A novel flow cytometric assay to quantify interactions between proteins and membrane lipids

Abstract

A diverse set of experimental systems has been developed to probe protein-lipid interactions. These include measurements with the headgroups of membrane lipids in solution, immobilized membrane lipids, and analysis of protein binding to membrane lipids reconstituted in liposomes. Each of these methodologies has strengths but also substantial limitations. For example, measurements between proteins and lipid headgroups or with immobilized membrane lipids do not probe interactions in their natural environment, the lipid bilayer. The use of liposomes, however, was so far mostly restricted to biochemical flotation experiments that do not provide quantitative and/or kinetic data. Here, we present a fast and sensitive flow cytometric method to detect protein-lipid interactions. This technique allows for quantitative measurements of interactions between multiple fluorescently labeled proteins and membrane lipids reconstituted in lipid bilayers. The assay can be used to quantify binding efficiencies and to determine kinetic constants. The method is further characterized by a short sampling time of only a few seconds that allows for high-content screening procedures. Finally, using light scatter measurements, the described method also allows for monitoring changes of membrane curvature as well as tethering of liposomes evoked by binding of proteins. A diverse set of experimental systems has been developed to probe protein-lipid interactions. These include measurements with the headgroups of membrane lipids in solution, immobilized membrane lipids, and analysis of protein binding to membrane lipids reconstituted in liposomes. Each of these methodologies has strengths but also substantial limitations. For example, measurements between proteins and lipid headgroups or with immobilized membrane lipids do not probe interactions in their natural environment, the lipid bilayer. The use of liposomes, however, was so far mostly restricted to biochemical flotation experiments that do not provide quantitative and/or kinetic data. Here, we present a fast and sensitive flow cytometric method to detect protein-lipid interactions. This technique allows for quantitative measurements of interactions between multiple fluorescently labeled proteins and membrane lipids reconstituted in lipid bilayers. The assay can be used to quantify binding efficiencies and to determine kinetic constants. The method is further characterized by a short sampling time of only a few seconds that allows for high-content screening procedures. Finally, using light scatter measurements, the described method also allows for monitoring changes of membrane curvature as well as tethering of liposomes evoked by binding of proteins. In recent years, a large number of protein domains have been identified that directly bind to membrane lipids. These include a variety of distinct lipid binding motifs, such as Pleckstrin Homology (PH), FYVE (Fab1, YOTB, Vac1, EEA1), PKC (Protein Kinase C), PX (Phox homology), and BAR (Bin, Amphiphysin, and Rvs) domains (1Seet L.F. Hong W. The Phox (PX) domain proteins and membrane traffic.Biochim. Biophys. Acta. 2006; 1761: 878-896Crossref PubMed Scopus (160) Google Scholar, 2Hurley J.H. Membrane binding domains.Biochim. Biophys. Acta. 2006; 1761: 805-811Crossref PubMed Scopus (124) Google Scholar, 3Lemmon M.A. Membrane recognition by phospholipid-binding domains.Nat. Rev. Mol. Cell Biol. 2008; 9: 99-111Crossref PubMed Scopus (1101) Google Scholar–4Hayakawa A. Hayes S. Leonard D. Lambright D. Corvera S. Evolutionarily conserved structural and functional roles of the FYVE domain.Biochem. Soc. Symp. 2007; 74: 95-105Crossref PubMed Scopus (31) Google Scholar) as well as basic and/or hydrophobic clusters (5Heo W.D. Inoue T. Park W.S. Kim M.L. Park B.O. Wandless T.J. Meyer T. PI(3,4,5)P3 and PI(4,5)P2 lipids target proteins with polybasic clusters to the plasma membrane.Science. 2006; 314: 1458-1461Crossref PubMed Scopus (525) Google Scholar, 6McLaughlin S. Murray D. Plasma membrane phosphoinositide organization by protein electrostatics.Nature. 2005; 438: 605-611Crossref PubMed Scopus (697) Google Scholar–7Temmerman K. Ebert A.D. Muller H.M. Sinning I. Tews I. Nickel W. A direct role for phosphatidylinositol-4,5-bisphosphate in unconventional secretion of fibroblast growth factor 2.Traffic. 