The dipole potential correlates with lipid raft markers in the plasma membrane of living cells
2017; Elsevier BV; Volume: 58; Issue: 8 Linguagem: Inglês
10.1194/jlr.m077339
ISSN1539-7262
AutoresTamás Kovács, Gyula Batta, Florina Zákány, János Szöllősi, Péter Nagy,
Tópico(s)Force Microscopy Techniques and Applications
ResumoThe dipole potential generating an electric field much stronger than any other type of membrane potential influences a wide array of phenomena, ranging from passive permeation to voltage-dependent conformational changes of membrane proteins. It is generated by the ordered orientation of lipid carbonyl and membrane-attached water dipole moments. Theoretical considerations and indirect experimental evidence obtained in model membranes suggest that the dipole potential is larger in liquid-ordered domains believed to correspond to lipid rafts in cell membranes. Using three different dipole potential-sensitive fluorophores and four different labeling approaches of raft and nonraft domains, we showed that the dipole potential is indeed stronger in lipid rafts than in the rest of the membrane. The magnitude of this difference is similar to that observed between the dipole potential in control and sphingolipid-enriched cells characteristic of Gaucher's disease. The results established that the heterogeneity of the dipole potential in living cell membranes is correlated with lipid rafts and imply that alterations in the lipid composition of the cell membrane in human diseases can lead to substantial changes in the dipole potential. The dipole potential generating an electric field much stronger than any other type of membrane potential influences a wide array of phenomena, ranging from passive permeation to voltage-dependent conformational changes of membrane proteins. It is generated by the ordered orientation of lipid carbonyl and membrane-attached water dipole moments. Theoretical considerations and indirect experimental evidence obtained in model membranes suggest that the dipole potential is larger in liquid-ordered domains believed to correspond to lipid rafts in cell membranes. Using three different dipole potential-sensitive fluorophores and four different labeling approaches of raft and nonraft domains, we showed that the dipole potential is indeed stronger in lipid rafts than in the rest of the membrane. The magnitude of this difference is similar to that observed between the dipole potential in control and sphingolipid-enriched cells characteristic of Gaucher's disease. The results established that the heterogeneity of the dipole potential in living cell membranes is correlated with lipid rafts and imply that alterations in the lipid composition of the cell membrane in human diseases can lead to substantial changes in the dipole potential. The eukaryotic cell membrane is a highly complex structure because of its lateral heterogeneity, the presence of membrane microdomains, and the "trinity" of membrane potentials, including transmembrane, surface, and dipole potentials (1.O'Shea P. Intermolecular interactions with/within cell membranes and the trinity of membrane potentials: kinetics and imaging.Biochem. Soc. Trans. 2003; 31: 990-996Crossref PubMed Google Scholar, 2.O'Shea P. Physical landscapes in biological membranes: physico-chemical terrains for spatio-temporal control of biomolecular interactions and behaviour.Philos. Trans. A Math. Phys. Eng. Sci. 2005; 363: 575-588Crossref PubMed Scopus (61) Google Scholar). The dipole potential is an intramembrane electrostatic potential that originates from the preferential alignment of interfacial water dipoles and dipolar segments of the lipid molecules with a negative contribution originating from phospholipid head group P−–N+ dipoles. Resulting from the arrangement of molecular dipoles, the interior part of the bilayer is characterized by a large positive dipole potential with a magnitude usually estimated in the range of several hundred millivolts, that is, a value that is much higher than that of transmembrane or surface potentials. As the dipole potential drops over a very short distance (2 and 3 nm, the approximate thickness of the