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. 2013 Dec 13;288(50):35660-70.
doi: 10.1074/jbc.M113.519116. Epub 2013 Oct 28.

Bile Acids Modulate Signaling by Functional Perturbation of Plasma Membrane Domains

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Free PMC article

Bile Acids Modulate Signaling by Functional Perturbation of Plasma Membrane Domains

Yong Zhou et al. J Biol Chem. .
Free PMC article

Abstract

Eukaryotic cell membranes are organized into functional lipid and protein domains, the most widely studied being membrane rafts. Although rafts have been associated with numerous plasma membrane functions, the mechanisms by which these domains themselves are regulated remain undefined. Bile acids (BAs), whose primary function is the solubilization of dietary lipids for digestion and absorption, can affect cells by interacting directly with membranes. To investigate whether these interactions affected domain organization in biological membranes, we assayed the effects of BAs on biomimetic synthetic liposomes, isolated plasma membranes, and live cells. At cytotoxic concentrations, BAs dissolved synthetic and cell-derived membranes and disrupted live cell plasma membranes, implicating plasma membrane damage as the mechanism for BA cellular toxicity. At subtoxic concentrations, BAs dramatically stabilized domain separation in Giant Plasma Membrane Vesicles without affecting protein partitioning between coexisting domains. Domain stabilization was the result of BA binding to and disordering the nonraft domain, thus promoting separation by enhancing domain immiscibility. Consistent with the physical changes observed in synthetic and isolated biological membranes, BAs reorganized intact cell membranes, as evaluated by the spatial distribution of membrane-anchored Ras isoforms. Nanoclustering of K-Ras, related to nonraft membrane domains, was enhanced in intact plasma membranes, whereas the organization of H-Ras was unaffected. BA-induced changes in Ras lateral segregation potentiated EGF-induced signaling through MAPK, confirming the ability of BAs to influence cell signal transduction by altering the physical properties of the plasma membrane. These observations suggest general, membrane-mediated mechanisms by which biological amphiphiles can produce their cellular effects.

Keywords: Bile Acid; Lipid Raft; Membrane Bilayer; Membrane Biophysics; Membrane Lipids; Membrane Structure; Ras.

