scholarly journals A plate reader-based method for cell water permeability measurement

2010 ◽  
Vol 298 (1) ◽  
pp. F224-F230 ◽  
Author(s):  
R. A. Fenton ◽  
H. B. Moeller ◽  
S. Nielsen ◽  
B. L. de Groot ◽  
M. Rützler
Keyword(s):  
Cell Volume ◽  
Water Permeability ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Phosphorylation Site ◽  
Type I ◽  
Volume Changes ◽  
Average Cell ◽  
Compound Libraries ◽  
Plate Reader

Cell volume and water permeability measurements in cultured mammalian cells are typically conducted under a light microscope. Many of the employed approaches are time consuming and not applicable to a study of confluent epithelial cell monolayers. We present here an adaptation of a calcein-quenching-based approach for a plate reader. A standard curve of fluorescence intensities at equilibrium has been recorded, following a shift from 285 mosmol/kgH2O to a series of altered extracellular osmolyte concentrations, ranging from final concentrations of 185 to 585 mosmol/kgH2O, by changing buffer d-mannitol concentrations. Similarly, according average cell volumes have been measured in suspension in a Coulter counter (particle-sizing device). Based on these measurements, we have derived an equation that facilitates the modeling of cell volume changes based on fluorescence intensity changes. We have utilized the method to study the role of a carboxyl-terminus aquaporin (AQP)-2 phosphorylation site, which is known to affect AQP2 membrane trafficking, in heterologous type I Madin-Darby canine kidney cells. We find that water permeability in cells expressing phosphorylation site mutants was in the following order: AQP2-S256D > AQP2 wild-type > AQP2-S256A. We propose that the method can be applied to study AQP function and more generally to study cell volume changes in adherent cell lines. Furthermore, it should be adaptable for AQP inhibitor screening in chemical compound libraries.

scholarly journals A Serine Residue in ClC-3 Links Phosphorylation–Dephosphorylation to Chloride Channel Regulation by Cell Volume

1999 ◽  
Vol 113 (1) ◽  
pp. 57-70 ◽  
Author(s):  
Dayue Duan ◽  
Suzanne Cowley ◽  
Burton Horowitz ◽  
Joseph R. Hume
Keyword(s):  
Cell Volume ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Phosphorylation Site ◽  
Cardiac Cells ◽  
Site Directed Mutagenesis ◽  
Cell Swelling ◽  
Transport Systems ◽  
Serine Residue ◽  
Channel Regulation

In many mammalian cells, ClC-3 volume-regulated chloride channels maintain a variety of normal cellular functions during osmotic perturbation. The molecular mechanisms of channel regulation by cell volume, however, are unknown. Since a number of recent studies point to the involvement of protein phosphorylation/dephosphorylation in the control of volume-regulated ionic transport systems, we studied the relationship between channel phosphorylation and volume regulation of ClC-3 channels using site-directed mutagenesis and patch-clamp techniques. In native cardiac cells and when overexpressed in NIH/3T3 cells, ClC-3 channels were opened by cell swelling or inhibition of endogenous PKC, but closed by PKC activation, phosphatase inhibition, or elevation of intracellular Ca2+. Site-specific mutational studies indicate that a serine residue (serine51) within a consensus PKC-phosphorylation site in the intracellular amino terminus of the ClC-3 channel protein represents an important volume sensor of the channel. These results provide direct molecular and pharmacological evidence indicating that channel phosphorylation/dephosphorylation plays a crucial role in the regulation of volume sensitivity of recombinant ClC-3 channels and their native counterpart, ICl.vol.

Monitoring of segmental intra- and extracellular volume changes using electrical impedance spectroscopy

1993 ◽  
Vol 74 (5) ◽  
pp. 2180-2187 ◽  
Author(s):  
D. C. Sasser ◽  
W. A. Gerth ◽  
Y. C. Wu
Keyword(s):  
Impedance Spectroscopy ◽  
Cell Volume ◽  
Equivalent Circuit ◽  
Electrical Impedance ◽  
Bioelectrical Impedance ◽  
Electrical Impedance Spectroscopy ◽  
Volume Changes ◽  
Average Cell ◽  
Fluid Shifts ◽  
Concentration Changes