2008; 9: 1204-1217Crossref PubMed Scopus (85) Google Scholar). Alongside the discovery of these domains, the spectrum of methods used to study protein-lipid interactions has also been diversifying. These methods can be classified according to the state and background in which the lipid of interest is presented. Immobilized lipids spotted onto nitrocellulose membranes are convenient to analyze. On the other hand, the results are only qualitative and lipids are in a nonphysiological state and environment (8Dowler S. Currie R.A. Downes C.P. Alessi D.R. DAPP1: a dual adaptor for phosphotyrosine and 3-phosphoinositides.Biochem. J. 1999; 342: 7-12Crossref PubMed Scopus (126) Google Scholar). Using surface plasmon resonance (SPR), lipids can be presented as bilayers (liposomes) to study interactions with immobilized proteins. This method allows for real-time measurements and results in quantitative data (9Narayan K. Lemmon M.A. Determining selectivity of phosphoinositide-binding domains.Methods. 2006; 39: 122-133Crossref PubMed Scopus (109) Google Scholar). However, a disadvantage of this technique is that either the protein or the membrane lipid in question must be immobilized and, therefore, is not presented in a physiologically relevant environment. SPR experiments are also inconvenient to analyze large numbers of individual samples, thus excluding high-content screening procedures. Additionally, competitive binding studies of multiple proteins cannot be performed. A standard method to quantitatively analyze protein-lipid interactions in suspension is isothermal titration calorimetry (ITC) (10Wiseman T. Williston S. Brandts J.F. Lin L.N. Rapid measurement of binding constants and heats of binding using a new titration calorimeter.Anal. Biochem. 1989; 179: 131-137Crossref PubMed Scopus (2407) Google Scholar). Protein binding to both lipid headgroups and liposomes has successfully been measured (11Lemmon M.A. Ferguson K.M. O'Brien R. Sigler P.B. Schlessinger J. Specific and high-affinity binding of inositol phosphates to an isolated pleckstrin homology domain.Proc. Natl. Acad. Sci. USA. 1995; 92: 10472-10476Crossref PubMed Scopus (470) Google Scholar). The main drawback remains the large amounts of protein (typically in the milligram range) being required for each individual measurement. Like for SPR experiments, ITC is not suited for competitive binding studies and high-content screening procedures. Finally, a widespread method to probe interactions between proteins and chemically defined liposomes are biochemical flotation experiments. Again, this approach does not allow for analyzing large numbers of samples in parallel and is basically a qualitative method. This is because it involves a complicated and time-consuming multistep procedure with a potential loss of protein by degradation along with other possible issues. The new experimental system described here is based on flow cytometry, which is a powerful tool to measure both light scatter and fluorescence properties of individual particles suspended in a continuous flow (12Shapiro H.M. Practical Flow Cytometry. Jon Wiley & Sons, New York1995Google Scholar). Cells, membrane vesicles, or particles, such as liposomes, are carried through a flow cell one at a time where they are illuminated by one or more lasers. In general, the forward scatter of laser light is measured to determine the size of the particle as well as the sideward scatter, roughly defining the granularity and internal complexity of the sample. Emitted fluorescence passes through several optical filters to reach the appropriate detector channel, termed FL1, FL2, etc., each measuring a specific emission range. The generated signal intensity is linearly dependent on the amount of molecules present. Typically, 10,000 events are measured and depicted as population histograms or, when two channels are directly compared, as dot plots. An average population signal can be extracted for the comparison of different samples. Flow cytometric analysis of liposomes has so far mostly been used to determine size and volume properties (13Sato K. Obinata K. Sugawara T. Urabe I. Yomo T. Quantification of structural properties of cell-sized individual liposomes by flow cytometry.J. Biosci. Bioeng. 2006; 102: 171-178Crossref PubMed Scopus (38) Google Scholar, 14Vorauer-Uhl K. Wagner A. Borth N. Katinger H. Determination of liposome size distribution by flow cytometry.Cytometry. 2000; 39: 166-171Crossref PubMed Scopus (66) Google Scholar–15Childers N.K. Michalek S.M. Eldridge J.H. Denys F.R. Berry A.K. McGhee J.R. Characterization of liposome suspensions by flow cytometry.J. Immunol. Methods. 