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Published as doi: 10.1096/fj.08-122192.
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(The FASEB Journal. 2009;23:1775-1785.)
© 2009 FASEB

Functional implications of the influence of ABCA1 on lipid microenvironment at the plasma membrane: a biophysical study

Ana Zarubica*,1, Anna Pia Plazzo{dagger},1, Martin Stöckl{dagger}, Tomasz Trombik*, Yannick Hamon*, Peter Müller{dagger}, Thomas Pomorski{dagger}, Andreas Herrmann{dagger} and Giovanna Chimini*,2

* Centre d’Immunologie de Marseille Luminy, Institut National de la Santè et de la Recherche Médicale, Centre National de la Recherche Scientifique, Université de la Méditerranée, Parc Scientifique de Luminy, Marseille, France; and

{dagger} Institut für Biologie/Biophysik, Humboldt-Universität zu Berlin, Berlin, Germany

2Correspondence: Centre d’Immunologie de Marseille Luminy, Parc Scientifique de Luminy case 906, 13288 Marseille, Cedex 09 France. E-mail: chimini{at}ciml.univ-mrs.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The ABCA1 transporter orchestrates cellular lipid homeostasis by promoting the release of cholesterol to plasmatic acceptors. The molecular mechanism is, however, unknown. We report here on the biophysical analysis in living HeLa cells of the ABCA1 lipid microenvironment at the plasma membrane. The modifications of membrane attributes induced by ABCA1 were assessed at both the outer and inner leaflet by monitoring either the lifetime of membrane inserted fluorescent lipid analogues by fluorescence lifetime imaging microscopy (FLIM) or, respectively, the membrane translocation of cationic sensors. Analysis of the partitioning of dedicated probes in plasma membrane blebs vesiculated from these cells allowed visualization of ABCA1 partitioning into the liquid disordered-like phase and corroborated the idea that ABCA1 destabilizes the lipid arrangement at the membrane. Specificity was demonstrated by comparison with cells expressing an inactive transporter. The physiological relevance of these modifications was finally demonstrated by the reduced membrane mobility and function of transferrin receptors under the influence of an active ABCA1. Collectively, these data assess that the control of both transversal and lateral lipid distribution at the membrane is the primary function of ABCA1 and positions the effluxes of cholesterol from cell membranes downstream to the redistribution of the sterol into readily extractable membrane pools.—Zarubica, A., Plazzo, A. P., Stöckl, M., Trombik, T., Hamon, Y., Müller, P., Pomorski, T., Herrmann, A., Chimini, G. Functional implications of the influence of ABCA1 on lipid microenvironment at the plasma membrane: a biophysical study.


Key Words: FLIM • TfR • cholesterol • phosphatidylserine • FRAP


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE ABCA1 TRANSPORTER IS A KEY controller of cellular lipid handling, and the loss of its function in humans leads to the development of a major perturbation of reverse cholesterol transport known as Tangier disease (1) . The physiological evidence attributes to ABCA1 the ability to control cellular effluxes of phospholipids and cholesterol to external acceptors such as apolipoproteins. This implies that ABCA1 acts as a lipid transporter (2) ; yet, formal evidence addressing the specific transport function is missing (3 , 4) .

It is generally accepted that an intact ABCA1 is required for significant lipid effluxes from the cell membrane to plasmatic acceptors. The prominent function of ABCA1 in this process is the phospholipidation of nascent apolipoproteins, which, only then, acquire competence to accept membrane cholesterol (5) . How the ABCA1-dependent lipid loading takes place and how it is related to cholesterol removal remain, however, unanswered questions.

Similarly, the contribution of the spatial distribution of cholesterol in the plasma membrane or of its embedding lipid environment to the efficiency of its own membrane desorption has been only partially elucidated (6 7 8 9 10) .

Previous work (11 12 13 14) has shown that the expression of ABCA1, or its loss, modifies the distribution of anionicphospholipids between membrane leaflets. These studies point out that lipidation of apolipoproteins requires not only the presence of ABCA1 but also the presence of specific ABCA1-dependent modifications of the lipid phase in the exoplasmic membrane leaflet.

Lipid flipping, the translocation of phospholipids from the inner to the outer leaflet, would, in this view, constitute the essential function of the transporter and lead, secondarily, to increased cholesterol bioavailabity; however, this has been recently challenged by other reports (15 , 16) .

In the attempt to further clarify the issue, we set out to assess the effect of ABCA1 on lipid architecture at the membrane by novel biophysical techniques applied to living cells.

The extent of the ABCA1-induced modification on the attributes of both the inner and outer membrane leaflets was assessed by the evaluation, in transfected HeLa cells, of membrane translocation of cationic probes developed by Yeung et al. (17) as sensors of the inner membrane potential and by fluorescence lifetime imaging (FLIM) analysis of lipid analogues as probes of the density of lipid arrangement in the exoplasmic membrane leaflet (18 19 20) . In both cases, significant modifications likely to affect the arrangement of lipid domains were evidenced. Indeed, in giant plasma membrane "blebs" vesiculated from the same cell systems, we quantitatively assessed a reduction in the size of the cholesterol-rich, tightly packed liquid ordered-like phases at the membrane related to the presence of an active transporter. These data, further corroborated by the widely used biochemical isolation of membrane domains, substantiate the evidence that the ATPase activity of ABCA1 provokes redistribution of lipids between contiguous phases at the membrane (21 , 22) . Repartitioning of membrane proteins, including the transporter itself, ensues; here the example of the physical and functional changes induced by the presence of ABCA1 on membrane transferrin receptor (TfR) was analyzed.

