−7 cm/s, with an average activation energy for water flow of 18.6 kcal/mol. The relatively low water permeabilities and high activation energies argue strongly against water flux via aquaporins. Comparison of membrane water and urea permeabilities with those of artificial liposomes and other isolated biological membranes indicates that the basolateral membrane urea permeability is fivefold lower than would be anticipated for its water permeability. These results indicate that the rectal gland maintains a selective barrier to urea in its basolateral membranes.

Airway epithelial tight junctions and binding and cytotoxicity ofPseudomonas aeruginosa

1999 ◽  
Vol 277 (1) ◽  
pp. L204-L217 ◽  
Author(s):  
Alfred Lee ◽  
Dar Chow ◽  
Brian Haus ◽  
Wanru Tseng ◽  
David Evans ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tight Junctions ◽  
Propidium Iodide ◽  
Time Course ◽  
Living Cells ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Basolateral Membranes ◽  
Free Edges

The role of tight junctions in the binding and cytoxicity of Pseudomonas aeruginosato apical or basolateral membranes of lung airway epithelial cells was tested with fluorescence microscopy on living cells. Binding of noncytotoxic P. aeruginosa strain O1 was assessed with P. aeruginosa that expressed green fluorescent protein. Binding of cytotoxic P. aeruginosa strain 6206 was assessed with FITC-labeled P. aeruginosa; cytotoxicity was determined from nuclear uptake of the impermeant dye propidium iodide. The role of direct contact of P. aeruginosa to epithelial cells was tested with filters with small (0.45-μm) or large (2.0-μm) pores. High transepithelial resistance ( Rt) Calu-3 and cultured bovine tracheal monolayers ( Rt> 1,000 Ω ⋅ cm2) bound P. aeruginosa very infrequently (<1 P. aeruginosa/100 cells) at the apical membrane, but P. aeruginosabound frequently to cells near “free edges” at holes, wounds, islands, and perimeters; cytotoxicity required direct interaction with basolateral membranes. Wounded high Rtepithelia showed increased P. aeruginosa binding and cytotoxicity at the free edges because basolateral membranes were accessible to P. aeruginosa, and dead and living cells near the wound bound P. aeruginosa similarly. Compared with high Rtepithelia, low RtCFT1 ( Rt= 100–200 Ω ⋅ cm2) and EGTA-treated Calu-3 monolayers were 25 times more susceptible to P. aeruginosa binding throughout the monolayer. Cytotoxicity to CFT1 cells (throughout the confluent monolayer, not only at the free edge) occurred after a shorter delay (0.25 vs. 2.0 h) and then five times faster than to Calu-3 cells, indicating that the time course of P. aeruginosa cytotoxicity may be limited by the rate of gaining access through tight junctions and that this occurred faster in low Rtthan in high Rtairway epithelia. Cytotoxicity appeared to occur in a sequential process that led first to a loss of fura 2 and a later uptake of propidium iodide. P. aeruginosa bound three times more frequently to regions between cells (tight junctions?) than to cell membranes of low RtCFT1 cells.

Na-K-Cl cotransport in nystatin-treated tracheal cells: regulation by isoproterenol, apical UTP, and [Cl]i

1994 ◽  
Vol 266 (5) ◽  
pp. C1440-C1452 ◽  
Author(s):  
M. Haas ◽  
D. G. McBrayer
Keyword(s):  
Epithelial Cells ◽  
Plasma Membranes ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Chloride Secretion ◽  
Airway Epithelial Cells ◽  
Tracheal Epithelial Cells ◽  
Cl Transport ◽  
Monovalent Ions ◽  
Stimulation Of

Chloride secretion in mammalian airway epithelia is stimulated by beta-adrenergic agonists via an adenosine 3',5'-cyclic monophosphate (cAMP)-dependent mechanism and by apical triphosphate nucleotides (ATP, UTP) via a cAMP-independent mechanism. Both types of secretagogues are known to stimulate apical Cl channels in airway cells; however, to maintain a stimulated rate of secretion, basolateral Cl influx via Na-K-Cl cotransport must be upregulated in parallel with apical Cl efflux. To examine the regulation of basolateral cotransport activity and its relationship to apical Cl efflux, we examined Cl transport in confluent primary cultures of dog tracheal epithelial cells treated with nystatin, an antibiotic that increases the permeability of plasma membranes to small monovalent ions, including Cl. By applying nystatin to the apical membrane of these cultures, apical Cl permeability could be increased to the point where transepithelial Cl transport is limited by transport across the basolateral membrane, which reflects primarily the activity of the cotransporter. In cultures of tracheal cells not treated with nystatin, transepithelial (basolateral-to-apical) 36Cl flux was increased two- to threefold by exposure to isoproterenol (5 microM, basolateral) or apical UTP (10 microM). Apical application of nystatin (400 units/ml) increased the basal level of transepithelial 36Cl flux approximately 1.5-fold and eliminated UTP stimulation of this flux, although an approximately twofold stimulation by isoproterenol persisted. Nystatin treatment also abolished UTP stimulation of saturable, basolateral [3H]bumetanide binding, a measure of functioning Na-K-Cl cotransporters in these cells; isoproterenol stimulation of binding was only mildly inhibited by nystatin treatment. Lowering intracellular Cl concentration ([Cl]i) by incubating cultures with apical media containing nystatin and reduced [Cl] (NO3 replacement) increased both basolateral-to-apical 36Cl flux and [3H]bumetanide binding in the absence of secretagogues or cell shrinkage. The results support our previous suggestion, based entirely on [3H]bumetanide binding [M. Haas, D. G. McBrayer, and J. R. Yankaskas. Am. J. Physiol. 264 (Cell. Physiol. 32): C189-C200, 1993], that UTP stimulation of basolateral Na-K-Cl cotransport in airway epithelial cells is entirely secondary to, and requires, an increase in apical Cl efflux, and further suggest that a decrease in [Cl]i may be a signal for cotransport activation in response to UTP. In addition, a cAMP-dependent cascade initiated by isoproterenol appears to directly stimulate the cotransporter.

