Abstract

The localization of many membrane proteins within cholesterol- and sphingolipid-containing microdomains is essential for proper cell signaling and function. These membrane domains, however, are too small and dynamic to be recorded, even with modern super-resolution techniques. Therefore, the association of membrane proteins with these domains can only be detected with biochemical assays that destroy the integrity of cells require pooling of many cells and take a long time to perform. Here, we present a simple membrane fluidizer–induced clustering approach to identify the phase-preference of membrane-associated molecules in individual live cells within 10–15 min. Experiments in phase-separated bilayers and live cells on molecules with known phase preference show that heptanol hyperfluidizes the membrane and stabilizes phase separation. This results in a transition from nanosized to micronsized clusters of associated molecules allowing their identification using routine microscopy techniques. Membrane fluidizer-induced clustering is an inexpensive and easy to implement method that can be conducted at large-scale and allows easy identification of protein partitioning in live cell membranes.

Supplementary keywordsAbbreviations: CTxB (cholera toxin subunit B), DiI-C18 (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate), DMEM (Dulbecco's Modified Eagle Medium), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), EGFR (epidermal growth factor receptor), EMCCD (electron multiplying charge-coupled device), FCS (fluorescence correlation spectroscopy), GPI (glycosylphosphatidylinositol), mEGFP (monomeric enhanced green fluorescence protein), MFIC (membrane fluidizer-induced clustering), PM (plasma membrane), trLAT (transmembrane domain of linker for activation of T-cells)Plasma membranes (PMs) are complex entities composed of a diversity of lipids and proteins that associate in different combinations, thereby resulting in PM heterogeneities. These heterogeneities exist in the form of protein clusters, lipid–lipid complexes, or combinations thereof, e.g., cholesterol sphingolipids or protein–lipid complexes (1Lipid rafts as a membrane-organizing principle., 2Lingwood D. Kaiser H.J. Levental I. Simons K. Lipid rafts as functional heterogeneity in cell membranes.). In addition to the preferential interaction of these molecules with each other, there is a dynamic cytoskeleton network and extracellular matrix that influences the spatial organization of heterogeneities in the PMs (3Kusumi A. Suzuki K.G.N. Kasai R.S. Ritchie K. Fujiwara T.K. Hierarchical mesoscale domain organization of the plasma membrane., 4Freeman S.A. Vega A. Riedl M. Collins R.F. Ostrowski P.P. Woods E.C. et al.Transmembrane pickets connect cyto- and pericellular skeletons forming barriers to receptor engagement.). PMs are extremely dynamic and are highly susceptible to change their physical properties and organization. Upon interaction with membrane-active compounds, such as peptides or anesthetics, membranes undergo a reversible modulation of spatial organization and membrane order (5Brown E.N. Purdon P.L. Van Dort C.J. General anesthesia and altered states of arousal: a systems neuroscience analysis., 6Chitilian H.V. Eckenhoff R.G. Raines D.E. Anesthetic drug development: novel drugs and new approaches., 7Mitchell D.C. Lawrence J.T.R. Litman B.J. Primary alcohols modulate the activation of the G protein-coupled receptor rhodopsin by a lipid-mediated mechanism., 8Hulse D. Kusel J.R. O'Donnell N.G. Wilkinson P.C. Effects of anaesthetics on membrane mobility and locomotor responses of human neutrophils., 9Gupta A. Marzinek J.K. Jefferies D. Bond P.J. Harryson P. Wohland T. The disordered plant dehydrin Lti30 protects the membrane during water-related stress by cross-linking lipids., 10The membrane