Dielectric Spectroscopy Based Detection of Specific and Nonspecific Cellular Mechanisms

Using radiofrequency dielectric spectroscopy, we have investigated the impact of the interaction between a G protein-coupled receptor (GPCR), the sterile2 α-factor receptor protein (Ste2), and its cognate agonist ligand, the α-factor pheromone, on the dielectric properties of the plasma membrane in living yeast cells (Saccharomyces cerevisiae). The dielectric properties of a cell suspension containing a saturating concentration of α-factor were measured over the frequency range 40Hz–110 MHz and compared to the behavior of a similarly prepared suspension of cells in the absence of α-factor. A spherical three-shell model was used to determine the electrical phase parameters for the yeast cells in both types of suspensions. The relative permittivity of the plasma membrane showed a significant increase after exposure to α-factor (by 0.06 ± 0.05). The equivalent experiment performed on yeast cells lacking the ability to express Ste2 showed no change in plasma membrane permittivity. Interestingly, a large change also occurred to the electrical properties of the cellular interior after the addition of α-factor to the cell suspending medium, whether or not the cells were expressing Ste2. We present a number of different complementary experiments performed on the yeast to support these dielectric data and interpret the results in terms of specific cellular reactions to the presence of α-factor.

[1]  H. Coster,et al.  The molecular organisation of bimolecular lipid membranes. The dielectric structure of the hydrophilic/hydrophobic interface. , 1981, Biochimica et biophysica acta.

[2]  Valerica Raicu,et al.  Dielectric properties of rat liver in vivo: a noninvasive approach using an open-ended coaxial probe at audio/radio frequencies , 1998 .

[3]  I. Chatterjee,et al.  Dielectric properties of isolated adrenal chromaffin cells determined by microfluidic impedance spectroscopy. , 2018, Bioelectrochemistry.

[4]  A. Hanyaloglu,et al.  Constitutive and Agonist-dependent Homo-oligomerization of the Thyrotropin-releasing Hormone Receptor , 2001, The Journal of Biological Chemistry.

[5]  D. A. G. Bruggeman.  Berechnung verschiedener physikalischer Konstanten von heterogenen Substanzen. I. Dielektrizitätskonstanten und Leitfähigkeiten der Mischkörper aus isotropen Substanzen , 1935 .

[6]  Jean-François Mercier,et al.  Quantitative assessment of beta 1- and beta 2-adrenergic receptor homo- and heterodimerization by bioluminescence resonance energy transfer. , 2002, The Journal of biological chemistry.

[7]  J. Gimsa,et al.  Dielectric spectroscopy of human erythrocytes: investigations under the influence of nystatin. , 1994, Biophysical journal.

[8]  Valerica Raicu,et al.  Dielectric properties of rat liver in vivo: analysis by modeling hepatocytes in the tissue architecture , 1998 .

[9]  E. Gheorghiu,et al.  Real-time monitoring of yeast cell division by dielectric spectroscopy. , 1999, Biophysical journal.

[10]  K. Kuder,et al.  Fluorescent GPCR ligands as new tools in pharmacology. , 2008, Current medicinal chemistry.

[11]  Nathan C Shaner,et al.  A guide to choosing fluorescent proteins , 2005, Nature Methods.

[12]  J. Rohrer,et al.  Identification of a novel sequence mediating regulated endocytosis of the G protein-coupled alpha-pheromone receptor in yeast. , 1993, Molecular biology of the cell.

[13]  C. D. Gelatt,et al.  Optimization by Simulated Annealing , 1983, Science.

[14]  Koji Asami,et al.  Effects of copper on dielectric properties of E. coli cells. , 2007, Colloids and surfaces. B, Biointerfaces.

[15]  C C Davis,et al.  Frequency domain impedance measurements of erythrocytes. Constant phase angle impedance characteristics and a phase transition. , 1992, Biophysical journal.

[16]  Susan Rae Smith-Baish,et al.  The dielectric properties of tissues , 1991 .

[17]  V. Raicu,et al.  Integrated molecular and cellular biophysics , 2008 .

[18]  R. Deschenes,et al.  Microfluidic device for trapping and monitoring three dimensional multicell spheroids using electrical impedance spectroscopy. , 2013, Biomicrofluidics.