Osmotically induced cellular volume changes in the perfused rat hindlimb were used to validate the use of bioelectrical impedance spectroscopy as a method for observing fluid shifts between the intracellular and extracellular spaces. Electrical impedance spectra were measured as cell volumes were manipulated by perfusion with Krebs-Henseleit solutions having different concentrations of NaCl. A simple equivalent circuit model of current conduction through the monitored tissue was fit to each measured spectrum to obtain segmental values of the equivalent intracellular resistance, membrane capacitance, and extracellular resistance. These parameters are theoretically governed by variations in the average cell volume fraction and ionic concentrations in the intra- and extracellular fluid spaces. In accord with this theoretical dependence, the parameters changed systematically and reversibly in conformance with both the magnitudes and directions of the perfusate concentration changes and the resultant cell volume changes. Results indicate that bioelectrical impedance spectroscopy, coupled with computer-aided equivalent circuit analysis, can be used to monitor segmental intercompartmental fluid shifts at minute-by-minute resolution.

scholarly journals Self-DNA Sensing by cGAS-STING and TLR9 in Autoimmunity: Is the Cytoskeleton in Control?

2021 ◽  
Vol 12 ◽  
Author(s):  
Roberto Amadio ◽  
Giulia Maria Piperno ◽  
Federica Benvenuti
Keyword(s):  
Autoimmune Diseases ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Defense Mechanism ◽  
Type I Interferons ◽  
Actin Dynamics ◽  
Primary Immune Deficiency ◽  
Type I ◽  
Dna Sensing

Modified or misplaced DNA can be recognized as a danger signal by mammalian cells. Activation of cellular responses to DNA has evolved as a defense mechanism to microbial infections, cellular stress, and tissue damage, yet failure to control this mechanism can lead to autoimmune diseases. Several monogenic and multifactorial autoimmune diseases have been associated with type-I interferons and interferon-stimulated genes (ISGs) induced by deregulated recognition of self-DNA. Hence, understanding how cellular mechanism controls the pathogenic responses to self-nucleic acid has important clinical implications. Fine-tuned membrane trafficking and cellular compartmentalization are two major factors that balance activation of DNA sensors and availability of self-DNA ligands. Intracellular transport and organelle architecture are in turn regulated by cytoskeletal dynamics, yet the precise impact of actin remodeling on DNA sensing remains elusive. This review proposes a critical analysis of the established and hypothetical connections between self-DNA recognition and actin dynamics. As a paradigm of this concept, we discuss recent evidence of deregulated self-DNA sensing in the prototypical actin-related primary immune deficiency (Wiskott-Aldrich syndrome). We anticipate a broader impact of actin-dependent processes on tolerance to self-DNA in autoimmune disorders.

PKC-ε regulates basolateral endocytosis in human T84 intestinal epithelia: role of F-actin and MARCKS

1999 ◽  
Vol 277 (6) ◽  
pp. C1239-C1249 ◽  
Author(s):  
Jaekyung Cecilia Song ◽  
Bruce J. Hrnjez ◽  
Omid C. Farokhzad ◽  
Jeffrey B. Matthews
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Basolateral Membrane ◽  
Phosphorylation Site ◽  
Fluid Phase ◽  
Cytochalasin D ◽  
Pkc Inhibitor ◽  
Intestinal Epithelia ◽  
Stimulation Of

Protein kinase C (PKC) and the actin cytoskeleton are critical effectors of membrane trafficking in mammalian cells. In polarized epithelia, the role of these factors in endocytic events at either the apical or basolateral membrane is poorly defined. In the present study, phorbol 12-myristate 13-acetate (PMA) and other activators of PKC selectively enhanced basolateral but not apical fluid-phase endocytosis in human T84 intestinal epithelia. Stimulation of basolateral endocytosis was blocked by the conventional and novel PKC inhibitor Gö-6850, but not the conventional PKC inhibitor Gö-6976, and correlated with translocation of the novel PKC isoform PKC-ε. PMA treatment induced remodeling of basolateral F-actin. The actin disassembler cytochalasin D stimulated basolateral endocytosis and enhanced stimulation of endocytosis by PMA, whereas PMA-stimulated endocytosis was blocked by the F-actin stabilizers phalloidin and jasplakinolide. PMA induced membrane-to-cytosol redistribution of the F-actin cross-linking protein myristoylated alanine-rich C kinase substrate (MARCKS). Cytochalasin D also induced MARCKS translocation and enhanced PMA-stimulated translocation of MARCKS. A myristoylated peptide corresponding to the phosphorylation site domain of MARCKS inhibited both MARCKS translocation and PMA stimulation of endocytosis. MARCKS translocation was inhibited by Gö-6850 but not Gö-6976. The results suggest that a novel PKC isoform, likely PKC-ε, stimulates basolateral endocytosis in model epithelia by a mechanism that involves F-actin and MARCKS.