actions of anesthetics and tranquilizers., 11Ingólfsson H.I. Andersen O.S. Alcohol’s effects on lipid bilayer properties., 12Modulation and dynamics of cell membrane heterogeneities.). Membrane proteins often reside in a specific lipid environment in their resting state, and they change their environment upon activation (13Bag N. Huang S. Wohland T. Plasma membrane organization of epidermal growth factor receptor in resting and ligand-bound states.). With increasing evidence, it is now realized that dynamic changes in the lipid environment of these proteins are essential for their functionality and regulation. The lipid environment of signaling proteins is hypothesized to influence the signal transduction originating at the PM (14Cheng P.C. Dykstra M.L. Mitchell R.N. Pierce S.K. A role for lipid rafts in B cell antigen receptor signaling and antigen targeting., 15Stone M.B. Shelby S.A. Nńñez M.F. Wisser K. Veatch S.L. Protein sorting by lipid phase-like domains supports emergent signaling function in B lymphocyte plasma membranes., 16Escribá P.V. Wedegaertner P.B. Goñi F.M. Vögler O. Lipid-protein interactions in GPCR-associated signaling.). It was recently shown that specific structural characteristics of proteins such as palmitoylation, length of the transmembrane segment, and type of amino acids in the transmembrane region could determine their preference for a certain phase (17Lorent J.H. Diaz-Rohrer B. Lin X. Spring K. Gorfe A.A. Levental K.R. et al.Structural determinants and functional consequences of protein affinity for membrane rafts.). However, the identity of the lipid environment surrounding the signaling proteins remains controversial, primarily because most of the existing literature relies on data obtained from indirect and artifact-prone methods (18Kwik J. Boyle S. Fooksman D. Margolis L. Sheetz M.P. Edidin M. Membrane cholesterol, lateral mobility, and the phosphatidylinositol 4,5-bisphosphate-dependent organization of cell actin., 19Have we become overly reliant on lipid rafts? Talking point on the involvement of lipid rafts in T-cell activation.). The reason why indirect methods have been used to detect membrane domains is that the size of domains is typically below the diffraction limit, and thus, they are inaccessible by routine imaging methods. Moreover, due to their dynamic nature, membrane domains typically last from microseconds to seconds and are often difficult to detect.To examine the complex PM structure and dynamics, artificially reconstituted model membranes have contributed significantly (20Maula T. Al Sazzad M.A. Slotte J.P. Influence of hydroxylation, chain length, and chain unsaturation on bilayer properties of ceramides., 21Benda A. Beneš M. Mareček V. Lhotský A. Hermens W.T. Hof M. How to determine diffusion coefficients in planar phospholipid systems by confocal fluorescence correlation spectroscopy., 22Honigmann A. Mueller V. Ta H. Schoenle A. Sezgin E. Hell S.W. et al.Scanning STED-FcS reveals spatiotemporal heterogeneity of lipid interaction in the plasma membrane of living cells., 23García-Sáez A.J. Chiantia S. Schwille P. Effect of line tension on the lateral organization of lipid membranes., 24Chiantia S. Kahya N. Ries J. Schwille P. Effects of ceramide on liquid-ordered domains investigated by simultaneous AFM and FCS., 25Maté S. Busto J.V. García-Arribas A.B. Sot J. Vazquez R. Herlax V. et al.N-Nervonoylsphingomyelin (C24:1) prevents lateral heterogeneity in cholesterol-containing membranes., 26Benda A. Fagul'ová V. Deyneka A. Enderlein J. Hof M. Fluorescence lifetime correlation spectroscopy combined with lifetime tuning: new perspectives in supported phospholipid bilayer research., 27Bacia K. Scherfeld D. Kahya N. Schwille P. Fluorescence correlation spectroscopy relates rafts in model and native membranes., 28Honigmann