[19]  Emile H. L. Aarts,et al.  Simulated Annealing: Theory and Applications , 1987, Mathematics and Its Applications.

[20]  Camelia Gabriel,et al.  Dielectric Properties of Biological Materials , 2018, Bioengineering and Biophysical Aspects of Electromagnetic Fields.

[21]  A. Irimajiri,et al.  A dielectric theory of "multi-stratified shell" model with its application to a lymphoma cell. , 1979, Journal of theoretical biology.

[22]  H. Fricke.  THE ELECTRIC CAPACITY OF SUSPENSIONS WITH SPECIAL REFERENCE TO BLOOD , 1925, The Journal of general physiology.

[23]  H. Schwan.  Electrical properties of tissue and cell suspensions. , 1957, Advances in biological and medical physics.

[24]  J. Maxwell.  A Treatise on Electricity and Magnetism , 1873, Nature.

[25]  Yosi Shacham-Diamand,et al.  Dielectric screening of early differentiation patterns in mesenchymal stem cells induced by steroid hormones. , 2010, Bioelectrochemistry.

[26]  Takao Shimizu,et al.  Specific ligands as pharmacological chaperones: The transport of misfolded G‐protein coupled receptors to the cell surface , 2010, IUBMB life.

[27]  F. Barnes,et al.  Handbook of biological effects of electromagnetic fields , 2007 .

[28]  R. Lefkowitz,et al.  Signalling: Seven-transmembrane receptors , 2002, Nature Reviews Molecular Cell Biology.

[29]  Shilpee Jain,et al.  Dielectric Relaxation in Biological Systems:Physical Principles, Methods and Applications , 2016 .

[30]  V. Raicu,et al.  Quaternary structure of the yeast pheromone receptor Ste2 in living cells. , 2017, Biochimica et biophysica acta. Biomembranes.

[31]  K. Blumer,et al.  G-protein-coupled receptors function as oligomers in vivo , 2000, Current Biology.

[32]  J. W. Wells,et al.  Determination of supramolecular structure and spatial distribution of protein complexes in living cells , 2009 .

[33]  U. Kaatze.  Complex Permittivity of Water as a Function of Frequency and Temperature , 1989 .

[34]  Mohammad Fallahi-Sichani,et al.  Lipid Raft-Mediated Regulation of G-Protein Coupled Receptor Signaling by Ligands which Influence Receptor Dimerization: A Computational Study , 2009, PloS one.

[35]  K. Kuder,et al.  Fluorescent GPCR ligands as new tools in pharmacology-update, years 2008-early 2014. , 2014, Current medicinal chemistry.

[36]  V. Raicu,et al.  Investigation of dielectric relaxation in systems with hierarchical organization: From time to frequency domain and back again , 2017 .

[37]  G. Milligan,et al.  Structural and biophysical characterisation of G protein-coupled receptor ligand binding using resonance energy transfer and fluorescent labelling techniques. , 2014, Biochimica et biophysica acta.

[38]  P. Fossier,et al.  Monitoring of Ligand-independent Dimerization and Ligand-induced Conformational Changes of Melatonin Receptors in Living Cells by Bioluminescence Resonance Energy Transfer* 210 , 2002, The Journal of Biological Chemistry.

[39]  D. Jenness,et al.  Homo-oligomeric complexes of the yeast alpha-factor pheromone receptor are functional units of endocytosis. , 2000, Molecular biology of the cell.

[40]  Valerică Raicu,et al.  Protein interaction quantified in vivo by spectrally resolved fluorescence resonance energy transfer. , 2005, The Biochemical journal.

[41]  V. Raicu,et al.  Development and Experimental Testing of an Optical Micro-Spectroscopic Technique Incorporating True Line-Scan Excitation , 2013, International journal of molecular sciences.

[42]  P. Conn,et al.  Chaperoning G protein-coupled receptors: from cell biology to therapeutics. , 2014, Endocrine reviews.

[43]  S. Rees,et al.  Monitoring Receptor Oligomerization Using Time-resolved Fluorescence Resonance Energy Transfer and Bioluminescence Resonance Energy Transfer , 2001, The Journal of Biological Chemistry.