Collectively, our data provide evidence that the ABCA1-induced changes in the transversal and lateral arrangement of membrane lipids are of consequence for both the spatial organization of proteins and the physicochemical properties of the plasma membrane. Furthermore, the study strongly corroborates the view of an essential role of lipid architecture in the priming of the cell surface for apolipoprotein-mediated cholesterol release and provides a novel insight into the mechanisms governing availability of cholesterol into chemically active and extractable pools (23) .


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals
All chemicals for sample preparation, as well as all solvents (CHCl3, CH3OH, and CH3CN) were obtained in highest commercially available purity from Fluka (Taufkirchen, Germany) and used as supplied.

Plasmids, cell culture, and transfection
HeLa Tet off cells (Clontech, Mountain View, CA, USA) were grown in Dulbecco modified Eagle medium (DMEM), 10% fetal calf serum (Gibco Life Technologies, Carlsbad, CA, USA), penicillin, streptomycin, and 1% sodium pyruvate (all from Invitrogen, Karlsruhe, Germany). Jurkat cells were grown in suspension below a concentration of 2 x 105 cells/ml in RPMI 1640 medium supplemented with 2 mM L-glutamine (both Biochrom, Berlin, Germany), 2 g/L sodium pyruvate, and 10% fetal calf serum. Plasmids containing ABCA1 or its variants, fused with enhanced green fluorescent protein (EGFP), enhanced yellow fluorescent protein (EYFP), or enhanced cyan fluorescent protein (ECFP), were generated in pBI vector (Clontech; ref. 12 ). Ced-7 cDNA (kindly provided by R. Horvitz, Massachusetts Institute of Technology, Boston, MA, USA) was engineered to include an EGFP tailpiece in pBI (24) . All constructs were verified by sequencing. Transient transfections were performed on a 60% confluent monolayer of HeLa cells with 5 µg of DNA in EXGEN 500 (Euromedex, Souffelweyersheim, France) (12) . Transfection efficiency, monitored at 24 h post-transfection by flow cytometry, was consistently at ~40%.

Multiple simultaneous transfections were performed with 5 µg of ABCA1 EYFP or its mutant (ABCA1MM) and 1.5 µg of cationic probes (a kind gift of S. Grinstein, University for Sick Children, Toronto, ON, Canada). For fluorescence recovery after photobleaching (FRAP) experiments, 5 µg of ABCA1-ECFP or its mutant and 1 µg of human transferrin receptor (hTfR) grafted with EGFP [a kind gift of D. Marguet, Centre d’Immunologie de Marseille Luminy (CIML), Marseille, France] were cotransfected. Surface binding of annexin V and ApoA-I was carried out at 60 h post-transfection as described previously (11) .

For treatment of Jurkat cells, 0.5 x 106 cells were pelleted (5 min; 200 g). For activation with OKT3 antibody, the pellet was resuspended in 25 µl medium and 25 µl OKT3 for 20 min at 37°C, washed, and incubated with 200 µl medium containing 4 µl TRITC-labeled anti-mouse IgG (diluted 1:100, Sigma, Taufkirchen, Germany) for 10 min. For sphingomyelinase (SMase) treatment, the pellet was resuspended in 200 µl medium containing 0.1 U/ml sphingomyelinase (from Staphylococcus aureus; Sigma) and incubated for 30 min at 37°C. Jurkat cells and the OKT3 antibody were kindly provided by Christian Freund (FMP, Berlin, Germany).

Antibodies
The molecules were detected with antibodies purchased as follows: flotillin-1, caveolin, and LAMP 2 (BD Biosciences; Le Pont de Claix, France; Pharmingen, San Diego, CA, USA); β-COP (Sigma); and calnexin (Stressgen, Ann Arbor, MI, USA). Antibodies against hTfR were a kind gift of P. Pierre (CIML, Marseille, France). Protein blots were probed with horseradish perioxidase-conjugated secondary antibodies (Jackson Research Laboratories, West Grove, PA, USA) and detected with ECL reagents (Amersham, Little Chalfont, UK).

Giant plasma membrane vesicles (GPMVs)
GPMVs or blebs were prepared from almost confluent HeLa cells either untransfected or expressing ABCA1 or ABCA1MM fused with EGFP or ECFP by chemically induced vesiculation as described previously (25 , 26) . Cells grown in T25 flasks were washed twice with GPMV buffer (2 mM CaCl2, 10 mM HEPES, and 0.15 M NaCl pH 7.4). To each flask, 1.5 ml of freshly prepared GPMV reagent (25 mM formaldehyde and 2 mM dithiothreitol in GPMV buffer) was added. Flasks were incubated for 1 h at 37°C while being slowly shaken (60–80 cycles/min). After incubation, GPMVs that had detached from the cells were gently decanted into a conical glass tube and allowed to settle on ice. Before imaging, R18 (octadecylrhodamin-B-chlorid; Invitrogen, Karlsruhe, Germany) was added at a concentration of ~0.5 µM to the GMPV suspension. Images of the equatorial plane of the blebs were taken at 25°C with temperature controlled with a water circulating bath. The relative domain size was calculated by approximating the vesicle to a circumference and calculating the length of the cord covered by the liquid-disordered (Ld)-like phase, where 1 is the length of the whole circumference.