monolayers in a bilayer) through the low dielectric hydrophobic interior of a membrane, it results in a large electrostatic dipole electric field (which is the spatial derivative of the potential) that a variety of experimental and computational techniques have estimated to be in the range of 108–109 V/m. This is significantly larger than either of the other two electrostatic fields associated with the transmembrane and surface potentials (estimated to be around 2.5 · 107, and 106 V/m, respectively) (1.O'Shea P. Intermolecular interactions with/within cell membranes and the trinity of membrane potentials: kinetics and imaging.Biochem. Soc. Trans. 2003; 31: 990-996Crossref PubMed Google Scholar, 2.O'Shea P. Physical landscapes in biological membranes: physico-chemical terrains for spatio-temporal control of biomolecular interactions and behaviour.Philos. Trans. A Math. Phys. Eng. Sci. 2005; 363: 575-588Crossref PubMed Scopus (61) Google Scholar, 3.Brockman H. Dipole potential of lipid membranes.Chem. Phys. Lipids. 1994; 73: 57-79Crossref PubMed Scopus (338) Google Scholar, 4.Wang L. Measurements and implications of the membrane dipole potential.Annu. Rev. Biochem. 2012; 81: 615-635Crossref PubMed Scopus (126) Google Scholar, 5.Richens J.L. Lane J.S. Bramble J.P. O'Shea P. The electrical interplay between proteins and lipids in membranes.Biochim. Biophys. Acta. 2015; 1848: 1828-1836Crossref PubMed Scopus (24) Google Scholar). Because of this large electric field, the dipole potential is considered to be essential for the conformation and the function of membrane proteins, and more generally for interactions between a lipid membrane and biological molecules embedded in a membrane, possibly influencing the distribution of proteins between different membrane regions and microdomains. Thus, the dipole potential was shown to influence the membrane permeability of large hydrophobic ions (6.Andersen O.S. Fuchs M. Potential energy barriers to ion transport within lipid bilayers. Studies with tetraphenylborate.Biophys. J. 1975; 15: 795-830Abstract Full Text PDF PubMed Scopus (167) Google Scholar), to affect membrane binding of drugs (7.Asawakarn T. Cladera J. O'Shea P. Effects of the membrane dipole potential on the interaction of saquinavir with phospholipid membranes and plasma membrane receptors of Caco-2 cells.J. Biol. Chem. 2001; 276: 38457-38463Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), to modulate the conductance and association of ionophores (8.Rokitskaya T.I. Kotova E.A. Antonenko Y.N. Membrane dipole potential modulates proton conductance through gramicidin channel: movement of negative ionic defects inside the channel.Biophys. J. 2002; 82: 865-873Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), to play a role in the gating mechanism and conformational changes of voltage-gated ion channels (9.Pearlstein R.A. Dickson C.J. Hornak V. Contributions of the membrane dipole potential to the function of voltage-gated cation channels and modulation by small molecule potentiators.Biochim. Biophys. Acta. 2017; 1859: 177-194Crossref Scopus (23) Google Scholar), and to alter the activity of Na+/K+ ATPase (10.Starke-Peterkovic T. Turner N. Else P.L. Clarke R.J. Electric field strength of membrane lipids from vertebrate species: membrane lipid composition and Na+-K+-ATPase molecular activity.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2005; 288: R663-R670Crossref PubMed Scopus (71) Google Scholar) and P-glycoprotein (11.Davis S. Davis B.M. Richens J.L. Vere K.A. Petrov P.G. Winlove C.P. O'Shea P. α-Tocopherols modify the membrane dipole potential leading to modulation of ligand binding by P-glycoprotein.J. Lipid Res. 2015; 56: 1543-1550Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). We have recently shown that alterations in the dipole potential change the ligand-binding affinity of epidermal growth factor receptor (ErbB1) and the ligand-induced clustering and activation of ErbB1 and ErbB2 and that the extent of these effects depends on whether the proteins are localized in or outside of lipid rafts (12.Kovács T. Batta G. Hajdu T. Szabó A. Váradi T. Zákány F. Csomós I. Szöllősi J. Nagy P. The dipole potential modifies the clustering and ligand binding affinity of ErbB proteins and their signaling efficiency.Sci. Rep. 2016; 6: 35850Crossref PubMed Scopus (18) Google Scholar). The dipole potential varies both longitudinally and laterally across the bilayer according to membrane composition and phospholipid packing density (1.O'Shea P. Intermolecular interactions with/within cell membranes and the trinity of membrane potentials: kinetics and imaging.Biochem. Soc. Trans. 