scholarly journals Basolateral membrane K+ channels in renal epithelial cells

2012 ◽  
Vol 302 (9) ◽  
pp. F1069-F1081 ◽  
Author(s):  
Kirk L. Hamilton ◽  
Daniel C. Devor
Keyword(s):  
Epithelial Cells ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Gene Families ◽  
Proper Function ◽  
Major Function ◽  
Basolateral Membranes ◽  
Family Approach ◽  
Membrane Transport Proteins ◽  
Significant Disease

The major function of epithelial tissues is to maintain proper ion, solute, and water homeostasis. The tubule of the renal nephron has an amazingly simple structure, lined by epithelial cells, yet the segments (i.e., proximal tubule vs. collecting duct) of the nephron have unique transport functions. The functional differences are because epithelial cells are polarized and thus possess different patterns (distributions) of membrane transport proteins in the apical and basolateral membranes of the cell. K+ channels play critical roles in normal physiology. Over 90 different genes for K+ channels have been identified in the human genome. Epithelial K+ channels can be located within either or both the apical and basolateral membranes of the cell. One of the primary functions of basolateral K+ channels is to recycle K+ across the basolateral membrane for proper function of the Na+-K+-ATPase, among other functions. Mutations of these channels can cause significant disease. The focus of this review is to provide an overview of the basolateral K+ channels of the nephron, providing potential physiological functions and pathophysiology of these channels, where appropriate. We have taken a “K+ channel gene family” approach in presenting the representative basolateral K+ channels of the nephron. The basolateral K+ channels of the renal epithelia are represented by members of the KCNK, KCNJ, KCNQ, KCNE, and SLO gene families.

scholarly journals Vesicular Stomatitis Virus Glycoprotein Does Not Determine the Site of Virus Release in Polarized Epithelial Cells

2002 ◽  
Vol 76 (8) ◽  
pp. 4103-4107 ◽  
Author(s):  
Gert Zimmer ◽  
Klaus-Peter Zimmer ◽  
Ina Trotz ◽  
Georg Herrler
Keyword(s):  
Epithelial Cells ◽  
Vesicular Stomatitis Virus ◽  
Plasma Membranes ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Vesicular Stomatitis Virus Glycoprotein ◽  
Recombinant Viruses ◽  
Virus Glycoprotein ◽  
Vesicular Stomatitis ◽  
Polarized Epithelial Cells

ABSTRACT In polarized epithelial cells, the vesicular stomatitis virus glycoprotein is segregated to the basolateral plasma membrane, where budding of the virus takes place. We have generated recombinant viruses expressing mutant glycoproteins without the basolateral-membrane-targeting signal in the cytoplasmic domain. Though about 50% of the mutant glycoproteins were found at the apical plasma membranes of infected MDCK cells, the virus was still predominantly released at the basolateral membranes, indicating that factors other than the glycoprotein determine the site of virus budding.

P2 purinoceptors regulate calcium-activated chloride and fluid transport in 31EG4 mammary epithelia

2003 ◽  
Vol 284 (4) ◽  
pp. C897-C909 ◽  
Author(s):  
Sasha Blaug ◽  
Jodi Rymer ◽  
Stephen Jalickee ◽  
Sheldon S. Miller
Keyword(s):  
Membrane Potential ◽  
Fluid Transport ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Fluid Secretion ◽  
Extracellular Nucleotides ◽  
Basolateral Membranes ◽  
Apical Side ◽  
Mammary Epithelia