Biochemical preparation and analysis of lipid rafts
This was performed accordingly to the detergent-free method (27) . Cells were scraped into base buffer (20 mM Tris-HCl, pH 7.8, and 250 mM sucrose) with 1 mM CaCl2 and 1 mM MgCl2 and then washed and lysed by mechanical shearing. The postnuclear supernatant was collected, diluted in 50% OptiPrep solution (Sigma), and overlaid with a 6–20% OptiPrep gradient in base buffer. After centrifugation at 52,000 g in a Beckman SW60 rotor (Beckman Instruments, Fullerton, CA, USA; 90 min, 4°C), 18 fractions were collected. Equal volumes per fraction were subjected to Western blot analysis for the indicated proteins. Densitometry was performed with AIDA 2.11 software (Raytest, Straubenhardt, Germany).

TfR-mediated endocytosis
HeLa cells (1x105) were labeled with 1:10 dilution of supernatant of OKT9 antibody against hTfR in cold DMEM containing 20 mM HEPES and 1% BSA (buffer A) for 20 min at 4°C. After being extensively washed, cells were incubated for 20 min at 37°C in buffer A (28) . After removal of the supernatant, cells were stained with the appropriate secondary antibody and fixed for 10 min on ice in the presence of 1% paraformaldehyde. Fluorescence-activated cell sorter (FACS) analysis was conducted after gating on EYFP fluorescent cells.

Radiolabeling and cholesterol efflux
Cells were labeled with 0.5 or 1 µCi/ml [3H]cholesterol (GE Healthcare, Freiburg, Germany) in DMEM and 1 mg/ml BSA for 24 h. After exposure to methyl-β-cyclodextrin (MβCD), the medium was collected and centrifuged (1000 g, 7 min). Cells were washed with Dulbecco phosphate-buffered saline and dissolved in 0.1 M NaOH for 30 min at room temperature. Radioactivity in the medium and cells was analyzed by liquid-scintillation counting, and effluxes were calculated as percentage of total counts.

Cholesterol and protein determination
Cholesterol levels were determined using the Amplex-Red cholesterol assay (Molecular Probes, Leiden, The Netherlands). Protein content was quantified with the Micro BCA protein assay kit (Pierce, Rockford, IL, USA).

Confocal microscopy and image analysis
Confocal microscopy was performed on a Zeiss LSM510 META (Carl Zeiss, Oberkochen, Germany) allowing spectral recording of signals or on an inverted Fluoview 1000 microscope (Olympus, Tokyo, Japan) with an x60 (numerical aperture 1.35) oil-immersion objective at 25°C. LSM Image Examiner (Carl Zeiss) or Image J software (U.S. National Institutes of Health, Bethesda, MD, USA) were used to quantify fluorescence intensities from raw images. Relative fluorescence intensity (RFI) was measured in regions of interests (ROIs) of equivalent size. For GPMV imaging, GFP fluorescence was excited with the 488 nm laser line of an Ar-ion laser, while the red R18 fluorescence was excited with a 559 nm diode laser. Emission of GFP and R18 was recorded between 500 and 530 nm and from 570 to 670 nm, respectively. Statistical analysis was performed with GraphPad Prism software (GraphPad, San Diego, CA, USA).

FRAP
Confocal FRAP was performed using a Zeiss LSM510 META allowing spectral recordings of signal (29 30 31) or an Olympus Fluoview 1000 microscope. To avoid spectral interference, the hTfR was transfected as an EGFP chimera in association with ABCA1 or ABCA1MM grafted with ECFP. Experiments were performed between 55–60 h post-transfection when satisfactory expression of both the TfR and the transporter can be achieved. Briefly, FRAP measurements were carried out with x63 (numerical aperture 1.4) Zeiss Plan-Neofluar objective at a digital zoom of 8, a scan speed of 8 µs/pixel, and the pinhole set at 294 µm. Five prebleach measurements of fluorescence intensity were taken using 1% laser power, after which a circular spot of radius 0.5 µm was photobleached using 100% laser power. Photobleaching of TfR was performed on the wild-type or mutant form of ABCA1-expressing cells with a 488-nm argon laser, and fluorescence emission was collected using a 505–530 bandpass filter. For FRAP of C6-NBD-PC, 8 prebleach frames were taken with 1% laser power before bleaching a circular spot of radius 1.2 µm for 10 ms using 100% laser power. Under these conditions, 70–80% of NBD fluorescence was bleached. Care was taken to compare cells with similar expression levels of the wild-type or mutant form of ABCA1 at the membrane. Recovery of fluorescence was recorded at low laser power (1%) until fluorescence reached a steady-state plateau. All FRAP measurements were performed at 25°C to minimize membrane deformations caused by temperature gradients and the influence of membrane traffic events on fluorescent recovery. The recovery time (t1/2), the initial fluorescence intensity (Ii), the postbleach fluorescence intensity (I0), and fluorescence intensity at infinite time after photobleaching (I{infty}) were determined for every experiment using IGORPro software and subsequently averaged. The diffusion coefficient (D) was calculated from the recovery time according to the formula D = {omega}2{gamma}/4t1/2 ({omega}: radius of the laser beam at its point of focus, {gamma}: correction factor for the amount of bleaching; ref. 32 ); the mobile fraction (Mf) was given by Mf = (I{infty}–I0/(Ii–I0) (31) . Statistical analyses were performed with GraphPad Prism software.