A. Sadeghi S. Keller J. Hell S.W. Eggeling C. Vink R. A lipid bound actin meshwork organizes liquid phase separation in model membranes., 29Sezgin E. Levental I. Grzybek M. Schwarzmann G. Mueller V. Honigmann A. et al.Partitioning, diffusion, and ligand binding of raft lipid analogs in model and cellular plasma membranes.). However, they cannot recapitulate every physiologically relevant attribute. For instance, although model membranes can be tuned to exhibit micron-sized domains of a specific phase (liquid disordered and liquid-ordered), it is nearly impossible to visualize domains directly in cell membranes as the domains in PMs are much smaller and are very sensitive, e.g., even giant plasma membrane vesicles do not keep the same organization as a live cell PM (1Lipid rafts as a membrane-organizing principle., 17Lorent J.H. Diaz-Rohrer B. Lin X. Spring K. Gorfe A.A. Levental K.R. et al.Structural determinants and functional consequences of protein affinity for membrane rafts., 30Su X. Ditlev J.A. Hui E. Xing W. Banjade S. Okrut J. et al.Phase separation of signaling molecules promotes T cell receptor signal transduction.). Therefore, there is a clear need of simple methods that can detect the phase preference of membrane proteins in live intact cell membranes. Biochemical methods that have been used to differentiate the proteins that localize in the raft and nonraft phases include detergent-resistant extraction (31Insolubility of lipids in Triton X-100: physical origin and relationship to sphingolipid/cholesterol membrane domains (rafts).), immunostaining (32Schnell U. Dijk F. Sjollema K.A. Giepmans B.N.G. Immunolabeling artifacts and the need for live-cell imaging.), and cell fractionation followed by mass spectrometry (33Applications of mass spectrometry to lipids and membranes.). However, these methods are artifact-prone as either they involve the use of nonphysiological experimental conditions or require fixed samples. Due to these reasons, fluorescence-based methods combined with live-cell imaging and spectroscopy (e.g., FCS diffusion law) are alternatives for determining the membrane heterogeneities (34Gupta A. Korte T. Herrmann A. Wohland T. Plasma membrane asymmetry of lipid organization: fluorescence lifetime microscopy and correlation spectroscopy analysis., 35Gupta A. Muralidharan S. Torta F. Wenk M.R. Wohland T. Long acyl chain ceramides govern cholesterol and cytoskeleton dependence of membrane outer leaflet dynamics., 36Huang S. Lim S.Y. Gupta A. Bag N. Wohland T. Plasma membrane organization and dynamics is probe and cell line dependent., 37Plasma Membrane Order; the Role of Cholesterol and Links to Actin Filaments., 38Gidwani A. Holowka D. Baird B. Fluorescence anisotropy measurements of lipid order in plasma membranes and lipid rafts from RBL-2H3 mast cells.). In previous studies, phase-specific fluorescent dyes and protein anchors have been used to understand the dynamic properties of the individual phases in live cell membranes (36Huang S. Lim S.Y. Gupta A. Bag N. Wohland T. Plasma membrane organization and dynamics is probe and cell line dependent., 39Klymchenko A.S. Kreder R. Fluorescent probes for lipid rafts: from model membranes to living cells.). Despite the wide usage of such methods, they have been difficult to implement due to the requirement of specialized instrumentation, and they also have some exceptions that pose problems in their interpretation (40Gupta A. Phang I.Y. Wohland T. To hop or not to hop: exceptions in the FCS Diffusion law., 41Šachl R. Bergstrand J. Widengren J. Hof M. Fluorescence correlation spectroscopy diffusion laws in the presence of moving nanodomains., 42Sevcsik E. Brameshuber M. Fölser M. Weghuber J. Honigmann A. Schütz G.J. GPI-anchored proteins do not reside in ordered domains in the live cell plasma membrane.).