2003; 31: 990-996Crossref PubMed Google Scholar, 5.Richens J.L. Lane J.S. Bramble J.P. O'Shea P. The electrical interplay between proteins and lipids in membranes.Biochim. Biophys. Acta. 2015; 1848: 1828-1836Crossref PubMed Scopus (24) Google Scholar, 13.Starke-Peterkovic T. Clarke R.J. Effect of headgroup on the dipole potential of phospholipid vesicles.Eur. Biophys. J. 2009; 39: 103-110Crossref PubMed Scopus (47) Google Scholar). This phenomenon is the basis for artificially decreasing and increasing the dipole potential by phloretin and 6-ketocholestanol, respectively (14.Clarke R.J. Kane D.J. Optical detection of membrane dipole potential: avoidance of fluidity and dye-induced effects.Biochim. Biophys. Acta. 1997; 1323: 223-239Crossref PubMed Scopus (121) Google Scholar, 15.Gross E. Bedlack Jr., R.S. Loew L.M. Dual-wavelength ratiometric fluorescence measurement of the membrane dipole potential.Biophys. J. 1994; 67: 208-216Abstract Full Text PDF PubMed Scopus (226) Google Scholar). Besides the experimental manipulation, the dependence of the dipole potential on the lipid composition is expected to lead to different dipole potential values in different microdomains of the membrane. Indeed, cholesterol has been shown to increase the dipole potential because of its intrinsic dipole moment and its effects on the compaction and physical properties of the membrane (16.Haldar S. Kanaparthi R.K. Samanta A. Chattopadhyay A. Differential effect of cholesterol and its biosynthetic precursors on membrane dipole potential.Biophys. J. 2012; 102: 1561-1569Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Apart from its effect on the dipole potential, cholesterol is known to induce the separation of liquid-ordered and -disordered domains in model membranes (17.Kahya N. Scherfeld D. Bacia K. Poolman B. Schwille P. Probing lipid mobility of raft-exhibiting model membranes by fluorescence correlation spectroscopy.J. Biol. Chem. 2003; 278: 28109-28115Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar). Although liquid-ordered domains were shown to correspond to lipid rafts in cell membranes (18.Bacia K. Scherfeld D. Kahya N. Schwille P. Fluorescence correlation spectroscopy relates rafts in model and native membranes.Biophys. J. 2004; 87: 1034-1043Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar), this correlation remains highly speculative and contentious. The source of the confusion is the striking difference between the stability and size of liquid-ordered domains in model membranes and lipid rafts in cell membranes (19.Sevcsik E. Schutz G.J. With or without rafts? Alternative views on cell membranes.BioEssays. 2016; 38: 129-139Crossref PubMed Scopus (79) Google Scholar). Most researchers agree that lipid rafts are thermodynamically unstable, with sphingolipid and cholesterol-enriched microdomains in cell membranes exhibiting certain dynamic properties similar to those of liquid-ordered, stable domains in model membranes. However, lipid-mediated interactions are most likely not primarily responsible for the generation of lipid rafts, because the cytoskeleton, membrane proteins, their interactions with lipids, and membrane turnover all contribute to the properties and existence of rafts (19.Sevcsik E. Schutz G.J. With or without rafts? Alternative views on cell membranes.BioEssays. 2016; 38: 129-139Crossref PubMed Scopus (79) Google Scholar, 20.Eggeling C. Ringemann C. Medda R. Schwarzmann G. Sandhoff K. Polyakova S. Belov V.N. Hein B. von Middendorff C. Schonle A. et al.Direct observation of the nanoscale dynamics of membrane lipids in a living cell.Nature. 