1989; 119: 135-143Crossref PubMed Scopus (34) Google Scholar). Here, we made use of it as a quantitative method to detect interactions between membrane lipids and peripheral membrane proteins in suspension, i.e., both interactions partners are presented in their natural environment. In addition to measuring direct interactions on a quantitative basis, this method is also suited to detect membrane surface effects that may be the result of proteins binding to specific membrane lipids. This includes clustering of liposomes by multivalent interactions and changes of membrane curvature. The experimental system is a single-step assay without the need for extensive washing procedures. It does not require large amounts of purified proteins and, provided that different fluorescent tags are used, allows for competitive binding studies. A general limitation in applications using liposomes is that membrane asymmetry between the two leaflets of a lipid bilayer cannot be reconstituted. Also, in the absence of membrane proteins, it is difficult to reconstitute microdomains with locally enriched membrane lipids. However, on the surface of liposomes, it is possible to mimic a certain leaflet, such as the cytoplasmic side of plasma membranes with a chemically defined composition of membrane lipids. Also, using a mixture of membrane lipids containing cholesterol and sphingomyelin, lipid microdomains can be reconstituted with local enrichments such as phosphoinositides. To establish the experimental method described here, we used liposomes with an overall lipid composition resembling plasma membranes of mammalian cells (16van Meer G. Lipids of the Golgi membrane.Trends Cell Biol. 1998; 8: 29-33Abstract Full Text PDF PubMed Scopus (122) Google Scholar). Because we used phosphoinositide binding proteins as a paradigm, the lipid mixture was designed to support the formation of cholesterol-dependent lipid microdomains in which the phosphoinositide phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is enriched (17McLaughlin S. Wang J. Gambhir A. Murray D. PIP(2) and proteins: interactions, organization, and information flow.Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 151-175Crossref PubMed Scopus (684) Google Scholar, 18Pike L.J. Miller J.M. Cholesterol depletion delocalizes phosphatidylinositol bisphosphate and inhibits hormone-stimulated phosphatidylinositol turnover.J. Biol. Chem. 1998; 273: 22298-22304Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). In native membranes, PI(4,5)P2 is a component of the inner leaflet of plasma membranes and has been shown to function in a number of cellular processes, including signaling and protein targeting (17McLaughlin S. Wang J. Gambhir A. Murray D. PIP(2) and proteins: interactions, organization, and information flow.Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 151-175Crossref PubMed Scopus (684) Google Scholar, G. in cell and membrane 2006; PubMed Scopus Google Scholar, of surface membrane Cell Biol. PubMed Scopus Google Scholar). To probe interactions of proteins with we two peripheral membrane proteins that bind PI(4,5)P2 with two different of recognition for phosphoinositides. the domain of was as a of a phosphoinositide binding domain (11Lemmon M.A. Ferguson K.M. O'Brien R. Sigler P.B. Schlessinger J. Specific and high-affinity binding of inositol phosphates to an isolated pleckstrin homology domain.Proc. Natl. Acad. Sci. USA. 1995; 92: 10472-10476Crossref PubMed Scopus (470) Google Scholar). we used a described of a protein with a polybasic that to K. Ebert A.D. Muller H.M. Sinning I. Tews I. Nickel W. A direct role for phosphatidylinositol-4,5-bisphosphate in unconventional secretion of fibroblast growth factor 2.Traffic. 2008; 9: 1204-1217Crossref PubMed Scopus (85) Google Scholar). A plasma mixture of membrane lipids, including PI(4,5)P2 was individual the of membrane lipids natural and The numbers are and at and used for experiments The lipid with to allow of liposomes by flow the of PI(4,5)P2 was The PI(4,5)P2 was either at the of or by with In this the of other membrane lipids the for of plasma in a new A lipid was by lipid in at for The lipid was in and with for particles with at for The lipid of the liposome suspension was of in and size using filters was liposomes with fluorescent protein by with liposomes to The liposomes to a lipid of measured by light at was by fluorescence to the liposome For liposome the was with a average of that the liposomes mostly not and in using the and was as described K. Ebert A.D. Muller H.M. Sinning I. Tews I. Nickel W. A direct role for phosphatidylinositol-4,5-bisphosphate in unconventional secretion of fibroblast growth factor 2.Traffic. 2008; 9: 1204-1217Crossref PubMed Scopus (85) Google Scholar, I. T. G. R. phosphatidylinositol-4,5-bisphosphate the size in 2005; PubMed Scopus Google Scholar). The tags and a further using either of or of and protein proteins at in using a with a cell and data with scatter and sideward scatter of light using The in which the was defined the size of defined the size where was which was further for sideward scatter so the not the for forward scatter the particle the range. fluorescence was by liposomes without to a of fluorescence for a in which fluorescence defined by a of for liposomes. between and was by liposomes in one and liposomes in the The for the and channels used for analysis the a of the data. 