Analytical Biochemistry 438 (2013) 133–135

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Analytical Biochemistry

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Separation of actin-dependent and actin-independent lipid rafts

Karin Klappe, Ina Hummel, Jan Willem Kok ⇑Department of Cell Biology, University Medical Center Groningen, University of Groningen, 9713 AV Groningen, The Netherlands

a r t i c l e i n f o a b s t r a c t

Article history:Received 27 November 2012Received in revised form 21 February 2013Accepted 13 March 2013Available online 26 March 2013

Keywords:Lipid raftSphingolipidCholesterolActinABC transporterSrc

0003-2697/$ - see front matter � 2013 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.ab.2013.03.018

⇑ Corresponding author. Fax: +31 50 3632728.E-mail address: j.w.kok@umcg.nl (J.W. Kok).

1 Abbreviations used: DRM, detergent-resistant msupernatant; DFM, detergent-free membrane; ABC,multidrug resistance-related protein 1; PGP, P-glycopr

Lipid rafts have been isolated on the basis of their resistance to various detergents and more recently byusing detergent-free procedures. The actin cytoskeleton is now recognized as a dynamic regulator of lipidraft stability. We carefully analyzed the effects of the cortical actin-disrupting agent latrunculin B on lipidraft markers of both protein and lipid nature and show that two detergent-free membrane subtypes canbe isolated and separated from each other on a one-step density gradient combined with pooling of theappropriate gradient fractions. These two subtypes differ in their dependence on the cortical actincytoskeleton.

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Various procedures have been developed to isolate lipid raftsbiochemically. For a long time, these methods have relied solelyon the use of detergents, such as Triton X-100 and Lubrol, isolatinglipid rafts on the basis of their resistance to these detergents. Hence,these isolated membrane domains were named detergent-resistantmembranes (DRMs),1 among other names. It became apparent thatthe composition of DRMs differed with the detergent of choice. Theuse of such methods resulted in ample discussion and criticism giventhe potential artifacts induced by these detergents. Fortunately,detergent-free isolation procedures also were developed, includingthe method developed by Macdonald and Pike [1].

In this procedure, confluent cells of two 75-cm2 flasks werewashed and subsequently scraped in base buffer (20 mM Tris–HCl [pH 7.8] and 250 mM sucrose) supplemented with 1 mM CaCl2

and 1 mM MgCl2. The collected cells were centrifuged at 250g for5 min. The resulting pellet was suspended in 1 ml of base buffersupplemented with 1 mM CaCl2, 1 mM MgCl2, and protease inhib-itors. After homogenization by passage through a 25-gauge needle20 times, another centrifugation step at 1000g for 10 min followed.The resulting post-nuclear supernatant (PNS) was collected andtransferred to a separate tube. The pellet was homogenized againin 1 ml of base buffer supplemented with 1 mM CaCl2, 1 mMMgCl2, and protease inhibitors, passed through the needle 20times, and centrifuged at 1000g for 10 min. The second PNS was

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embrane; PNS, post-nuclearATP-binding cassette; MRP1,otein.

combined with the first. Protein content of the combined PNSwas determined, and samples were processed for gradient analysisbased on equal amounts of protein adjusted to 2-ml volumes. Sub-sequently, 2 ml of base buffer containing 50% OptiPrep was addedto this 2 ml of PNS. By using a gradient mixer, an 8-ml gradient of0–20% OptiPrep in base buffer was poured on top of this 4 ml in acentrifugation tube. After centrifugation at 22,000 rpm for 90 minat 4 �C in a Beckman SW41 rotor, 18 fractions were collected fromtop to bottom and stored at �80 �C [1].