2009; 457: 1159-1162Crossref PubMed Scopus (1182) Google Scholar, 21.Honigmann A. Mueller V. Ta H. Schoenle A. Sezgin E. Hell S.W. Eggeling C. Scanning STED-FCS reveals spatiotemporal heterogeneity of lipid interaction in the plasma membrane of living cells.Nat. Commun. 2014; 5: 5412Crossref PubMed Scopus (211) Google Scholar, 22.Kraft M.L. Plasma membrane organization and function: moving past lipid rafts.Mol. Biol. Cell. 2013; 24: 2765-2768Crossref PubMed Scopus (113) Google Scholar). Despite this controversy, a huge variety of biological functions, including transmembrane signaling and membrane trafficking, have been linked to lipid rafts in health and disease (23.Simons K. Toomre D. Lipid rafts and signal transduction.Nat. Rev. Mol. Cell Biol. 2000; 1: 31-39Crossref PubMed Scopus (5152) Google Scholar, 24.Simons K. Ehehalt R. Cholesterol, lipid rafts, and disease.J. Clin. Invest. 2002; 110: 597-603Crossref PubMed Scopus (915) Google Scholar). Because the dipole potential is entirely located within the low dielectric, hydrophobic interior of the plasma membrane, it is difficult to measure directly. All of the methods described for the estimation of the dipole potential, including the measurement of the permeability of large hydrophobic ions (25.Flewelling R.F. Hubbell W.L. The membrane dipole potential in a total membrane potential model. Applications to hydrophobic ion interactions with membranes.Biophys. J. 1986; 49: 541-552Abstract Full Text PDF PubMed Scopus (334) Google Scholar), cryoelectron microscopy (26.Wang L. Bose P.S. Sigworth F.J. Using cryo-EM to measure the dipole potential of a lipid membrane.Proc. Natl. Acad. Sci. USA. 2006; 103: 18528-18533Crossref PubMed Scopus (88) Google Scholar), molecular dynamics simulations (27.Harder E. Mackerell Jr., A.D. Roux B. Many-body polarization effects and the membrane dipole potential.J. Am. Chem. Soc. 2009; 131: 2760-2761Crossref PubMed Scopus (93) Google Scholar, 28.Ding W. Palaiokostas M. Wang W. Orsi M. Effects of lipid composition on bilayer membranes quantified by all-atom molecular dynamics.J. Phys. Chem. B. 2015; 119: 15263-15274Crossref PubMed Scopus (52) Google Scholar), atomic force microscopy (29.Yang Y. Mayer K.M. Wickremasinghe N.S. Hafner J.H. Probing the lipid membrane dipole potential by atomic force microscopy.Biophys. J. 2008; 95: 5193-5199Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), and vibrational Stark effect spectroscopy (30.Shrestha R. Anderson C.M. Cardenas A.E. Elber R. Webb L.J. Direct measurement of the effect of cholesterol and 6-ketocholestanol on the membrane dipole electric field using vibrational stark effect spectroscopy coupled with molecular dynamics simulations.J. Phys. Chem. B. 2017; 121: 3424-3436Crossref PubMed Scopus (12) Google Scholar), are characterized by serious difficulties and disadvantages considering their applicability to living cells. Therefore, we opted for the fast-sensing voltage-sensitive fluorophores for measuring the dipole potential of living cells. The response of di-8-ANEPPS, a 4-p-aminostyryl-1-pyridinium derivative, to changes in the local electric field is based on electrochromism, and its emission measured at two different excitation wavelengths is sensitive to the dipole potential (12.Kovács T. Batta G. Hajdu T. Szabó A. Váradi T. Zákány F. Csomós I. Szöllősi J. Nagy P. The dipole potential modifies the clustering and ligand binding affinity of ErbB proteins and their signaling efficiency.Sci. Rep. 2016; 6: 35850Crossref PubMed Scopus (18) Google Scholar, 14.Clarke R.J. Kane D.J. Optical detection of membrane dipole potential: avoidance of fluidity and dye-induced effects.Biochim. Biophys. Acta. 1997; 1323: 223-239Crossref PubMed Scopus (121) Google Scholar, 15.Gross E. Bedlack Jr., R.S. Loew L.M. Dual-wavelength ratiometric fluorescence measurement of the membrane dipole potential.Biophys. J. 1994; 67: 208-216Abstract Full Text PDF PubMed Scopus (226) Google Scholar). The sensitivity of di-8-ANEPPS to changes in membrane fluidity (14.Clarke R.J. Kane D.J. Optical detection of membrane dipole potential: avoidance of fluidity and dye-induced effects.Biochim. Biophys. Acta. 1997; 1323: 223-239Crossref PubMed Scopus (121) Google Scholar) required the