Figures

FIGURE 1.
FIGURE 1.
Bile acid-induced cell injury correlates with plasma membrane vesicle dissolution. A, cell toxicity quantified by leakage of the cytoplasmic enzyme LDH is sensitive to millimolar cholic and deoxycholic acid treatment. B, synthetic liposome leakage, measured by release of calcein, correlates with cytotoxicity. C, phase contrast imaging (at room temperature) of isolated GPMVs shows that treatment of GPMVs with 1 mm DCA or 5 mm CA leads to near-complete dissolution of membranes. Higher BA concentrations lead to no observable vesicles (insets show magnifications of representative areas). The quantitative agreement between these results strongly suggests a mechanistic link between membrane disruption and cytotoxicity. All results are representative of at least three independent experiments.
FIGURE 2.
FIGURE 2.
Bile acids stabilize phase separation in GPMVs. A, exemplary fluorescent images of GPMVs (labeled with FAST DiO) at various temperatures show that whereas phase separation in untreated vesicles is observable only below 15 °C, treatment with 1 mm CA increases phase separation temperature to ∼17 °C and 0.5 mm DCA above 20 °C. B, Tmisc is calculated by counting the percentage of phase-separated vesicles at several temperatures and fitting to a sigmoidal curve. C, bile acid treatment dramatically increased Tmisc, with DCA more effective than CA. Effect of higher BA concentrations could not be evaluated because of GPMV dissolution. D, bile acid-mediated stabilization of phase separation was observed regardless of whether GPMVs were isolated using PFA/DTT (PD) or NEM. E, the domain-stabilizing effect of bile acids was observable (although somewhat attenuated compared with treatment of isolated membranes) by pretreating cells with BAs prior to GPMV isolation. Data in B--D are average ± S.D. (error bars) from three independent experiments.
FIGURE 3.
FIGURE 3.
Stabilized phase separation does not affect protein partitioning. A, exemplary images of phase partitioning of a model transmembrane protein (trLAT) in untreated and 0.5 mm DCA-treated GPMVs. Red images show the localization of trLAT, which is approximately equal in both phases, relative to the fluorescent nonraft marker FAST DiO (green). B, partitioning quantified by Kp,raft (the ratio of fluorescent intensity in the raft divided by nonraft phase) was slightly, but not significantly, reduced by DCA for trLAT, a peripheral raft phase protein (GPI-anchored GFP), and an integral nonraft protein (TfR-GFP). Inset, Kp,raft of trLAT was not significantly different in control versus DCA-treated GPMVs isolated with either PFA/DTT or NEM. The higher raft phase partitioning in NEM GPMVs was expected (28). Data are mean ± S.D. (error bars) from 10–20 vesicles/condition, representative of three independent experiments.
FIGURE 4.
FIGURE 4.
Stabilization of domain separation by bile acid-induced disordering of the nonraft phase. A, DCA induces a progressive, concentration-dependent decrease in membrane order of DOPC:cholesterol (Chol) (1:1) synthetic liposomes, as evidenced by the red shift of normalized fluorescence emission of the order-sensitive fluorophore C-Laurdan. Higher DCA concentrations are possible without complete membrane dissolution in this context because of high lipid concentration (∼1 mm) relative to GPMV suspensions. B, generalized polarization (GP) of C-Laurdan calculated from the data in A shows the quantitative trend of membrane disordering. C and D, disordering effect of DCA is much more pronounced in liquid-disordered phase membranes (DOPC:Chol, 1:1) than liquid-ordered phase (DPPC:Chol, 1:1), with an intermediate effect on an intermediate order membrane (POPC:Chol, 1:1); a.u., arbitrary units. E, a fluorescent analog of bile acid (NBD-lithocholic acid) preferentially binds to the disordered (nonraft) domain in phase separated GPMVs, evidenced by co-partitioning between the nonraft marker rhodamine-DOPE and the bile acid derivative. F and G, treatment of isolated GPMVs with 0.2 mm DCA leads to further disordering of the disordered domains, without a notable effect on raft phase order (mean ± S.D. (error bars) of 5–9 vesicles/condition). The greater difference between the coexisting domains (ΔGP = 0.23 ± 0.02 in DCA-treated compared with 0.14 ± 0.04 in untreated) is likely responsible to the DCA-enhanced stability of phase separation.
FIGURE 5.
FIGURE 5.
Bile acid specifically enhances the formation of nanoclusters of GFP-tK and full-length K-Ras. A, plasma membrane sheets from cells expressing GFP-tK, the GFP-tagged membrane-anchoring domain of K-Ras, were attached to EM grids and labeled with anti-GFP. Nanoclustering evaluated by EM (see “Experimental Procedures” for details of data analysis) was significantly increased in cells exposed to DCA compared with untreated controls (p = 0.001 for both 50 μm and 150 μm DCA). B, DCA at 150 μm did not have a statistically significant effect on nanoclustering of GFP-tH. C, representative FLIM data show fixed BHK cells expressing GFP-tK alone, or in combination with RFP-tK either untreated, treated with 50 μm, or 200 μm DCA. Images reveal a reduction of GFP lifetime with RFP coexpression due to FRET in nanoclusters and an enhancement of FRET (nanoclustering) by DCA. D, quantification of FLIM imaging reveals that DCA doses ≥50 μm significantly increased FRET (*, p < 0.05 compared with 0 μm DCA) indicative of enhanced nanoclustering of both the minimal anchor tK and the oncogenic full-length protein K-Ras.G12V. E, the clustering of tH and full-length H-Ras.G12V was not significantly affected by DCA treatment (ns; p > 0.1 compared with 0 μm DCA). All data are shown as mean ± S.E. (error bars) of at least ≥60 cells representative of three independent experiments.
FIGURE 6.
FIGURE 6.
Bile acid enhances MAPK signaling induced by EGF. BHK cells were serum-starved for 2 h, treated with DCA for 1 h, stimulated with 2.5 ng/ml EGF for 5 min, and harvested. Whole cell lysates were used to blot for phosphorylated MEK, ERK, or Akt. A, representative Western blots with antibodies against pMEK, pERK, or pAkt473. B–D, normalized band intensity values (see “Experimental Procedures”) for pMEK (B), ppERK (C), and pAkt (D) in the form of means ± S.E. (error bars) as a function of bile acid concentrations from three independent experiments.

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