Based on this method, we refer to these isolated membrane do-mains as detergent-free membranes (DFMs). Macdonald and Pikefound lipid rafts markers to reside primarily in fractions 1–3 [1].However, caveolin was found mostly in fractions 7–10, where cho-lesterol was also enriched, suggesting a separation of caveolar andnoncaveolar DFMs [1]. During recent years, it has become apparentthat certain lipid rafts can be dynamically regulated by the corticalactin cytoskeleton that affects their stability [2–4]. However, thereis no method to separate different subtypes of DFMs with respect todependence on cortical actin. We adapted the DFM isolation proce-dure by fractionating the OptiPrep density gradient into 9 fractionsand carefully analyzed the protein and lipid distributions over thedensity gradient. Thus, we obtained a method to separate actin-dependent and actin-independent DFMs. The relative abundanceof specific proteins in these 9 fractions was determined by process-ing equal volumes of the gradient fractions for Western blotting(Fig. 1A). After quantification of the specific protein bands (Odyssey,LI-COR Biosciences), the percentage of a given protein in each gra-dient fraction was calculated relative to the total in the entire gra-dient (= sum of individual gradient fractions). These percentageswere summed as follows: 1–2, 3–4, 5–6, and 7–9 (Fig. 1B). For lipid

Fig.1. Gradient fractions 1–2, as well as fractions 3–4, are enriched in cholesterol and sphingolipids in both Neuro-2a and NIH 3T3 MDR1 G185 cells. DFMs were isolated fromNeuro-2a cells (A–D) or NIH 3T3 MDR1 G185 cells (E, F). (A, B) The proteins MRP1/ABCC1, actin, src, and RhoGDI were detected on Western blot in the 9 gradient fractions (A)and were quantified, expressed as percentages, and pooled for gradient fractions 1–2, 3–4, 5–6, and 7–9 (B). For each marker protein, these percentages add up to 100%. Blackbars: MRP1/ABCC1; white bars: actin; dark gray bars: src; light gray bars: RhoGDI. (C, E) In the pooled gradient fractions, cholesterol (black bars) and sphingolipids (whitebars) were determined (expressed in nmol), as was the protein content (gray bars, expressed in lg � 10�2). (D, F) Enrichment indicates the ratio of cholesterol/protein (blackbars) or sphingolipid/protein (white bars). To calculate these ratios, the percentage values in the gradient of each of cholesterol, sphingolipids, and protein were used.Therefore, these ratios are unit-less and represent the enrichment in gradient fractions of cholesterol or sphingolipids relative to protein enrichment in the respectivefractions. Data in the right panels (B, D, F) represent the means ± standard deviations of three independent experiments, and data in the left panels (A, C, E) represent typicalexamples taken from three independent experiments. ⁄,#Values are significantly (P < 0.05) different from those of fractions 5–6 (membrane fractions considered to be of non-DFM nature) as determined by Student’s t test.

134 Notes & Tips / Anal. Biochem. 438 (2013) 133–135

analysis, gradient fractions were pooled (1–2, 3–4, 5–6, and 7–9)immediately after fractionation of the gradient, and these pooledgradient fractions were subjected to lipid extraction and quantifica-tion, focusing on sphingolipids and cholesterol, the two lipid classesknown to be enriched in lipid rafts. Lipids were extracted as de-scribed previously [5]. The cholesterol content was determined byspectrophotometry using a cholesterol oxidase/peroxidase assay[6]. The sphingolipid content was analyzed by liquid chromatogra-phy–electrospray ionization tandem mass spectrometry as de-scribed previously [7]. The amounts of individual sphingolipidspecies were summed to obtain the total sphingolipid content.From the absolute amounts, the percentage of a given lipid in eachpool of gradient fractions was calculated relative to the total in theentire gradient. In addition, a determination of total protein in thepooled gradient fractions was performed, allowing us to calculatethe enrichment of the above-mentioned lipids relative to protein.