design of a new class of dipole potential-sensitive dyes. 3-Hydroxyflavone derivatives are characterized by two well-separated bands in their emission spectra belonging to normal (N*) and tautomer (T*) excited states of their flavone chromophore. Species T* appears as a result of an excited-state intramolecular proton transfer (ESIPT) reaction that is very sensitive to changes in the local electric field. The relative intensities of N* and T* fluorescence emission bands can be used to measure the dipole potential. Various 3-hydroxyflavone derivatives have been designed in which the chromophore is oriented in opposite directions with respect to the bilayer plane. Consistently, the N*:T* emission ratio of PPZ8 and of its related analog, F66, change in opposite directions upon modifying the dipole potential (31.Shynkar V.V. Klymchenko A.S. Duportail G. Demchenko A.P. Mely Y. Two-color fluorescent probes for imaging the dipole potential of cell plasma membranes.Biochim. Biophys. Acta. 2005; 1712: 128-136Crossref PubMed Scopus (67) Google Scholar, 32.Klymchenko A.S. Duportail G. Mely Y. Demchenko A.P. Ultrasensitive two-color fluorescence probes for dipole potential in phospholipid membranes.Proc. Natl. Acad. Sci. USA. 2003; 100: 11219-11224Crossref PubMed Scopus (128) Google Scholar, 33.M'Baye G. Shynkar V.V. Klymchenko A.S. Mely Y. Duportail G. Membrane dipole potential as measured by ratiometric 3-hydroxyflavone fluorescence probes: accounting for hydration effects.J. Fluoresc. 2006; 16: 35-42Crossref PubMed Scopus (20) Google Scholar). Alterations in the lipid composition of the membrane are associated with certain diseases (24.Simons K. Ehehalt R. Cholesterol, lipid rafts, and disease.J. Clin. Invest. 2002; 110: 597-603Crossref PubMed Scopus (915) Google Scholar). Gaucher's disease is a lysosomal storage disorder in which glucosylceramide accumulates because of a deficiency of glucocerebrosidase (34.Butters T.D. Gaucher disease.Curr. Opin. Chem. Biol. 2007; 11: 412-418Crossref PubMed Scopus (97) Google Scholar). Although symptoms are usually believed to be caused by the storage of glucosylceramide, weak or no correlation among the severity of symptoms, residual enzyme activity, and the amount of stored lipid casts doubt on the causative relationship (35.Sidransky E. Gaucher disease: insights from a rare Mendelian disorder.Discov. Med. 2012; 14: 273-281PubMed Google Scholar). Gross alterations in lysosomal degradation of various substances due to "jamming" of the endosomal pathway and endoplasmic reticulum stress have also been invoked to explain the development of disease symptoms (36.Simons K. Gruenberg J. 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Selective labeling of lipid rafts is based on the preferential concentration of certain membrane components in them. The incorporation of glycosylphosphatidylinositol (GPI)-anchored proteins into lipid rafts is driven by their lipid moiety. Various studies suggest that GPI-anchored proteins might rather be entrapped into "transient confinement zones" generated by the barrier effects of the cortical matrix, including the actin cytoskeleton and spectrin meshwork. The link between these compartments and lipid rafts remains unclear, but according to the operational definition of lipid rafts given above, cytoskeleton-mediated effects also contribute to the formation of raft-like microdomains (40.Ritchie K. Iino R. Fujiwara T. Murase K. Kusumi A. The fence and picket structure of the plasma membrane of live cells as revealed by single molecule techniques (review).Mol. Membr. Biol. 2003; 20: 13-18Crossref PubMed Scopus (167) Google Scholar, 41.Varma R. Mayor S. GPI-anchored proteins are organized in submicron domains at the cell surface.Nature. 