In this work, we present a novel membrane fluidizer–induced clustering (MFIC) methodology to determine the localization of molecules in live cell membranes. In this study, we use heptanol as a membrane-fluidizing agent and show using total internal reflection fluorescence microscopy (TIRFM) that there is reversible reorganization in the cell membrane as a result of heptanol treatment. The molecules that reside in cholesterol-dependent domains segregate into micron-sized clusters, while molecules that reside outside the cholesterol-dependent domains stay dispersed. We test this assay in both model membranes and live intact cell membranes using several molecules with known phase preference. Moreover, we use this method to probe the localization of signaling-related proteins such as epidermal growth receptor factor (EGFR), IL-2Rɑ, K-Ras, and H-Ras. Furthermore, we test this method on other cell lines to ensure the universality of this method across different cell membranes. This method is an inexpensive, fast (∼min), and minimally invasive way to determine whether a molecule resides within lipid domains in live cells.

Materials and methodsReagents

The lipids 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and cholesterol (Chol) were used in this work. Head group–labeled rhodamine dye 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (14:0 Liss Rhod PE) was used as the fluorophore to label supported lipid bilayers. The lipids and dye were purchased from Avanti Polar Lipids (Alabama) and dissolved in chloroform.

1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI-C18, #D3911), octadecyl rhodamine B chloride (#O246), and CTxB-555 (cholera toxin subunit B [recombinant] with Alexa Fluor 555 conjugate, #C34776) were purchased from Invitrogen (Thermo Fisher Scientific, Singapore). They were dissolved in anhydrous dimethyl sulfoxide (#276855, Sigma-Aldrich, Singapore) to prepare the stock solutions. The fluidizer 1-heptanol 98% (#H2805) was purchased from Sigma-Aldrich (Singapore).

An Alexa Fluor 488–conjugated EGFR monoclonal antibody was purchased from Cell Signaling Technology (EGF Receptor [D38B1] XP Rabbit mAb [Alexa Fluor 488 Conjugate], #5616S, MA).

Supported lipid bilayer preparation

All glassware (slides, coplin jars, and round-bottom flasks) were cleaned thoroughly with an alkaline cleaning solution (Hellmanex III, Hellma Analytics, Müllheim, Germany) using sonication (Elmasonic S30H, Elma Schmidbauer GmbH, Singen, Germany) for 30 min. They were then washed with ultrapure water (Milli-Q, Merck, NJ), submerged in 2 M sulfuric acid, and sonicated again for 30 min. After washing the glassware with deionized water and immersing them in the water, a final sonication was done for another 30 min.

A silicone elastomer (SYLGARD 184 Silicone Elastomer Kit, Dow, MI) was filled in an O-ring mold and cured at 65°C overnight. The O-rings (1.5 cm inner diameter) were carefully removed using forceps and attached to a slide using the silicone elastomer, followed by curing at 65°C for 3 h.

DOPC:DPPC:Chol (4:3:3) solution (500 μM) and 100 nM 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) were mixed thoroughly in a round-bottom flask and evaporated in a rotary evaporator (Rotavapor R-210, Büchi, Flawil, Switzerland) for 3–4 h until a thin lipid film was formed. The lipid film was dissolved in 2 ml of a buffer solution (10 mM Hepes, 150 mM NaCl, pH 7.4) and sonicated until the solution became clear, indicating the formation of large unilamellar vesicles. Lipid solution (200 μl) was added into an O-ring attached to a slide and incubated at 65°C for 1 h to allow vesicle fusion and formation of the supported lipid bilayer (SLB). The SLB was cooled to room temperature (25°C) for 30 min and then washed with the buffer solution multiple times to eliminate the unfused vesicles. SLB measurements were done at 37°C.