[44]  V. Raicu,et al.  Protein influence on the plasma membrane dielectric properties: in vivo study utilizing dielectric spectroscopy and fluorescence microscopy. , 2007, Bioelectrochemistry.

[45]  K Asami,et al.  Dielectric behavior of wild-type yeast and vacuole-deficient mutant over a frequency range of 10 kHz to 10 GHz. , 1996, Biophysical journal.

[46]  J. Vilardaga.  ENDOSOMAL GENERATION OF cAMP in GPCR SIGNALING , 2014, Nature chemical biology.

[47]  Graeme Milligan,et al.  Applications of bioluminescence- and fluorescence resonance energy transfer to drug discovery at G protein-coupled receptors. , 2004, European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences.

[48]  Ru Zhang,et al.  Tools for GPCR drug discovery , 2012, Acta Pharmacologica Sinica.

[49]  A. Irimajiri,et al.  Dielectric monitoring of rouleaux formation in human whole blood: a feasibility study. , 1996, Biochimica et biophysica acta.

[50]  George Khelashvili,et al.  Quantitative modeling of membrane deformations by multihelical membrane proteins: application to G-protein coupled receptors. , 2011, Biophysical journal.

[51]  V. Raicu,et al.  Effects of cetyltrimethylammonium bromide (CTAB) surfactant upon the dielectric properties of yeast cells. , 1998, Biochimica et biophysica acta.

[52]  R. Eglen,et al.  Functional G protein-coupled receptor assays for primary and secondary screening. , 2005, Combinatorial chemistry & high throughput screening.

[53]  Paul R. Selvin,et al.  The renaissance of fluorescence resonance energy transfer , 2000, Nature Structural Biology.

[54]  K. Blumer,et al.  Oligomerization of G-Protein-Coupled Receptors: Lessons from the Yeast Saccharomyces cerevisiae , 2005, Eukaryotic Cell.

[55]  Hywel Morgan,et al.  High Speed Single Cell Dielectric Spectroscopy. , 2020, ACS sensors.

[56]  J. Galzi,et al.  Fluorescence resonance energy transfer to probe human M1 muscarinic receptor structure and drug binding properties , 2003, Journal of neurochemistry.

[57]  Elizabeth Mathew,et al.  Fluorescent approaches for understanding interactions of ligands with G protein coupled receptors. , 2014, Biochimica et biophysica acta.

[58]  Graeme Milligan,et al.  G Protein-Coupled Receptor Dimerization: Function and Ligand Pharmacology , 2004, Molecular Pharmacology.

[59]  Experimental verification of the kinetic theory of FRET using optical microspectroscopy and obligate oligomers. , 2015, Biophysical journal.

[60]  Ali Arif,et al.  A Low-Cost Multiple Complementary Split-Ring Resonator-Based Microwave Sensor for Contactless Dielectric Characterization of Liquids , 2020, IEEE Sensors Journal.

[61]  H. Schiöth,et al.  The Repertoire of G-Protein–Coupled Receptors in Fully Sequenced Genomes , 2005, Molecular Pharmacology.

[62]  Marcel Hibert,et al.  Biased agonist pharmacochaperones of the AVP V2 receptor may treat congenital nephrogenic diabetes insipidus. , 2009, Journal of the American Society of Nephrology : JASN.

[63]  K. O’Malley,et al.  GPCR signalling from within the cell , 2018, British journal of pharmacology.

[64]  S. Angers,et al.  Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[65]  E. Vitols,et al.  STUDIES ON THE OXIDATIVE METABOLISM OF SACCHAROMYCES CEREVISIAE , 1961, The Journal of biophysical and biochemical cytology.

[66]  D D Jenness,et al.  Direct evidence for ligand-induced internalization of the yeast alpha-factor pheromone receptor. , 1994, Molecular and cellular biology.

[67]  Barrie Kellam,et al.  Fluorophore-tagged GPCR ligands. , 2005, Current opinion in chemical biology.

[68]  H. Pauly,et al.  Über die Impedanz einer Suspension von kugelförmigen Teilchen mit einer Schale , 1959 .

[69]  Karl Willy Wagner,et al.  Erklärung der dielektrischen Nachwirkungsvorgänge auf Grund Maxwellscher Vorstellungen , 1914 .