Phospholipid labeling, FLIM, and determination of fluorescence lifetimes
Palmitoyl-{6-[(7-nitro-2–1,3-benzoxadiazol-4-yl)amino]hexanoyl}-snglycero-3-phospho choline(C6-NBD-PC) and N-{6-[(7-nitro-2–1,3-benzoxadiazol-4yl)amino]hexanoyl}-D-lactosylb1–1'-sphingosine (C6-NBD lactosyl ceramide) were from Avanti Polar Lipids (Alabaster, AL, USA). HeLa cells (105) grown in glass bottom microwell dishes (MatTek, Ashland, MA, USA) were labeled with a 0.25 µM solution of NBD lipid analog in PBS (or 1.5 µM for FRAP measurements) for 20 min on ice, washed with phosphate buffered saline at 25°C, and immediately analyzed. For labeling of Jurkat cells, cell pellets were resuspended with 200 µl solution of 2.5 µM C6-NBD-PC in RPMI 1640 and incubated for 10 min on ice. To remove the staining solution, the cells were again pelleted and resuspended in 200 µl PBS. Cholesterol depletion was performed by 1 min incubation on ice with 5 mM MβCD (Sigma) before being labeled (22 , 33 , 34) .

Images were taken by confocal laser scanning microscopy using an inverted Fluoview 1000 microscope (Olympus) and an x60 (numerical aperture 1.35) oil-immersion objective at 25°C, if not stated otherwise. Images with a frame size of 512 x 512 pixels were acquired. FLIM images were acquired by a commercial FLIM upgrade kit (PicoQuant, Berlin, Germany). For excitation of NBD, a pulsed diode laser with a wavelength of 468 nm was used (pulse width: 60 ps; pulse frequency: 10 MHz; 4 µs/pixel). Emission was recorded using a 540/40 bandpass filter. Single photons were registered with a single photon avalanche photodiode. For each image, 50–70 frames were acquired; the average photon count rate was ~2–4 x 104 counts/s.

For the analysis of the fluorescence lifetime parameters of NBD analogues in cell membranes, compartments were selected by applying an intensity threshold to exclude fluorescence from background or cytoplasm. If necessary, the selection was refined manually to exclude regions not associated with the membrane. For the selected ROI, an overall fluorescence decay curve was generated by summing up the photons registered for that region. From the decay curve, only the part not affected by the instrument response function was used ("tail-fit"; approximately from 3 ns after beginning of the pulse). With the use of a nonlinear least squares, iterative fitting procedure fluorescence decay curves were fitted as a sum of exponential terms to obtain the fluorescence lifetimes of the NBD group:

Formula
where F(t) is the fluorescence intensity at time t and {alpha}i is a preexponential factor representing the intensity of the time-resolved decay of the component with lifetime {tau}i. Typically, two lifetimes provided sufficient quality of fit when considering fluorescence decays originating from analogues in the plasma membrane. Due to the tail-fit method, the short component {tau}1 detected for NBD might be underestimated. Quality of fits was judged by the distribution of the residuals and the {chi}2 value. For construction of lifetime histograms, 3 x 3 pixels in the selected regions of interest were binned and the fitting procedure described above was repeated for each binned pixel using the lifetime parameters obtained from the overall decay curve as starting parameters. In the histograms, the intensity weighted frequencies ({alpha}i x {tau}i) are plotted.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
ABCA1 reduces the inner membrane potential
The outward redistribution of anionic phospholipids previously associated with the expression of ABCA1 at the cell surface (12 13 14) should in principle affect the inner membrane potential by sequestering negative charges (35) . To test this, we took advantage of the fluorescent cationic probes engineered by Yeung et al. (17) . These modified versions of K-Ras (Fig. 1A ) associate to the inner leaflet of the plasma membrane as a function of the negativity of its charge.


Figure 1
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  		Figure 1. ABCA1 induces a shift in the inner membrane potential. Coexpression of an intact ABCA1 transporter (A1) and the cationic probes K-pre and R-pre reduces their association to the inner membrane leaflet. A) Subcellular localization of surface potential sensitive probes mRFP K-pre and mRFP R-pre expressed in HeLa cells by transfection and detected by confocal imaging. Schematic diagram of the structure and sequence of the probes is at bottom. Amino acids are given in single letter code. B) Effect of ABCA1 expression on the efficiency of membrane translocation of mRFP K-pre and mRFP R-pre. Calculations are detailed in Results. Values represent means ± SE. Table 1 summarizes the whole set of data, including the nonfunctional mutant ABCA1MM.

We relied on our well-established transfection system in HeLa cells and on the availability of the nonfunctional variant of the transporter ABCA1MM, largely studied in the literature (11 , 12 , 36 , 37) . This mutant bears the transition K to M known to interfere with ATP hydrolysis in both nucleotide binding domains. The wild-type transporter and ABCA1MM were expressed as chimeras with fluorescent proteins (EGFP, EYFP, or ECFP) grafted as a C-terminal tailpiece.

To avoid spectral interference in the measurements of membrane association parameters, the cationic probes (R-pre and K-pre) were cotransfected as mRFP chimeras in association with ABCA1 or ABCA1MM tagged with EYFP. Considering the different cellular half-life of the transfected products, we analyzed the cells by fluorescence microscopy at 48 h post-transfection, when satisfactory expression of both the cationic probe and the transporter can be achieved (13 , 17 , 36) . In expressing cells, ROIs were defined at the membrane and in the cytosol to assess the association of the cationic probes in the presence or absence of an intact ABCA1 (Fig. 1B ). Membrane association was estimated according to the equation (RFIPM – RFIcytosol)/RFIPM (17) , where RFI is relative fluorescence intensity. Care was taken to compare cells with similar expression levels of the wild-type or mutant form of ABCA1 at the membrane [intensity of fluorescence signal (expressed as mean±SE; arbitrary units) for K-pre cotransfection: A1=2287±100 (n=24), A1MM=2398±138 (n=19); for R-pre cotransfection: A1=2572±165 (n=31), A1MM=2290±155 (n=18)].