PlasmidsThe green fluorescent protein–tagged glycosylphosphatidylinositol-anchored protein (GPI-GFP plasmid) was a kind gift of John Dangerfield (Anovasia Pte Ltd., Singapore). The plasmids IL2Rα-EGFP (Addgene plasmid #86055), mEGFP-HRas (Addgene plasmid #18662), and pLVET-HA-K-RasG12V-IRES-GFP (Addgene plasmid #107140) were purchased from Addgene (MA). The construction of EGFR-mApple has been previously described (43Sankaran J. Balasubramanian H. Tang W.H. Ng X.W. Röllin A. Wohland T. Simultaneous spatiotemporal super-resolution and multi-parametric fluorescence microscopy.). The EGFR-mEGFP plasmid was constructed in the same way as EGFR-mApple. Lifeact-mRFPruby (Addgene plasmid #51009; a gift originally from Rusty Lansford) was gifted by Wu Min (CBIS, NUS) (44Riedl J. Crevenna A.H. Kessenbrock K. Yu J.H. Neukirchen D. Bista M. et al.Lifeact: a versatile marker to visualize F-actin.).The sequences of the transmembrane domain of linker for activation of T-cells (WT-trLAT) and a mutant with all the transmembrane domain (TMD) amino acids (except the palmitoylation sites) mutated to leucines (allL-trLAT) have been previously described (45Diaz-Rohrer B.B. Levental K.R. Simons K. Levental I. Membrane raft association is a determinant of plasma membrane localization.). DNA duplexes were designed with the trLAT sequences flanked by AgeI and SpeI restriction sites on the 5′ and 3′ ends, respectively, with a 6-base linker between them. The duplex DNA sequences were synthesized and purchased from Integrated DNA Technologies Pte. Ltd. (Singapore). The EGFR-mApple and EGFR-mEGFP plasmids were digested with AgeI (AgeI-HF, R3552S, New England BioLabs, MA) and SpeI (SpeI-HF, R3133S, New England BioLabs) to create the plasmid backbones. The WT-trlAT and allL-trLAT sequences were also digested with AgeI and SpeI to create the inserts. The backbones and inserts were ligated using T4 DNA ligase (M0202S; New England BioLabs) to create four plasmids—WT-trLAT-mEGFP, WT-trLAT-mApple, allL-trLAT-mEGFP, and allL-trLAT-mApple.Cell cultureThe protocol detailing the steps in the preparation of live cell samples for fluorescence applications is provided in Protocol Exchange (46Sankaran J. Balasubramanian H. Tang W.H. Ng X.W. Röllin A. Wohland T. Preparation of live cell samples for fluorescence spectroscopy and computational super-resolution imaging.). SH-SY5Y (#CRL-2266) and HeLa (#CCL-2) cells were obtained from ATCC (Manassas, VA). They were cultivated in Dulbecco's Modified Eagle Medium (DMEM/High glucose with L-glutamine, without sodium pyruvate—#SH30022.FS) (HyClone, GE Healthcare Life Sciences, UT) supplemented with 10% fetal bovine serum (FBS; #10270106, Gibco, Thermo Fisher Scientific, Singapore) and 1% penicillin-streptomycin (#15070063, Gibco, Thermo Fisher Scientific), at 37°C in a 5% (v/v) CO2 humidified environment (Forma Steri-Cycle CO2 incubator, Thermo Fisher Scientific).

Cell cultures that were ∼90% confluent were passaged. The spent media were removed from the culture flask and 5 ml 1× PBS (phosphate-buffered saline; without Ca2+ and Mg2+) was used to wash the cells twice. TrypLE Express Enzyme (2 ml) (1×; #12604021, Gibco, Thermo Fisher Scientific, Singapore) was added, and the flask was placed in the CO2 incubator for 2–3 min. Upon detachment of the cells, 5 ml culture media were added to the flask to inhibit trypsin. The cell suspension was centrifuged (#5810, Eppendorf, Hamburg, Germany) at 200 g for 3 min. The supernatant was discarded, and the cell pellet was resuspended in 5 ml 1× PBS. An automated cell counter (TC20, Bio-Rad, Singapore) was used to count the cells, and the required number of cells was used for the next step of cell membrane staining or transfection.

Cell membrane staining

After passaging, the required number of cells were seeded onto culture dishes (#P35G-1.5-20-C, MatTek, MA) containing culture media and allowed to attach for 24 h. DiI-C18, R18, and CTxB-555 stock solutions were diluted to 100 nM working concentration in imaging medium (DMEM with no phenol red, #21063029, Gibco, Thermo Fisher Scientific) supplemented with 10% FBS. They were used for cell membrane staining. The media were removed and replaced with the working dye solution. The cells were placed in the CO2 incubator for 20 min. Then, the dye solution was removed, and the cells were washed with 1× HBSS (Hank’s Balanced Salt Solution, with Ca2+ and Mg2+; #14025134; Gibco, Thermo Fisher) twice. DMEM without phenol red (#21063029; Gibco, Thermo Fisher Scientific, MA), hereafter called imaging media, was supplemented with 10% FBS and added to the cells before measurements.