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Chem. Full Text PDF PubMed Google Scholar). effects to be and in further analysis not Flow allows for the analysis of particles, such as liposomes, with to both fluorescent and light In this liposomes be in two liposomes on a size by forward and sideward scatter of measurements of used to determine the of liposome used to liposomes lipid identified background that the analysis These measurements defined size that for a fluorescent membrane lipid was in the membrane and used to detect liposomes using can be by a laser and in the in parallel to A was set so that this with the defined size for a and analysis of liposomes data through these filters further with a defined lipid composition generated by size The use of a large size generated liposomes with a size as shown by measurements light the average size of with a to The size of liposome and the to analyze these for the analysis of a potential role of membrane curvature in protein binding to membrane lipids. To measure protein-lipid interactions in flow cytometry, a fluorescent is This can be either by the of with fluorescently labeled protein with or by of the protein of interest with fluorescent proteins. In this we used as a fluorescent to the domain of and a liposomes with measured not that the of is to detect these amounts of fluorescent and with a PI(4,5)P2 binding protein K. Ebert A.D. Muller H.M. Sinning I. Tews I. Nickel W. A direct role for phosphatidylinositol-4,5-bisphosphate in unconventional secretion of fibroblast growth factor 2.Traffic. 2008; 9: 1204-1217Crossref PubMed Scopus (85) Google Scholar). In it is data A and a titration the binding of to liposomes with a of the of the Each size a fluorescence which is for background binding of the of for possible liposome or membrane effects was by the fluorescence signal by a This was termed the fluorescence A standard to determine the number of fluorescent molecules was generated using and These fluorescence to and fluorescence using a and two standard that these are specific for the and the The domain is one of the and lipid binding domain M.A. Membrane recognition by phospholipid-binding domains.Nat. Rev. Mol. Cell Biol. 2008; 9: 99-111Crossref PubMed Scopus (1101) Google Scholar). To we the domain of that to was with liposomes at different was measured using the experimental described for each size and for the background as described shown in we and binding at protein protein of binding was For liposomes containing a of for and for was in with data on binding properties of the PI(4,5)P2 to these proteins K. Ebert A.D. Muller H.M. Sinning I. Tews I. Nickel W. A direct role for phosphatidylinositol-4,5-bisphosphate in unconventional secretion of fibroblast growth factor 2.Traffic. 2008; 9: 1204-1217Crossref PubMed Scopus (85) Google Scholar, M.A. Ferguson K.M. O'Brien R. Sigler P.B. Schlessinger J. Specific and high-affinity binding of inositol phosphates to an isolated pleckstrin homology domain.Proc. Natl. Acad. Sci. USA. 1995; 92: 10472-10476Crossref PubMed Scopus (470) Google Scholar, R. S. S. S. The pleckstrin homology domain of with to phosphatidylinositol in bilayer 1995; PubMed Scopus Google Scholar). The for is with data with or and for and (11Lemmon M.A. Ferguson K.M. O'Brien R. Sigler P.B. Schlessinger J. Specific and high-affinity binding of inositol phosphates to an isolated pleckstrin homology domain.Proc. Natl. Acad. Sci. USA. 1995; 92: 10472-10476Crossref PubMed Scopus (470) Google Scholar, R. S. S. S. The pleckstrin homology domain of with to phosphatidylinositol in bilayer 1995; PubMed Scopus Google Scholar, A. G. I. Wang J. Murray D. S. of on membranes by of J. Full Text Full Text PDF PubMed Scopus Google K. S. H. of inositol that 1999; PubMed Scopus Google Scholar). with other the by the method may be to the to for potential These data the of the method described binding liposome was dependent on size as Also, for both and the data not a in of binding to liposomes with a that both proteins do not bind membranes in a The of binding at be by for the protein to amounts of protein molecules using the defined standard This was for of liposome the fluorescence be to the number of lipid molecules present in liposomes of the that more PI(4,5)P2 was present A of for the binding of to PI(4,5)P2 was the that of the lipids is present on the side and for that data the size analysis because the fluorescence intensity was a results of and binding to of molecules in The was using the standard in more PI(4,5)P2 of which was to be