1998; 394: 798-801Crossref PubMed Scopus (1026) Google Scholar). Subunit B of cholera toxin (CTX-B) has also been widely used as a marker of lipid rafts because of its selective binding to GM1 ganglioside (42.Harder T. Scheiffele P. Verkade P. Simons K. Lipid domain structure of the plasma membrane revealed by patching of membrane components.J. Cell Biol. 1998; 141: 929-942Crossref PubMed Scopus (1045) Google Scholar). Because of its multivalence, it can induce lipid reorganization into coexisting liquid-ordered and liquid-disordered fractions due to GM1 crosslinking (43.Hammond A.T. Heberle F.A. Baumgart T. Holowka D. Baird B. Feigenson G.W. Crosslinking a lipid raft component triggers liquid ordered-liquid disordered phase separation in model plasma membranes.Proc. Natl. Acad. Sci. USA. 2005; 102: 6320-6325Crossref PubMed Scopus (270) Google Scholar, 44.Raghunathan K. Wong T.H. Chinnapen D.J. Lencer W.I. Jobling M.G. Kenworthy A.K. Glycolipid crosslinking is required for cholera toxin to partition into and stabilize ordered domains.Biophys. J. 2016; 111: 2547-2550Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Although based on the differences in the lipid composition of raft-like microdomains and the bulk phase of the membrane, the assumption of a larger dipole potential in rafts seems logical, but this relationship has not been demonstrated directly in living cell membranes. Heterogeneity observed in the dipole potential has been assumed to be caused by lipid rafts, but explicit proof and quantitative analysis of this correlation has not been presented (1.O'Shea P. Intermolecular interactions with/within cell membranes and the trinity of membrane potentials: kinetics and imaging.Biochem. Soc. Trans. 2003; 31: 990-996Crossref PubMed Google Scholar, 7.Asawakarn T. Cladera J. O'Shea P. Effects of the membrane dipole potential on the interaction of saquinavir with phospholipid membranes and plasma membrane receptors of Caco-2 cells.J. Biol. Chem. 2001; 276: 38457-38463Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 45.Duggan J. Jamal G. Tilley M. Davis B. McKenzie G. Vere K. Somekh M.G. O'Shea P. Harris H. Functional imaging of microdomains in cell membranes.Eur. Biophys. J. 2008; 37: 1279-1289Crossref PubMed Scopus (29) Google Scholar). On the other hand, the correspondence between electrostatic and topographic maps created by atomic-force microscopy performed with model membranes suggested that the dipole potential is larger is liquid-ordered domains (29.Yang Y. Mayer K.M. Wickremasinghe N.S. Hafner J.H. Probing the lipid membrane dipole potential by atomic force microscopy.Biophys. J. 2008; 95: 5193-5199Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). On the basis of the correlation between the dipole potential reported by three different, ratiometric, dipole potential-sensitive dyes and the distribution of lipid rafts labeled by CTX-B, GPI-anchored green fluorescent protein (GFP), or an anticholesterol antibody, we show that the dipole potential is significantly larger in lipid rafts than in the rest of the membrane. The fact that the magnitude of this difference is similar to the alteration in the dipole potential brought about by the increased sphingolipid concentration in Gaucher's disease points to a potential pathophysiological role for these findings. The human breast cancer cell line SKBR-3, the human epithelial carcinoma cell line A431, and the human acute monocytic leukemia-derived cell line THP-1 were obtained from the American Type Culture Collection (Manassas, VA) and grown according to their specifications. AlexaFluor647-tagged CTX-B was purchased from ThermoFisher (Waltham, MA). The GFP-GPI plasmid was a kind gift from Jennifer Lippincott-Schwartz (National Institutes of Health, Bethesda, MD), and the anticholesterol antibody was provided by János Matkó (Eötvös Loránd University, Budapest, Hungary). AlexaFluor647-transferrin was purchased from ThermoFisher. 6-Ketocholestanol (3β-hydroxy-5α-cholestan-6-1) and Pluronic F-127 were purchased from Sigma Aldrich (St. Louis, MO), and di-8-ANEPPS (4-(2-[6-(dioctylamino)-2-naphtalenyl]ethenyl)-1-(3-sulfopropyl)pyridinium inner salt) was acquired from ThermoFisher. F66 (N-[3-(40-dihexylamino-3-hydroxy-flavonyl-6-oxy)-propyl]N,N-dimethyl-N-(3-sulfopropyl)-ammonium, inner salt) and PPZ8 (3-[4-(4-[4V-(3-hydroxy-6-octyloxyflavonyl)phenyl]piperazino)-1-pyridiniumyl]-1-propanesulfonate) were kind gifts from Andrey Klymchenko (Université de Strasbourg, Strasbourg, France). The protein kinase C activator phorbol 12-myristate 13-acetate (PMA) and the