Transfection

After passaging, the required number of cells was centrifuged at 200 g for 3 min. The supernatant was discarded, and the cells were resuspended in R buffer (Neon Transfection Kit, Thermo Fisher Scientific). Suitable amounts of plasmids were mixed with the cells for transfection. The cells were electroporated according to the manufacturer’s protocol (electroporation settings: SH-SY5Y – pulse voltage = 1,200 V, pulse width = 20 ms, pulses = 3; HeLa – pulse voltage = 1,005 V, pulse width = 35 ms, pulses = 2) using Neon Transfection system (Thermo Fisher Scientific). After transfection, the cells were seeded onto culture dishes containing DMEM (with 10% FBS; no antibiotics). The cells were incubated in the CO2 incubator for 20–48 h before the measurements.

Cell measurements

The transfected cells were washed twice with HBSS, and imaging media (with 10% FBS) were added before measurements. EGFR-transfected cells were starved for a few hours in imaging media (without FBS to prevent aberrant activation of EGFR) before measurements. Stock heptanol solution was filtered with a 0.2 μm syringe filter and added to the imaging media to obtain the working concentration of 5 mM. Measurements were done after 10–20 min of incubation.

For the two-color EGFR antibody measurements, the antibody was diluted 1:200 in imaging media and added to EGFR-transfected cells. The cells were incubated for 3 h in the CO2 incubator. Subsequently, they were washed with HBSS twice, and imaging media were added. After initial imaging of the antibody labeling, 5 mM heptanol was added to the cells, and they were imaged.

Cell viability determined through trypan blue staining

To determine fraction of live cells in the sample before and after heptanol treatment, the samples were trypsinized, and trypan blue stain was mixed with the cells (Bio-Rad). The cells were counted using an automated cell counter (TC20, Bio-Rad) which provided the live cell fraction values.

Western blotting analysis of EGFR phosphorylation

Four 10 cm cell culture dishes (#353003, Corning, NY) were each seeded with 2 × 105 CHO-K1 cells transfected with 10 μg of EGFR-mApple. A mock transfection without any plasmid was also done. The transfected cells were incubated at 37°C with 5% CO2 for 36 h. The cells were then washed with 1× HBSS and serum-starved in imaging DMEM for 4 h. Following this, three of the transfected samples were treated with 5 mM heptanol for 15 min, 100 ng/ml EGF for 20 min, and 5 mM heptanol for 15 min followed by 100 ng/ml EGF for 20 min, respectively.

A Western blot kit (#12957, Western Blotting Application Solutions Kit, Cell Signaling Technology) was used for performing the Western blots as per the manufacturer’s protocol. All the cells were lysed using the provided cell lysis buffer, and the cell extracts were sonicated using an ultrasonicator (VC 505, Sonics, CT). Sodium dodecyl sulphate–polyacrylamide gel electrophoresis was used to separate the proteins in the samples. Two 4%–20% precast polyacrylamide gels (#4561093, Mini-PROTEAN TGX Precast Protein Gels (10-well, 30 μl), Bio-Rad, CA) were processed in parallel—one gel was for probing with total EGFR primary antibody and the other gel with phosphorylated EGFR primary antibody. A 10–250 kDa protein ladder (#1610373, Precision Plus Protein All Blue Prestained Protein Standards, Bio-Rad, CA) was also loaded along with the samples.

The protein bands were wet-transferred from each PAGE gel to a PVDF membrane (0.45 μm pore size). In each sample set of two membranes, one membrane was incubated in a primary antibody solution containing total EGFR polyclonal antibody (#2232S, Cell Signaling Technology) and β-actin polyclonal antibody (#4967S, Cell Signaling Technology). The other membrane was incubated in a primary antibody solution containing phospho-EGFR (Y1173) monoclonal antibody (#4407S, Cell Signaling Technology) and β-actin polyclonal antibody. This was followed by washing, blocking, and incubation in the secondary antibody (#7074S, Cell Signaling Technology). An enhanced chemiluminescent substrate solution (#6883, SignalFire ECL Reagent, Cell Signaling Technology) was used, and the chemiluminescence was detected using an imager (ImageQuant LAS 4000, GE Healthcare Bio-Sciences AB, Uppsala, Sweden) equipped with a CCD camera. The CCD camera was operated after cooling to −25°C, and the images were saved as 16 bit tiff files.

Calculation of EGFR phosphorylation levelsThe intensity counts were of the bands were first corrected for the background (area on membrane with no bands).

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