To study cortical actin dependence of DFMs, cells were treatedfor 45 min with 10 lM latrunculin B (for details, see Refs. [4,8]).Two different cell lines were employed: Neuro-2a cells expressing

the ATP-binding cassette (ABC) transporter multidrug resistance-related protein 1 (MRP1/ABCC1) and NIH 3T3 MDR1 G185 cellsexpressing the ABC transporter P-glycoprotein (PGP/ABCB1). Thesecell lines offer the advantage that we can measure the activity of aprotein (i.e., the efflux activity of the transporter) as a function ofits DFM association as well as its interaction with cortical actin.First, we validated the DFM analysis procedure in terms of enrich-ment of specific lipids in the pooled gradient fractions. This isbased on the following definition of DFMs: structures that (i) floatin a density gradient due to a high lipid/protein ratio and ensuinglow density and (ii) are enriched in sphingolipids and cholesterol.The pooled gradient fractions 1–2, as well as fractions 3–4, con-tained floating material with a high lipid/protein ratio (not shown;see Ref. [7]). Indeed, both the pooled gradient fractions 1–2 and 3–4 turned out to be strongly enriched in sphingolipids as well ascholesterol in both Neuro-2a cells (Fig. 1D) and NIH 3T3 MDR1G185 cells (Fig. 1F) and, thus, can be designated as DFMs. In abso-lute amounts, sphingolipids and cholesterol were distributed moreor less evenly along the gradient, in contrast to the protein content,

Notes & Tips / Anal. Biochem. 438 (2013) 133–135 135

which was very low in gradient fractions 1–2 and 3–4 representingDFMs (Fig. 1C and E). As expected, the non-DFM marker RhoGDIwas found mostly in gradient fractions 7–9, and very little wasfound in fractions 1–2 and 3–4 (Fig. 1A and B; see also Ref. [8]).

Next, the association of various proteins, as well as sphingoli-pids and cholesterol, with the pooled gradient fractions in bothcontrol and latrunculin-treated cells was determined. If sphingoli-pids, cholesterol, or certain proteins are in DFMs that are actindependent, the latrunculin B treatment is likely to disrupt theseDFMs, resulting in a shift of such proteins or lipids out of the gra-dient fractions. To reveal such a shift, we expressed the data as foldvalues that were calculated as follows. In the first step, the amountof a certain protein in each gradient fraction or the amount of a cer-tain lipid in each pool of gradient fractions (e.g., fractions 1–2) wasmeasured as described above. Second, the amount was expressedas a percentage relative to the total of that protein or lipid in theentire gradient (= sum of amounts in individual [pools of] gradientfractions). Third, the first two steps were performed in both latrun-culin-treated and control cells. Fourth, for each pool of gradientfractions, the fold value (= percentage of a given protein or lipidin latrunculin-treated cells divided by that in control cells) was cal-culated. For example, if a protein or lipid shifts out of DFM frac-tions 1–2, the fold value is lower than 1. Our results show thatthis is consistently the case for the pooled gradient fractions 1–2,from which both sphingolipids and cholesterol shift on latrunculinB treatment in the two cell lines (Table 1), although the shift ofcholesterol from these gradient fractions in Neuro-2a cells justdid not reach significance. It appears that these lipids shift to gra-dient fractions 3–4, suggesting that they remain in DFMs that are,however, no longer actin associated. In Neuro-2a cells, the proteins

Table 1Gradient distribution of lipid raft markers on cortical actin disruption by latrunculin Btreatment.

Neuro-2a MRP1 Actin Src Cholesterol Sphingolipidfold fold fold fold fold

Fractions 1–2 0.30 0.42 0.50 0.73 0.53Fractions 3–4 0.92 1.02 1.34 1.37 1.45Fractions 5–6 1.13 0.99 1.22 1.04 1.19Fractions 7–9 1.86 1.21 0.90 0.86 0.77

NIH 3T3 MDR1 PGP Actin Src Cholesterol Sphingolipidfold fold fold fold fold

Fractions 1–2 0.62 0.51 0.68 0.57 0.60Fractions 3–4 0.89 1.22 1.15 1.46 1.58Fractions 5–6 1.06 1.04 1.14 1.12 1.08Fractions 7–9 1.19 1.05 0.70 0.63 0.41

Neuro-2a MRP1 Actin Src Cholesterol SphingolipidP value P value P value P value P value