scholarly journals Osmotically Induced Cell Volume Changes Alter Anterograde and Retrograde Transport, Golgi Structure, and COPI Dissociation

1999 ◽  
Vol 10 (5) ◽  
pp. 1445-1462 ◽  
Author(s):  
Tina H. Lee ◽  
Adam D. Linstedt
Keyword(s):  
Cell Volume ◽  
Mammalian Cells ◽  
Brefeldin A ◽  
Retrograde Transport ◽  
Volume Changes ◽  
Hypertonic Medium ◽  
Osmotic Stress Response ◽  
Mediate Cell ◽  
Independent Effects ◽  
Er Export

Physiological conditions that impinge on constitutive traffic and affect organelle structure are not known. We report that osmotically induced cell volume changes, which are known to occur under a variety of conditions, rapidly inhibited endoplasmic reticulum (ER)-to-Golgi transport in mammalian cells. Both ER export and ER Golgi intermediate compartment (ERGIC)-to-Golgi trafficking steps were blocked, but retrograde transport was active, and it mediated ERGIC and Golgi collapse into the ER. Extensive tubulation and relatively rapid Golgi resident redistribution were observed under hypo-osmotic conditions, whereas a slower redistribution of the same markers, without apparent tubulation, was observed under hyperosmotic conditions. The osmotic stress response correlated with the perturbation of COPI function, because both hypo- and hyperosmotic conditions slowed brefeldin A-induced dissociation of βCOP from Golgi membranes. Remarkably, Golgi residents reemerged after several hours of sustained incubation in hypotonic or hypertonic medium. Reemergence was independent of new protein synthesis but required PKC, an activity known to mediate cell volume recovery. Taken together these results indicate the existence of a coupling between cell volume and constitutive traffic that impacts organelle structure through independent effects on anterograde and retrograde flow and that involves, in part, modulation of COPI function.

scholarly journals Plasma Membrane Water Permeability of Cultured Cells and Epithelia Measured by Light Microscopy with Spatial Filtering

1997 ◽  
Vol 110 (3) ◽  
pp. 283-296 ◽  
Author(s):  
Javier Farinas ◽  
Malea Kneen ◽  
Megan Moore ◽  
A.S. Verkman
Keyword(s):  
Light Microscopy ◽  
Cell Volume ◽  
Water Permeability ◽  
Signal Intensity ◽  
Spatial Filtering ◽  
Water Channels ◽  
Molecular Water ◽  
Toad Urinary Bladder ◽  
Volume Changes ◽  
Cell Layers

A method was developed to measure the osmotic water permeability (Pf) of plasma membranes in cell layers and applied to cells and epithelia expressing molecular water channels. It was found that the integrated intensity of monochromatic light in a phase contrast or dark field microscope was dependent on relative cell volume. For cells of different size and shape (Sf9, MDCK, CHO, A549, tracheal epithelia, BHK), increased cell volume was associated with decreased signal intensity; generally the signal decreased 10–20% for a twofold increase in cell volume. A theory relating signal intensity to relative cell volume was developed based on spatial filtering and changes in optical path length associated with cell volume changes. Theory predictions were confirmed by signal measurements of cell layers bathed in solutions of various osmolarities and refractive indices. The excellent signal-to-noise ratio of the transmitted light detection permitted measurement of cell volume changes of <1%. The method was applied to characterize transfected cells and tissues that natively express water channels. Pf in control Chinese hamster ovary cells was low (0.0012 cm/s at 23°C) and increased more than fourfold upon stable transfection with aquaporins 1, 2, 4, or 5. Pf in apical and basolateral membranes in polarized epithelial cells grown on porous supports was measured. Pfbl and Pfap were 0.0011 and 0.0024 cm/s (MDCK cells), and 0.0039 and 0.0052 cm/s (human tracheal cells) at 23°C. In intact toad urinary bladder, basolateral Pf was 0.036 cm/s and apical membrane Pf after vasopressin stimulation was 0.025 cm/s at 23°C. The results establish light microscopy with spatial filtering as a technically simple and quantitative method to measure water permeability in cell layers and provide the first measurement of the apical and basolateral membrane permeabilities of several important epithelial cell types.

