Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol

Abstract

Controlled distribution of lipids across various cell membranes is crucial for cell homeostasis and regulation. We developed an imaging method that allows simultaneous in situ quantification of cholesterol in two leaflets of the plasma membrane (PM) using tunable orthogonal cholesterol sensors. Our imaging revealed marked transbilayer asymmetry of PM cholesterol (TAPMC) in various mammalian cells, with the concentration in the inner leaflet (IPM) being ∼12-fold lower than that in the outer leaflet (OPM). The asymmetry was maintained by active transport of cholesterol from IPM to OPM and its chemical retention at OPM. Furthermore, the increase in the IPM cholesterol level was triggered in a stimulus-specific manner, allowing cholesterol to serve as a signaling lipid. We found excellent correlation between the IPM cholesterol level and cellular Wnt signaling activity, suggesting that TAPMC and stimulus-induced PM cholesterol redistribution are crucial for tight regulation of cellular processes under physiological conditions.

Figure 1. Simultaneous quantification of OPM ([Chol]_o) and IPM cholesterol ([Chol]_i) of HeLa cells by orthogonal cholesterol sensors

(a) Basic strategy. Binding of our cholesterol sensor (an engineered D4 domain labeled with a solvatochromic fluorophore, such as NR3) to the cholesterol-containing membrane led to major changes in spectral properties of the fluorophore. (b,c) Cholesterol dependence of DAN- (b) and NR3-labeled (c) sensors in binding to POPC/POPS/cholesterol (80–x/20/x: x = 0–40 mol%) large unilamellar vesicles. For sensors, we used QYDA, YDA, D434A/A463W, D434A and WT (from left to right). Vesicle binding of the proteins was monitored in terms of an increase in fluorescence intensity (ΔF) that was normalized against the maximal ΔF (ΔF_max). (d) Simultaneous quantification of [Chol]_o and [Chol]_i in resting HeLa cells. (e,f) Effects of 1-h cholesterol depletion by 5 mM MβCD and cholesterol enrichment by 5 mM MβCD-cholesterol (1:1) adduct on [Chol]_o and [Chol]_i. Extracellularly added DAN-D434A and microinjected NR3-YDA were used for OPM and IPM sensors, respectively. Each image shows spatially resolved [Chol]_o or [Chol]_i on the cross-section of a representative cell at a given time. A pseudo-coloring scheme with red representing the highest and blue the lowest concentration is used to illustrate the spatial concentration heterogeneity. Scale bars represent 5 µm. Spatiotemporally averaged [Chol]_o and [Chol]_i are displayed in the right panels. Cholesterol quantification was performed in triplicate in multiple cells. All data represent mean ± s.d. n = 6 (b,c), 225 (d), 125 (e) and 116 (f).

Figure 2. Stimulus-induced increases in [Chol]_i (D[Chol]_i) in HeLa cells

(a) Wnt3a dose dependence of Δ[Chol]_i. Spatially averaged [Chol]_i in response to 50, 25 and 12.5 ng/ml of Wnt3a (blue circles from top to bottom) was plotted as a function of time. 50 ng/ml of Wnt5a (green triangles) and Wnt11 (red squares) were used as controls. (b) Time course of spatially averaged [Chol]_i (orange) and [Chol]₀ (blue) in response to 50 ng/ml Wnt3a. Notice the excellent synchronization and quantitative correlation between Δ[Chol]_i and Δ[Chol]₀. (c) Angular profiles (that is, plots of the relative fluorescence intensity using a single PM location as a reference point) of [Chol]_i (orange) and PM-bound EGFP-Dvl2 (blue) at 0, 15 and 30 min after Wnt3a stimulation. The EGFP fluorescence intensity of PM-bound Dvl2 was normalized using the maximal intensity value at 30 min. Notice the good spatial correlation between Δ[Chol]_i and Dvl2 PM recruitment. All data represent mean ± s.d. from quintuplicate measurements using multiple cells. n = 135 (a), 108 (b) and 49 (c) cells.

Figure 3. Mechanisms for PM transbilayer asymmetry of cholesterol and its physiological relevance

(a) Effects of lipid transporter RNAi and SMase treatment on [Chol]_i. Spatial distribution of [Chol]_i in HeLa cells before and after ABCACA1/G1 double knockdown (KD) and ABCACA1/G1 double KD + SMase treatment (0.2 U/ml) is shown. Scale bars represent 5 µm. (b) Effects of various treatments on the [Chol]_o/[Chol]_i ratio. Simultaneous quantification of [Chol]_o and [Chol]_i was performed as described in Figure 1. The red line indicates [Chol]_o/[Chol]_i = 1.0. P < 0.001 for all data pairs. (c) Time courses of spatially averaged [Chol]_i in HEK293 WT, ABCACA1 knockout (KO), ABCACA1 KO transfected with ABCACA1 WT, ABCACA1 K939M/K1952M, ABCACA1 S884A and ABCACA1 S884E, respectively, in response to 50 ng/ml of Wnt3a. (d) Time courses of spatially averaged [Chol]_i in ABCACA1 KO transfected with ABCACA1 S1296A, ABCACA1 S1296E, ABCACA1 S884A/S1296A and ABCACA1 S884E/S1296E in response to 50 ng/ml of Wnt3a. (e) Linear correlation between [Chol]_i and the basal Wnt signaling activity in HeLa cells. The Wnt signaling activity in WT (1), ABCG1 KD (2), ABCACA1 KD (3) and ABCACA1/G1 double KD (4) HeLa cells was measured using the TOP-FLA SH assay. Relative activity was calculated as the ratio of the observed activity to that of HeLa WT. Linear regression was used to fit the data. (f) Wnt3a-induced (50 ng/ml) Wnt signaling activity for various HeLa cells. Fold increase in signaling activity was calculated as the ratio of the observed activity to that of unstimulated WT HeLa cells. P < 0.001 for all data pairs. All imaging data (a–d) represent mean ± s.d. from triplicate measurements in multiple cells. n = 225 (HeLa), 121(ABCACA1 KD), 116 (ABCG1 KD), 115 (ABCACA1/G1 KD), 134 (SMase), 102 (ABCACA1 KD + SMase), 109 (ABCG1 KD + SMase) and 107 (ABCACA1/G1 KD + SMase). n = 60 for c and d cells. The activity data are mean ± s.d. from quadruplicate measurements.