[70]  Dielectric monitoring of biological cell sedimentation , 1992 .

[71]  R. Pepperkok,et al.  Spectral imaging and linear un‐mixing enables improved FRET efficiency with a novel GFP2–YFP FRET pair , 2002, FEBS letters.

[72]  K. O’Malley,et al.  Intracellular GPCRs Play Key Roles in Synaptic Plasticity. , 2018, ACS chemical neuroscience.

[73]  C. Cametti,et al.  Reduction of the contribution of electrode polarization effects in the radiowave dielectric measurements of highly conductive biological cell suspensions. , 2001, Bioelectrochemistry.

[74]  Yuri Feldman,et al.  Fractal-polarization correction in time domain dielectric spectroscopy , 1998 .

[75]  W. D. Gregory,et al.  Correction of electrode polarization contributions to the dielectric properties of normal and cancerous breast tissues at audio/radiofrequencies , 2007, Physics in medicine and biology.

[76]  Jie Zhuang,et al.  Microfluidic impedance spectroscopy as a tool for quantitative biology and biotechnology. , 2012, Biomicrofluidics.

[77]  J. Poznański,et al.  Interactions of α-Factor-1, a Yeast Pheromone, and Its Analogue with Copper(II) Ions and Low-Molecular-Weight Ligands Yield Very Stable Complexes. , 2016, Inorganic chemistry.

[78]  Y. Kubo,et al.  Ligand-induced Rearrangements of the GABAB Receptor Revealed by Fluorescence Resonance Energy Transfer* , 2010, The Journal of Biological Chemistry.

[79]  R. Zare,et al.  Structure and conformational changes in the C-terminal domain of the beta2-adrenoceptor: insights from fluorescence resonance energy transfer studies. , 2007, The Journal of biological chemistry.

[80]  V. Raicu,et al.  Relaxation in systems with hierarchical organization: Analytical derivation of the relaxation and dispersion functions , 2019, Physics Letters A.

[81]  D. Piston,et al.  Fluorescent protein FRET: the good, the bad and the ugly. , 2007, Trends in biochemical sciences.

[82]  M. Bouvier,et al.  Ligands act as pharmacological chaperones and increase the efficiency of δ opioid receptor maturation , 2002, The EMBO journal.

[83]  V. Raicu,et al.  In-Cell Detection of Conformational Substates of a G Protein-Coupled Receptor Quaternary Structure: Modulation of Substate Probability by Cognate Ligand Binding. , 2020, The journal of physical chemistry. B.

[84]  George Khelashvili,et al.  Ligand-Dependent Conformations and Dynamics of the Serotonin 5-HT2A Receptor Determine Its Activation and Membrane-Driven Oligomerization Properties , 2012, PLoS Comput. Biol..

[85]  J. Becker,et al.  Identification of ligand binding regions of the Saccharomyces cerevisiae alpha-factor pheromone receptor by photoaffinity cross-linking. , 2004, Biochemistry.

[86]  Koji Asami,et al.  Characterization of biological cells by dielectric spectroscopy , 2002 .

[87]  S. Mitaku,et al.  Identification of G protein‐coupled receptor genes from the human genome sequence , 2002, FEBS letters.

[88]  Leigh A. Stoddart,et al.  Fluorescence‐ and bioluminescence‐based approaches to study GPCR ligand binding , 2015, British journal of pharmacology.

[89]  A. Burkhalter,et al.  Activation of Metabotropic Glutamate Receptor mGlu5 on Nuclear Membranes Mediates Intranuclear Ca2+ Changes in Heterologous Cell Types and Neurons* , 2003, Journal of Biological Chemistry.

[90]  G. Fontgalland,et al.  PCB-integrated non-destructive microwave sensor for liquid dielectric spectroscopy based on planar metamaterial resonator , 2020 .

[91]  V. Raicu,et al.  Dielectric properties of yeast cells as simulated by the two-shell model. , 1996, Biochimica et biophysica acta.

[92]  Valerica Raicu,et al.  Dielectric relaxation in biological systems , 2015 .

[93]  M. Hauser,et al.  Study of the binding environment of alpha-factor in its G protein-coupled receptor using fluorescence spectroscopy. , 2008, The journal of peptide research : official journal of the American Peptide Society.