In the case of both R-pre and K-pre, a reduction of membrane association of the probe was evidenced in the presence of an active transporter (Table 1 ). A decreased membrane association of K-pre was also observed in the presence of ABCA1MM. This is likely to derive from overexpression and does not reduce the significance of the ABCA1-specific effect (P<0.0001, A1 vs. A1MM).


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  		Table 1. Analysis of membrane translocation of the charge sensitive probes R-pre and K-pre in the presence of the indicated transporters

These results indicate a lowering in the net negative charge at the inner membrane leaflet specifically generated by ABCA1. Although the formal cause cannot be established in our experimental setup, the loss of negative charge is consistent with removal of anionic phospholipids, such as phosphatidylserine (PS), from the inner leaflet (12 , 35) . This is in turn in agreement with the enhanced exposure of PS on the outer leaflet detected as enhanced annexin-V binding in ABCA1-expressing cells (see below and refs. 12 13 14 ).

ABCA1 activity affects the physicochemical properties of the cell surface
To analyze the effect of ABCA1 on the physical properties of the outer membrane leaflet, we monitored the fluorescence lifetime of C6-NBD lipid analogues incorporated in the plasma membrane of HeLa cells expressing an intact or mutant ABCA1 transporter. It has indeed been shown that the fluorescence lifetime of NBD-labeled lipids can be used to characterize heterogeneous lateral lipid organization and physical properties of the plasma membrane of living cells (18 , 38) .

On incubation of cells with C6-NBD-PC on ice, lipid analogues inserted essentially into the outer leaflet of the plasma membrane (Fig. 2A ). Subsequently, fluorescence decay curves of C6-NBD analogues located in the plasma membrane were measured. Proper fitting of the decay curves was achieved with two lifetime components {tau}1 and {tau}2 centered at ~3 and 11.5 ns, respectively, as previously described (18) . The short component {tau}1, originating from the red edge excitation shift of NBD in membranes (18 , 39) , was of very small amplitude (<5%) and similar in all samples and conditions. Conversely, {tau}2 that is sensitive to lipid environment (18) shifted toward longer values on ABCA1 expression and in comparison to nontransfected HeLa cells or cells expressing ABCA1MM (Fig. 2B and Table 2 ). Moreover, an increase of the width of the distribution of NBD lifetimes was observed in ABCA1 containing plasma membrane, suggesting increased heterogeneity in the microenvironment of the probe (Fig. 2B ). A broader distribution was also observed in the presence of ABCA1MM. This is again likely to derive from overexpression. Indeed the presence of membrane proteins in lipid bilayers can broaden fluorescence lifetime distribution (40 , 41) . A similar behavior was found for C6-NBD lactosyl ceramide (Table 2) .


Figure 2
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  		Figure 2. ABCA1 influences lifetime and mobility of C6-NBD-PC. A) Intensity (top panel) and average lifetime image shown as pseudocolor image as indicated in scale (bottom panel) of HeLa cells labeled with C6-NBD-PC as described in Materials and Methods. Scale bars = 10 µm. B) Histograms of lifetime component {tau}2 of C6-NBD-PC in plasma membrane of untransfected HeLa cells and cells expressing wild-type ABCA1 or its inactive mutant ABCA1MM. Histograms were normalized by setting maximum to 1. Average ± SD of the NBD lifetime calculated as described in Materials and Methods are reported in Table 2 . C) Diffusion of C6-NBD-PC measured by FRAP is slower in the presence of an active ABCA1 as compared with both HeLa and ABCA1MM-expressing cells. D) Fluorescence lifetime of C6-NBD-PC is different in resting (control) and activated Jurkat cells. Cells were activated by incubation with OKT3 antibody or by sphingomyelinase (SMase) treatment as described in Materials and Methods. For each sample, 23 cells were analyzed. Differences between control and treated cells are highly significant (unpaired t test; P<10–7 in each case).


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  		Table 2. Fluorescence lifetimes {tau} of the lipid analogues C6-NBD-PC and C6-NBD lactosyl ceramide

As cholesterol content is a major determinant of lipid packing, we next monitored NBD lifetimes after depletion of cholesterol by MβCD-mediated extraction. This treatment induced a shift of the lifetime distribution toward lower values and reverted the lifetimes to similar values in all groups (Table 2) .

In parallel, the subcellular distribution of cholesterol and its extractability by MβCD were measured after cellular labeling with [3H]cholesterol. An increase in MβCD-extractable cholesterol was found for ABCA1-expressing cells despite an equivalent distribution of cholesterol in cellular membranes [percentage of total [3H]cholesterol extracted (mean±SE; n=2): from 20.4±0.9 and 27.5±2.0% in mock and ABCA1MM-transfected cells, respectively, to 33.4±2.5% in ABCA1-expressing cells; equivalent to a 5.4-fold increase after normalization for protein expression; also see Supplemental Fig. 1 to illustrate that subcellular distribution of radioactive cholesterol is unaffected in the presence of ABCA1].

Taken together, these results suggest that the C6-NBD probes are sensing a more packed lipid environment on ABCA1 expression. Indeed, when we monitored the lateral mobility of C6-NBD-PC at the plasma membrane under the same conditions by FRAP analysis, we found a slower diffusion of the lipid analog in the presence of ABCA1 (Fig. 2C ) [diffusion coefficient, mean±SE: 0.102±0.009 µm2/s (14 cells) for HeLa cells, 0.080±0.007 µm2/s (13 cells) for ABCA1MM-expressing cells, and 0.033±0.002 µm2/s (16 cells) for ABCA1-expressing cells].