It has been reported that secretory mammary epithelial cells (MEC) release ATP, UTP, and UDP upon mechanical stimulation. Here we examined the physiological changes caused by ATP/UTP in nontransformed, clonal mouse mammary epithelia (31EG4 cells). In control conditions, transepithelial potential (apical side negative) and resistance were −4.4 ± 1.3 mV (mean ± SD, n = 12) and 517.7 ± 39.4 Ω · cm2, respectively. The apical membrane potential was −43.9 ± 1.7 mV, and the ratio of apical to basolateral membrane resistance ( R A/ R B) was 3.5 ± 0.2. Addition of ATP or UTP to the apical or basolateral membranes caused large voltage and resistance changes with an EC50 of ∼24 μM (apical) and ∼30 μM (basal). Apical ATP/UTP (100 μM) depolarized apical membrane potential by 17.6 ± 0.8 mV ( n = 7) and decreased R A/ R B by a factor of ≈3. The addition of adenosine to either side (100 μM) had no effect on any of these parameters. The ATP/UTP responses were partially inhibited by DIDS and suramin and mediated by a transient increase in free intracellular Ca2+ concentration (427 ± 206 nM; 15–25 μM ATP, apical; n = 6). This Ca2+ increase was blocked by cyclopiazonic acid, by BAPTA, or by xestospongin C. 31EG4 MEC monolayers also secreted or absorbed fluid in the resting state, and ATP or UTP increased fluid secretion by 5.6 ± 3 μl · cm−2 · h−1( n = 10). Pharmacology experiments indicate that 31EG4 epithelia contain P2Y2 purinoceptors on the apical and basolateral membranes, which upon activation stimulate apical Ca2+-dependent Cl channels and cause fluid secretion across the monolayer. This suggests that extracellular nucleotides could play a fundamental role in mammary gland paracrine signaling and the regulation of milk composition in vivo.

Imaging of filter-grown epithelial cells: MDCK Na(+)-H+ exchanger is basolateral

1991 ◽  
Vol 260 (4) ◽  
pp. C868-C876 ◽  
Author(s):  
S. O. Rosenberg ◽  
P. A. Berkowitz ◽  
L. Li ◽  
V. L. Schuster
Keyword(s):  
Epithelial Cells ◽  
Mdck Cells ◽  
Single Cells ◽  
Basolateral Membrane ◽  
Simple Method ◽  
Apical Side ◽  
Inverted Microscope ◽  
Permeable Supports ◽  
Darby Canine Kidney

We report a simple method for growing epithelial cells on permeable supports and for imaging the cells from the apical side using an inverted microscope. Madin-Darby canine kidney (MDCK) cells were either seeded onto the conventional side of Millipore-CM filters or onto “inverted” filters. The peak transepithelial resistance of confluent monolayers was the same with either system. Cells on inverted filters that were stained with various dyes and imaged by epifluorescence exhibited more distinct intercellular spaces, cell margins, nuclei, and subapical vesicles. We also perfused both sides of inverted filters with HCO3/CO2-free saline and measured intracellular pH (pHi) using 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) and digital imaging. The intrinsic buffer capacity of MDCK cells increased exponentially as pHi decreased. After an NH4Cl load, the H+ extrusion rate (JH+) in control saline was 2.42 +/- 0.62 mM/min. JH+ was completely blocked by 1 mM basolateral amiloride. In contrast, 1 mM apical amiloride had no effect. We conclude that 1) growth of epithelial cells on an inverted filter system is useful for the microspectrofluorimetric determination of pHi in single cells and for the imaging of apical/subapical structures, and 2) the Na(+)-H+ exchanger of MDCK cells is functionally polarized to the basolateral membrane.

scholarly journals How we discovered fluorescent speckle microscopy

2011 ◽  
Vol 22 (21) ◽  
pp. 3940-3942 ◽  
Author(s):  
E. D. Salmon ◽  
Clare M. Waterman
Keyword(s):  
Epithelial Cells ◽  
Fluorescence Intensity ◽  
Focal Adhesions ◽  
Time Lapse ◽  
Living Cells ◽  
Variable Intensity ◽  
Macromolecular Assemblies ◽  
Dynamic Assembly ◽  
Fluorescence Images

Fluorescent speckle microscopy (FSM) is a method for measuring the movements and dynamic assembly of macromolecular assemblies such as cytoskeletal filaments (e.g., microtubules and actin) or focal adhesions within large arrays in living cells or in preparations in vitro. The discovery of the method depended on recognizing the importance of unexpected fluorescence images of microtubules obtained by time-lapse recording of vertebrate epithelial cells in culture. In cells that were injected with fluorescent tubulin at ∼10% of the cytosol pool, microtubules typically appeared as smooth threads with a nearly constant fluorescence intensity. One day, when an unusually low concentration of fluorescent tubulin was injected into cells, the images from a sensitive cooled charge-coupled detector camera showed microtubules with an unusual “speckled” appearance—there were fluorescent dots with variable intensity and spacing along the microtubules. A first thought was that the speckles were an artifact. With further thought, we surmised that the speckles could be telling us something about stochastic association of tubulin dimers with the growing end of a microtubule. Numerous experiments confirmed the latter hypothesis. Subsequently the method we call FSM has proven to be very valuable. The speckles turned out not to be a meaningless artifact, but rather a serendipitous find.

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