Fractions 1–2 0.013 0.040 0.007 0.053 0.001Fractions 3–4 0.417 0.926 0.100 0.105 0.011Fractions 5–6 0.405 0.948 0.157 0.636 0.270Fractions 7–9 0.032 0.194 0.060 0.727 0.538

NIH 3T3 MDR1 PGP Actin Src Cholesterol SphingolipidP value P value P value P value P value

Fractions 1–2 0.291 0.047 0.375 0.003 0.002Fractions 3–4 0.572 0.011 0.248 0.003 0.007Fractions 5–6 0.580 0.569 0.426 0.161 0.353Fractions 7–9 0.398 0.577 0.346 0.041 0.030

Note. Neuro-2a or NIH 3T3 MDR1 G185 cells were treated with latrunculin B andsubjected to lipid raft analysis. Various protein and lipid markers were determinedin the pooled gradient fractions 1–2, 3–4, 5–6, and 7–9 and were expressed aspercentages. These percentages were used to calculate the fold, which is defined asthe ratio of the percentage of a given marker in latrunculin B-treated cells dividedby the percentage in control samples (see text for details). A value less than 1indicates that the respective marker shifts out of this fraction on cortical actindisruption. Data represent the means of three to five independent experiments.Bold type indicates that fold values are significant (P < 0.05) as determined using aStudent’s t test.

MRP1/ABCC1, actin, and src significantly shifted out of the gradientfractions 1–2 on latrunculin B treatment (Table 1). MRP1/ABCC1did not shift out of gradient fractions 3–4. Therefore, the loss ofMRP1/ABCC1 activity observed (not shown; see Ref. [4]) is corre-lated with a shift out of the (actin-dependent) DFM gradient frac-tions 1–2. In NIH 3T3 MDR1 G185 cells, actin significantly shiftedout of gradient fractions 1–2, consistent with the result obtainedin Neuro-2a cells. PGP/ABCB1 and src were only marginally associ-ated with gradient fractions 1–2 in control NIH 3T3 MDR1 G185cells and were more abundantly associated with the gradient frac-tions 3–4 (data not shown; see Ref. [8]). As a consequence, a poten-tial shift of these proteins out of gradient fractions 1–2 onlatrunculin B treatment did not reach significance due to intrinsicvariation. As expected, latrunculin B treatment did not shift PGP/ABCB1 out of the (actin-independent) gradient fractions 3–4 (Ta-ble 1), and consequently its activity was not affected (not shown;see Ref. [8]).

In conclusion, with our DFM analysis procedure, both thepooled gradient fractions 1–2 and 3–4 contain material that fitsthe criteria for the definition of DFMs, that is, flotation on a densitygradient and enrichment in sphingolipids and cholesterol. More-over, the biochemically isolated and separated DFMs in gradientfractions 1–2 are actin dependent, in contrast to those in gradientfractions 3–4. This is evidenced by the observation that actin shiftsout of the gradient fractions 1–2, but not out of fractions 3–4, onlatrunculin B treatment. Moreover, the DFM-specific lipid classsphingolipids and cholesterol shift out of gradient fractions 1–2,but not out of fractions 3–4, on latrunculin B treatment. Finally,the ABC transporter MRP1/ABCC1 shifts out of gradient fractions1–2 and loses activity. In contrast, PGP/ABCB1 remains associatedwith gradient fractions 3–4 and is fully active on latrunculin Btreatment. Thus, with this procedure, we biochemically isolateand separate two types of DFMs that are different concerning theirdependence on the cortical actin cytoskeleton. As shown elsewhere[4,8], this has functional consequences for protein activity, as re-vealed by the efflux function of ABC transporters.

Acknowledgments

Annie van Dam and Hjalmar Permentier of the Mass Spectrom-etry Core Facility (University of Groningen) are kindly acknowl-edged for their help in the analysis of sphingolipids by liquidchromatography–electrospray ionization tandem mass spectrome-try. Michael Gottesman of the National Institutes of Health iskindly acknowledged for providing the NIH 3T3 MDR1 G185mouse fibroblast cell line.

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