To explore the possibility that enhanced lifetime measured was associated with formation of condensed, raft-like domains, we analyzed the C6-NBD-PC lifetime in the plasma membrane of Jurkat cells after activation of the T-cell receptor via anti-CD3 antibody. This treatment has been shown to trigger formation of condensed membrane domains at the activation sites, as assessed by the fluorescent probes Laurdan (42) and PMI-COOH (19) . The analysis revealed a sensible shortening of {tau}2 in activated Jurkat cells as compared with untreated cells (Fig. 2D ). Jurkat cells were also incubated with SMase to induce hydrolysis of sphingomyelin and production of ceramide, a process also able to reproduce domain condensation (43) . Again, a decreased NBD lifetime was observed for the longer component {tau}2 (Fig. 2D ).

On this ground, we excluded that the increment in lifetime observed on expression of ABCA1 originates from condensation of lipid domains.

ABCA1 partitions into Ld domain and modulates organization of lipid domains
Next, we analyzed the lipid domain organization and ABCA1 partition in giant vesicles obtained by blebbing of plasma membranes. Such vesicles have been shown to form large, visible Ld- and liquid-ordered (Lo)-like domains at temperatures approximately ≤25°C and can be used to characterize the phase preference of lipids and proteins (25 , 26) .

GPMVs were generated from HeLa cells expressing ABCA1 or ABCA1MM fused with the EGFP or ECFP. At 25°C, >50% of the vesicles exhibit phase separation as indicated by selective probe enrichment. As shown in Fig. 3A , in vesicles with coexisting phases, both ABCA1 and ABCA1MM are almost exclusively present in the Ld-like phase colabeled with the single chain amphiphilic fluorophore R18. R18, already known to partition almost exclusively into the Ld phase in giant unilamellar vesicles (GUVs)(44) , was confirmed to be preferentially enriched in the same phase also in GPMVs (not shown). We observed a larger Ld-like phase at 25°C in GPMVs prepared from ABCA1-expressing cells as compared with both nontransfected cells (HeLa) and ABCA1MM-expressing cells (Fig. 3B ). Quantification of the relative extent of the Ld-like phase is shown in Fig. 3 , inset.


Figure 3
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  		Figure 3. ABCA1 partitions into the Ld-like phase in GPMVs and modulates its size. A) GPMVs were prepared from untransfected HeLa cells and cells expressing ABCA1 and ABCA1MM tagged with EGFP. Both ABCA1 and ABCA1MM are almost exclusively enriched in the Ld-like phase costained with R18. Images were taken at the equatorial plane of the vesicles at 25°C. Scale bars = 5 µm. B) Boxplot displaying distribution of size of the Ld-like phase in different samples (whiskers: 90th and 10th percentile; box: first quartile, median, and third quartile) at 25°C. In the presence of ABCA1, this phase is larger as compared with that in vesicles prepared from nontransfected HeLa or ABCA1MM-expressing cells. *P < 0.001; unpaired two-tailed t tests. Inset: data represented as means ± SE; number of domains analyzed in parenthesis. Typically, 1–3 domains/vesicle were found.

To complement the biophysical measurements with more widely used biochemical approaches, we analyzed the partitioning behavior of ABCA1 in membrane domains isolated according to the detergent-free method developed by Macdonald and Pike (27) . This method, based on floatation on OptiPrep gradients, allows analysis of domains at the plasma membrane, since including a subcellular fractionation step.

The approach was first validated on untransfected HeLa cells (Fig. 4A ) and on RAW 294.7 cells induced to express high levels of ABCA1 after treatment with 9-cis retinoic acid and 22-hydroxy cholesterol (45 ; Supplemental Fig. 2). ABCA1 was recovered as expected in fractions corresponding to plasma membrane (fractions 1 to 6), and also Golgi apparatus and endolysosomal vesicles, witnessed by enrichment in β-COP and LAMP 2, respectively (Fig. 4A and Supplemental Fig. 2). Interestingly, a minor, but consistent, fraction of the transporter partitioned in the lighter fractions of the plasma membrane (fractions 1 to 3) corresponding to microdomains (Fig. 4B and not shown).


Figure 4
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  		Figure 4. ATPase activity of ABCA1 modifies its partitioning in rafts isolated by detergent-free methods. Homogenates of HeLa cells (A), HeLa cells expressing ABCA1 (B) or its mutant ABCA1MM (C) were fractionated on detergent-free OptiPrep gradients. Fractions, numbered from the top to the bottom of the gradients, were analyzed for the distribution of marker proteins (A) and ABCA1 (A1) by immunoblotting. While in A all the gradient is shown, in B and C only the first 6 fractions corresponding to the plasma membrane are shown. ABCA1MM is enriched in the first 3 fractions (corresponding to microdomains) as compared with the active transporter. Partitioning of flotillin-1 (FL) appears unmodified, while distribution of TfR is altered by concomitant expression of an active transporter. Calnexin (CNX) and β-COP are significantly detectable from fraction 7 onward. Data represent at least 4 independent experiments.

As shown in Fig. 4B, C , on floatation on OptiPrep gradient, an altered partitioning of ABCA1MM vs. the wild-type form was detected in the plasma membrane fractions (fractions 1 to 6). Quantitative assessment was performed by densitometry analysis of hybridization signals in the first three fractions as compared with the total signal at the membrane; this revealed that while 32.5 ± 6.2% (mean±SE; n=4) of total ABCA1 at the membrane partitions in fractions 1 to 3, 65.2 ± 5.1% (n=3) of ABCA1MM showed similar floatation characteristics (P<0.05; Table 3 ).