for of molecules in The was using the standard in more PI(4,5)P2 of which was to be for and with when the titration to with the and the protein of the sample. the of the binding of molecules in The was using the standard in more PI(4,5)P2 of which was to be for was by the amount of or by the amount of PI(4,5)P2 and with when the titration to with the and the protein of the sample. the of the binding of molecules in The was using the standard in more PI(4,5)P2 of which was to be for was by the amount of or by the amount of PI(4,5)P2 and with when the titration to with the and the protein of the sample. the of the binding and with when the titration to with the and the protein of the sample. the of the binding the average of experiments standard data liposomes with a analysis because the fluorescence are the and with when the titration to with the and the protein of the sample. the of the binding of molecules in The was using the standard in more PI(4,5)P2 of which was to be for The was by the amount of or by the amount of PI(4,5)P2 in a new the average of experiments standard data liposomes with a analysis because the fluorescence are the of the of the method described here is that it allows for monitoring both binding based on different of membrane curvature and changes in liposome In the protein binding to liposomes be by in the as well as when size are shown in as this is not the for or In the protein binding changes the a liposome can to a different size in the size the size are defined for particles and result in a different scatter signal and size a liposome in signal intensity of the fluorescent membrane a These membrane can be by defining a based on fluorescence that a in lipid molecules liposome size fluorescence directly with the amount of lipid molecules in liposomes of a protein binding changes this this that a liposome of more lipids a liposome of the measured for example, curvature or liposome tethering has a may the of membrane for example, liposomes have a different as liposomes. The of the is the average of the for measured size For a on the of the events with a can be enriched through a and to further analysis for this by the titration of and to liposomes, including as a the overall is liposomes are at protein This is with the of to membranes, when containing amounts of and Lemmon M.A. Membrane of the pleckstrin homology domain.Biochem. J. 2005; PubMed Scopus Google Scholar). also a for The in size at protein be as either an liposome is to or liposome The was in the of by methods not a a few events in the are by a These events have a and a In a distribution of events is between size the changes in membrane curvature. in lipid protein interactions can a variety of each of which with both and The assay described in this study a as it of the of experimental systems but of their Membrane lipids are presented in their natural environment as of a bilayer immobilized on of a A of membrane lipids natural and can be used to bilayers of a defined lipid composition to mimic lipid of membranes on the surface of liposomes. The new assay does not or flotation This loss of multistep and allows for of protein binding to liposomes. measurements are a few seconds sample. a flow with a allows for the analysis for example, samples in a in this of for example, can be to of a protein-lipid The other large of proteins can be for potential lipid binding The amount of required is For example, the procedure to the of with liposomes only PI(4,5)P2 and of protein A measurement required an of is for individual measurements in the method is qualitative in an titration can be such measurements, constants can be the quantitative binding can be as In this we measured one protein at a time using proteins. on the use of fluorescent such as to proteins and the of multiple channels in flow cytometry, competitive binding studies on are Additionally, using such as fluorescent protein and fluorescent by binding to membranes be measured by fluorescence resonance using a flow distinct liposome containing different fluorescently labeled lipids or for analysis are A of two distinct liposome each containing specific membrane lipids, be to tethering of liposomes. to the of fluorescent proteins and lipids as well as light assay has the potential to the of protein binding to membranes, such as the of membrane curvature and membrane For further liposomes with specific properties can be enriched through the use of a In we a system to directly binding and surface of multiple proteins membrane lipids, without the suited for applications and with only in of amounts of proteins and lipids. Using appropriate the assay also allows for the of The and for the and for the of are to and for the analysis of liposomes by and to for of the