β-glucosidase inhibitor conduritol B epoxide (CBE) were purchased from Sigma Aldrich. Lipid rafts were labeled with one of three different methods. i) GM1-enriched membrane rafts were labeled by incubating cells grown on 8-well chambered coverglass in the presence of 8 μg/ml AlexaFluor647-CTX-B for 20 min on ice to prevent internalization of CTX-B. ii) For labeling lipid rafts with GFP-GPI, SKBR-3 and A431 cells grown on 8-well chambered coverglass were transfected with 0.5 μg GFP-GPI plasmid/well using Lipofectamine2000 (ThermoFisher) at a lipid to DNA ratio of 2:1 (v/w). The transfection protocols were otherwise according to the manufacturer's specifications. iii) The cholesterol component of lipid rafts was visualized with AC8, a cholesterol-specific monoclonal antibody (46.Bíró A. Cervenak L. Balogh A. Lőrincz A. Uray K. Horváth A. Romics L. Matkó J. Füst G. László G. Novel anti-cholesterol monoclonal immunoglobulin G antibodies as probes and potential modulators of membrane raft-dependent immune functions.J. Lipid Res. 2007; 48: 19-29Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), followed by secondary labeling with the Fab fragment of Cy5-conjugated goat anti-mouse IgG (ThermoFisher) to minimize crosslinking. Alternatively, transferrin residing outside lipid rafts (47.Pike L.J. Han X. Gross R.W. Epidermal growth factor receptors are localized to lipid rafts that contain a balance of inner and outer leaflet lipids: a shotgun lipidomics study.J. Biol. Chem. 2005; 280: 26796-26804Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar) was labeled with 25 μg/ml AlexaFluor647-conjugated transferrin (ThermoFisher) on ice. After labeling cells with one of the aforementioned indicators and a dipole potential-sensitive dye, images were taken with an LSM880 confocal laser-scanning microscope (Carl Zeiss AG, Jena, Germany). AlexaFluor647 and Cy5 were excited at 633 nm, and their emission was detected between 649 and 759 nm, whereas GFP was excited at 488 nm, and its emission was detected between 506 and 555 nm. The dipole potential was increased by treating SKBR-3 and A431 cells with 6-ketocholestanol at a concentration of 100 μM for 10 min at room temperature in the presence of 0.05% (v/v) Pluronic F-127 (12.Kovács T. Batta G. Hajdu T. Szabó A. Váradi T. Zákány F. Csomós I. Szöllősi J. Nagy P. The dipole potential modifies the clustering and ligand binding affinity of ErbB proteins and their signaling efficiency.Sci. Rep. 2016; 6: 35850Crossref PubMed Scopus (18) Google Scholar, 14.Clarke R.J. Kane D.J. Optical detection of membrane dipole potential: avoidance of fluidity and dye-induced effects.Biochim. Biophys. Acta. 1997; 1323: 223-239Crossref PubMed Scopus (121) Google Scholar). For measuring the dipole potential with di-8-ANEPPS, we grew SKBR-3 and A431 cells, as well as control and CBE-treated THP-1-derived macrophages, on 8-well chambered coverglass, then incubated them with 2 μM di-8-ANEPPS (with or without CTX-B) for 20 min on ice, and their fluorescence was measured with a ratiometric assay shown to be responsive to changes in the dipole potential (15.Gross E. Bedlack Jr., R.S. Loew L.M. Dual-wavelength ratiometric fluorescence measurement of the membrane dipole potential.Biophys. J. 1994; 67: 208-216Abstract Full Text PDF PubMed Scopus (226) Google Scholar). The dye was excited at 458 and 514 nm, and its emission was measured between 584 and 686 nm. Image acquisition was performed using an LSM880 confocal laser-scanning microscope. Image processing was carried out with the DipImage toolbox (Delft University of Technology, Delft, The Netherlands) under MATLAB (MathWorks, Natick, MA). The fluorescence intensity ratio (exc458/514) of the cell membrane pixels, calculated after background subtraction, is expected to show positive correlation with the dipole potential (15.Gross E. Bedlack Jr., R.S. Loew L.M. Dual-wavelength ratiometric fluorescence measurement of the membrane dipole potential.Biophys. J. 1994; 67: 208-216Abstract Full Text PDF PubMed Scopus (226) Google Scholar, 48.Zhang J. Davidson R.M. Wei M.D. Loew L.M. Membrane electric properti
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