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  		Table 3. Functional parameters of ABCA1 and related transporters and comparison to raft partitioning

We then investigated the correlation between the partitioning in membrane domains and the surface phenotypes elicited by the expression of ABCA1, i.e., the binding of ApoA-I and annexin V, both prerequisite for cellular effluxes of phospholipids and cholesterol (11 , 13) . We took advantage of two ABCA1-related molecules, CED-7 and HA819, showing potentially informative patterns of phenotype dissociation. CED-7 is an ABC transporter of the A class, functional ortholog of ABCA1 in the worm (24 , 46) , whereas HA819 is a previously described gain of function variant of the transporter (13) . When expressed in HeLa cells, ABCA1, CED-7, and the HA819 variant elicit binding of annexin V to similar values, while the binding of ApoA-I varies broadly (Table 3) . CED-7 fails to elicit any binding or significant efflux of lipids, while HA819 is more efficient than native ABCA1 in inducing both binding and efflux (13 ; Table 3 ). The biochemical analysis of partitioning evidenced that the three molecules, ABCA1, CED-7, and the HA819 mutant, floated similarly (Table 3) . This excluded a causal link between the induction of ApoA-I binding or cholesterol effluxes and membrane partitioning. Rather, the partitioning in lighter fractions correlates strongly with exposure of PS on the cell surface, assessed here as annexin V binding. This positioned the destabilization of the membrane lipid microenvironment as a direct consequence of ABCA1-dependent PS redistribution and upstream to the final effluxes of membrane cholesterol.

To ascertain more precisely the organization of membrane cholesterol under the influence of ABCA1, we analyzed the lipid composition of isolated membrane fractions prepared from bulk cultures of HeLa cells (~25x106) expressing ABCA1 or ABCA1MM. Cholesterol content and percent distribution, measured on plasma membrane fractions in three independent experiments, were similar [cholesterol (nanomol/mg of proteins loaded onto the gradient): 5.1±1.3 and 6.2±2.3 for A1 and A1MM, respectively, and Table 4 ]. This supports the conclusion that the ABCA1, while not affecting membrane cholesterol content, provokes its redistribution into pools readily accessible to external acceptors.


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  		Table 4. Lipid distribution in isolated membrane fractions

ABCA1 affects lateral organization and function of TfR
To assess whether the ABCA1-mediated modulation of the plasma membrane lipid phase affected the organization of membrane proteins, we analyzed in the same preparations the partitioning of other classical markers. While flotillin-1 distribution was uninfluenced by the presence of either the active or mutant ABCA1, an ABCA1-dependent shift in the distribution of the endogenous TfR was evidenced (Fig. 4B, C ). In nontransfected HeLa cells and, similarly, in cells expressing ABCA1MM, TfR floated in the lighter fractions (fractions 1 2 3 ) of OptiPrep gradients (quantified, as percentage of total content in fraction 1 to 6, at 50.7±4.3%, n=5 in ABCA1MM-expressing cells and at 49.9±6.3%, n=5 in HeLa cells). Conversely, in cells expressing a functionally intact ABCA1, the recovery of TfR in lighter fractions was reduced (34.6±4.9%; n=6; P<0.05 A1 vs. A1MM). This indicated that the transport function of ABCA1 can influence the partitioning of membrane proteins with similar floatation characteristics.

In light of the previously reported finding of ABCA1-dependent variations in the rate of TfR-mediated endocytosis (14 , 47) , we analyzed the mobility of transfected TfR-EGFP at the membrane by FRAP (48) while confirming the functional defect in our cellular system. In the presence of an active transporter, an increased time of recovery of TfR was evidenced (Fig. 5A, B and Table 5 ); this was due to a large increase in the fraction of TfR molecules with low mobility (Fig. 5B ) consistent with an ABCA1-induced modification of their embedding lipid environment. In parallel, endocytosis mediated by endogenous TfR was analyzed by flow cytometry in HeLa cells transiently transfected with ABCA1-EYFP or ABCA1MM-EYFP (Fig. 5C ). In each sample, cells expressing the transporter were selected by gating on EYFP signal. TfR-mediated endocytosis was reduced, in the presence of ABCA1, ~20% [28.8% decrease as compared with nontransfected cells in the same sample (Fig. 5C , A1NEG), 20.6 and 25.1% to cells, respectively, expressing or not ABCA1MM (Fig, 5C , A1MM POS and NEG); P<0.05]. This suggested that the modified lipid environment in which TfR is embedded may underlie the functional impairments induced by the coexpression of ABCA1.


Figure 5
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  		Figure 5. ABCA1 affects biophysical and functional attributes of TfR. A) FRAP analysis of the effect of ABCA1 (A1) or ABCA1MM (A1MM) on the mobility of TfR at the membrane of HeLa cells. Diffusion behavior of TfR receptor was analyzed as described in Materials and Methods. A reduction of time of recovery t1/2 in the presence of an active ABCA1 is detected from the FRAP recovery curves. From these curves, t1/2 used to the calculate diffusion coefficient and the mobile fraction were determined (Table 5) . B) Reduction of t1/2 of TfR in the presence of an active ABCA1 reflects an increase in the fraction of TfR molecules (38.5%) repopulating the bleached area at t1/2 longer than 5 s. C) TfR-mediated endocytosis is influenced by ABCA1. FACS analysis of TfR-mediated endocytosis was performed on HeLa cells transiently transfected with ABCA1-EYFP (A1) or ABCA1MM-EYFP (A1MM). Expressing (POS) and nonexpressing (NEG) cells in the same sample were identified by gating on EYFP fluorescent signal. Estimation of TfR-mediated endocytosis in the individual cell populations is monitored by comparing surface TfR signal at 4°C (empty histogram) and after incubation at 37°C (filled histogram; for statistical analysis see Results).