Liver cell hydration and integrin signaling

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Michele Bonus ◽  
Dieter Häussinger ◽  
Holger Gohlke
Keyword(s):  
Cell Volume ◽  
Liver Cell ◽  
Cell Function ◽  
The Other ◽  
Signaling Cascades ◽  
Integrin Signaling ◽  
Volume Changes ◽  
The One ◽  
And Oxidative Stress

Abstract Liver cell hydration (cell volume) is dynamic and can change within minutes under the influence of hormones, nutrients, and oxidative stress. Such volume changes were identified as a novel and important modulator of cell function. It provides an early example for the interaction between a physical parameter (cell volume) on the one hand and metabolism, transport, and gene expression on the other. Such events involve mechanotransduction (osmosensing) which triggers signaling cascades towards liver function (osmosignaling). This article reviews our own work on this topic with emphasis on the role of β1 integrins as (osmo-)mechanosensors in the liver, but also on their role in bile acid signaling.

scholarly journals ADP-Ribosylation Factor (ARF) Interaction Is Not Sufficient for Yeast GGA Protein Function or Localization

2002 ◽  
Vol 13 (9) ◽  
pp. 3078-3095 ◽  
Author(s):  
Annette L. Boman ◽  
Paul D. Salo ◽  
Melissa J. Hauglund ◽  
Nicole L. Strand ◽  
Shelly J. Rensink ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Carboxypeptidase Y ◽  
Minor Role ◽  
Temperature Sensitive ◽  
Adp Ribosylation ◽  
And Function ◽  
Adp Ribosylation Factor

Golgi-localized γ-ear homology domain, ADP-ribosylation factor (ARF)-binding proteins (GGAs) facilitate distinct steps of post-Golgi traffic. Human and yeast GGA proteins are only ∼25% identical, but all GGA proteins have four similar domains based on function and sequence homology. GGA proteins are most conserved in the region that interacts with ARF proteins. To analyze the role of ARF in GGA protein localization and function, we performed mutational analyses of both human and yeast GGAs. To our surprise, yeast and human GGAs differ in their requirement for ARF interaction. We describe a point mutation in both yeast and mammalian GGA proteins that eliminates binding to ARFs. In mammalian cells, this mutation disrupts the localization of human GGA proteins. Yeast Gga function was studied using an assay for carboxypeptidase Y missorting and synthetic temperature-sensitive lethality between GGAs andVPS27. Based on these assays, we conclude that non-Arf-binding yeast Gga mutants can function normally in membrane trafficking. Using green fluorescent protein-tagged Gga1p, we show that Arf interaction is not required for Gga localization to the Golgi. Truncation analysis of Gga1p and Gga2p suggests that the N-terminal VHS domain and C-terminal hinge and ear domains play significant roles in yeast Gga protein localization and function. Together, our data suggest that yeast Gga proteins function to assemble a protein complex at the late Golgi to initiate proper sorting and transport of specific cargo. Whereas mammalian GGAs must interact with ARF to localize to and function at the Golgi, interaction between yeast Ggas and Arf plays a minor role in Gga localization and function.

Sterol and lipid trafficking in mammalian cells

2006 ◽  
Vol 34 (3) ◽  
pp. 335-339 ◽  
Author(s):  
F.R. Maxfield ◽  
M. Mondal
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Vesicular Transport ◽  
Cholesterol Content ◽  
Cellular Functions ◽  
Lipid Trafficking ◽  
Sterol Transport ◽  
A Cell ◽  
Asymmetrically Distributed

The pathways involved in the intracellular transport and distribution of lipids in general, and sterols in particular, are poorly understood. Cholesterol plays a major role in modulating membrane bilayer structure and important cellular functions, including signal transduction and membrane trafficking. Both the overall cholesterol content of a cell, as well as its distribution in specific organellar membranes are stringently regulated. Several diseases, many of which are incurable at present, have been characterized as results of impaired cholesterol transport and/or storage in the cells. Despite their importance, many fundamental aspects of intracellular sterol transport and distribution are not well understood. For instance, the relative roles of vesicular and non-vesicular transport of cholesterol have not yet been fully determined, nor are the non-vesicular transport mechanisms well characterized. Similarly, whether cholesterol is asymmetrically distributed between the two leaflets of biological membranes, and if so, how this asymmetry is maintained, is poorly understood. In this review, we present a summary of the current understanding of these aspects of intracellular trafficking and distribution of lipids, and more specifically, of sterols.

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