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  		Table 5. FRAP analysis of TfR mobility in the presence of the indicated transporters


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have presented in this article a complementary biophysical and biochemical analysis of the effect of ABCA1 on membrane lipid environment. This allowed us to visualize, in intact membrane, the preference of ABCA1 for Ld-like domains and to highlight and detail the specific destabilizing effect of the transporter on the preexisting membrane arrangement.

Our study indicates lipid packing as a primary target of ABCA1 function. The increase in the fluorescence lifetime of NBD lipid analogues inserted into the outer plasma membrane leaflet provides evidence for an enhanced lipid packing (18) and reveals differences in lipid arrangements on recipient membranes that concern, at least, the compartment where the probe partitions.

Considering the correlation between lifetime and cholesterol content, the increment could stem from increased amounts of cholesterol in the outer membrane leaflet. However, since the distribution of cholesterol in ABCA1 expressing membranes is unchanged, redistribution between different pools in the plasma membrane appears most likely.

We surmise that the destabilization of raft domains, induced by the activity of ABCA1, results either in enhanced partition of the NBD analogues to these domains or in a redistribution of cholesterol outside rafts enhancing lipid packing in the nonraft domains. Both situations would give rise to an increase of {tau}2. The influence of ABCA1 on phase behavior in GPMVs and the evidence for a smaller Lo-like phase corroborate this hypothesis and indicate an ABCA1-mediated effect on the miscibility properties of phospholipids and cholesterol. Thus, our data are consistent with the hypothesis prompted by Landry et al. (22) and Koseki et al. (21) of an ABCA1-induced destabilization of raft domains at the membrane.

To investigate how this destabilization takes place, we analyzed the physicochemical attributes of the inner plasma membrane leaflet. The presence of ABCA1 reduces negative charges at the endofacial membrane surface. This is consistent with both a loss of anionic phospholipids from the inner membrane leaflet and the ABCA1-dependent increase in the outer exposure of PS, demonstrated by annexin V binding. Indeed, the contribution of PS to the recruitment of charge biosensors to the inner leaflet of the plasma membrane has been recently documented (35) . However, although the loss of negative charges from the inner membrane leaflet suggests flopping of anionic phospholipids, it is technically unfeasible, at this point, to ascertain whether transversal movements of PS are the underlying cause. Nevertheless, outward displacement of PS, a polar phospholipid bearing unsaturated fatty acyl chains, would justify the changes in lipid microenvironment. It has in fact been previously demonstrated that chemical stresses able to generate outward movements of PS lead to an increase in cholesterol availability for MβCD-mediated effluxes (8 9 10) . The increased extractability observed both after chemical stresses or in the presence of ABCA1 would reflect a PS-mediated enhanced miscibility of cholesterol and phospholipids in the outer leaflet leading to a lateral redistribution of cholesterol (49) . It is worth reminding, however, that the order of magnitude of the outward PS translocation in these two cases is considerably different.

Finally, our results are consistent with the model (23) proposing that the ABCA1 ability to promote cholesterol effluxes is independent from and precedes its actual binding to specific plasmatic acceptors such as ApoA-I. Rather they proposed, in line with our results, that ABCA1 intrinsically redistributes cholesterol to cell-surface domains readily accessible to apolipoproteins. To conclude, it is important to stress that the effect of ABCA1 on lateral lipid distribution is not only determinant to prime cellular effluxes of cholesterol but in addition alters the lateral membrane distribution of selected groups of receptors. Whether these share as a common denominator the location in structurally distinct lipid domains is notoriously difficult to determine (50) . At any rate, the disrupting action of ABCA1 on lipid architecture appears physiologically influential; its presence sensibly reduces the membrane mobility and the function of TfR, as assessed by receptor-mediated endocytosis (48) . Likewise, under the influence of ABCA1 activity the contingent of cytokine receptors associated to rafts may as well be reduced (51 , 52 ; Lydie Pradel, CIML, unpublished results). This can actually underlie the immunological deviations observed in ABCA1 knockout mice (53) .


   ACKNOWLEDGMENTS
 
We thank Olivier Chambenoit and Veronique Rigot for participating in the initial experimental setup and Hai Tao HE and Doriane Trompier for advice; Sergio Grinstein and Tony Yeung for providing the cationic probes, and R. H. Horvitz for providing the CED-7 cDNA. We also thank the Plateforme d’Imagerie Commune Scientifique de Luminy and especially Mathieu Fallet for expert technical assistance. This work was financially supported by EU contract MRTN-CT-2004-005330; at the Centre d’Immunologie de Marseille Luminy, support was provided by specific institutional grants from CNRS and INSERM, the Mucoviscidose ABCF Foundation, and Foundation pour la Recherche Médicale; and at Humboldt University, the work was financially supported by grants from the Deutsche Forschungsgemeinschaft to A.H. (FG 475) and to P.M. and T.P. (Mu 1017/5). No conflicts of interests are related to the manuscript.


   FOOTNOTES
 
1 These authors contributed equally to this work.

Received for publication October 27, 2008. Accepted for publication December 18, 2008.


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