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. 2021 Nov 29;4(1):164.
doi: 10.1038/s42004-021-00603-1.

Acyl-chain saturation regulates the order of phosphatidylinositol 4,5-bisphosphate nanodomains

Luís Borges-Araújo  1   2 , Marco M Domingues  3 , Alexander Fedorov  1 , Nuno C Santos  3 , Manuel N Melo  2 , Fábio Fernandes  4   5
Affiliations

Acyl-chain saturation regulates the order of phosphatidylinositol 4,5-bisphosphate nanodomains

Luís Borges-Araújo et al. Commun Chem. .

Abstract

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) plays a critical role in the regulation of various plasma membrane processes and signaling pathways in eukaryotes. A significant amount of cellular resources are spent on maintaining the dominant 1-stearoyl-2-arachidonyl PI(4,5)P2 acyl-chain composition, while less abundant and more saturated species become more prevalent in response to specific stimuli, stress or aging. Here, we report the impact of acyl-chain structure on the biophysical properties of cation-induced PI(4,5)P2 nanodomains. PI(4,5)P2 species with increasing levels of acyl-chain saturation cluster in progressively more ordered nanodomains, culminating in the formation of gel-like nanodomains for fully saturated species. The formation of these gel-like domains was largely abrogated in the presence of 1-stearoyl-2-arachidonyl PI(4,5)P2. This is, to the best of our knowledge, the first report of the impact of PI(4,5)P2 acyl-chain composition on cation-dependent nanodomain ordering, and provides important clues to the motives behind the enrichment of PI(4,5)P2 with polyunsaturated acyl-chains. We also show how Ca2+-induced PI(4,5)P2 nanodomains are able to generate local negative curvature, a phenomenon likely to play a role in membrane remodeling events.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Cation-induced clusters are formed independently of PI(4,5)P2 acyl-chain composition as seen by homo-FRET of the TF-PI(4,5)P2 analog.
PI(4,5)P2 cluster formation was determined through the incorporation of 0.1 mol% of TF-PI(4,5)P2 in LUVs containing POPC and increasing concentrations of unlabeled PI(4,5)P2. The experiments were carried out for the three unlabeled PI(4,5)P2 species, with different acyl-chain composition. TF-PI(4,5)P2 fluorescence anisotropy (<r>) values were measured in the presence (400 µM Ca2+, red) and absence (5 mM EDTA, blue) of calcium. Error bars represent the SD from N = 3 independent experiments. A significant decrease in TF-PI(4,5)P2 fluorescence anisotropy in the presence of calcium is observed for the three PI(4,5)P2 species ((16:0)2 PI(4,5)P2: F(1,12) = 17.87, p = 0.0012; (18:1)2 PI(4,5)P2: F(1,12) = 96.84; p < 0.0001; (18:0 20:4) PI(4,5)P2: F(1,12) = 107.7, p < 0.0001).
Fig. 2
Fig. 2. Cation-induced clusters are formed independently of PI(4,5)P2 acyl-chain composition, as seen by AFM measurements of supported lipid bilayers.
PI(4,5)P2 cluster formation was detected through the AFM measurement of SLBs containing DOPC and 5% PI(4,5)P2. Experiments were carried out for the three PI(4,5)P2 species in study and figures are labeled according to the acyl-chain composition of the PI(4,5)P2 species employed. Topographical images were acquired (a) and analyzed with first- or second-level flattening, using the JPK data processing software, from which the membrane heigh profiles (b) were obtained. Dark patches correspond to defects in the supported membrane. The height values of each pixel (higher than 0 nm) were transformed into log10 value, avoiding the influence of the height values of defects on the histograms. The frequency count axis represents the number of data points grouped into each bin on the height histograms.
Fig. 3
Fig. 3. TMA-DPH fluorescence spectroscopy shows that calcium-induced PI(4,5)P2 nanodomains are more ordered than monodisperse PI(4,5)P2, even for unsaturated acyl-chain compositions.
PI(4,5)P2 local membrane order was determined through the incorporation of TMA-DPH at a 1:300 lipid ratio in MLVs containing POPC and increasing concentrations of unlabeled PI(4,5)P2. The experiments were done for the three acyl-chain compositions in study. For all acyl-chain compositions analyzed here, an increase in local membrane order takes place upon Ca2+-induced PI(4,5)P2 nanodomain formation ((16:0)2 PI(4,5)P2: F(1,16) = 148.3, p = 0.0001; (18:1)2 PI(4,5)P2: F(1,16) = 19.71, p = 0.0004; (18:0 20:4) PI(4,5)P2: F(1,16) = 12.23, p < 0.0030). TMA-DPH fluorescence anisotropy (<r>) (a) was measured in the presence (400 µM Ca2+, red) and absence (5 mM EDTA, blue) of calcium. The TMA-DPH response to a temperature gradient (b) was measured in samples composed of POPC:PI(4,5)P2 95:5. Pure POPC samples were measured as controls. All samples were measured both in the presence and absence of calcium. Error bars for all measurements represent the SD from N = 3 independent experiments.
Fig. 4
Fig. 4. (16:0)2 PI(4,5)P2 forms gel-like nanodomains upon undergoing calcium-induced clustering.
PI(4,5)P2 gel-like properties were detected through the incorporation of tPnA at a 1:300 lipid ratio in MLVs containing POPC and increasing concentrations of unlabeled PI(4,5)P2. Experiments were done for the three acyl-chain compositions in study. tPnA fluorescence anisotropy (a) and fluorescence intensity weighed lifetime (τ¯) (b) were measured in the presence (400 µM Ca2+, red) and absence (5 mM EDTA, blue) of calcium. In the presence of calcium, we observed no effect on tPnA fluorescence lifetime for both (18:1)2 PI(4,5)P2 (F(1,12) = 1.564, p = 0.2349) and (18:0 20:4) PI(4,5)P2 (F(1,12) = 0.7426, p = 0.4057). For (16:0)2 PI(4,5)P2 in the presence of calcium, we observe a steep increase in both fluorescence anisotropy and fluorescence lifetime (F(1,12) = 708.1, p ≤ 0.0001). The thermal profile of tPnA anisotropy (c) was measured in samples composed of POPC:PI(4,5)P2 90:10. Controls were also carried out with samples of pure POPC. The impact of increasing concentrations of (18:0 20:4) PI(4,5)P2 on the formation of (16:0)2 PI(4,5)P2 gel-like nanodomains (d) was detected in MLVs, containing POPC: PI(4,5)P2 (95:5) at several (16:0)2 to (18:0 20:4) PI(4,5)P2 ratios, through the incorporation of tPnA at a 1:300 lipid ratio. Error bars for fluorescence anisotropy measurements represent the SD from N = 3 independent experiments.
Fig. 5
Fig. 5. CG MD simulations showcase the impact of acyl-chain composition on PI(4,5)P2 and PI(4,5)P2 nanodomain biophysical properties.
a Average PI(4,5)P2 cluster size over the course of the simulation for the three acyl-chain compositions studied, both in the presence and absence of calcium. Final simulation snapshots of the large membrane systems are also shown. PI(4,5)P2 lipid headgroups are depicted in gray, with the phosphates discriminated in orange. PI(4,5)P2 acyl chains are colored according to the corresponding color code. Ca2+ ions are represented in blue. The bulk POPC lipids are represented by the translucent gray surface. b PI(4,5)P2 acyl-chain S-value order parameter for all acyl-chain beads of each composition, in the presence (red) and absence (blue) of calcium. c Dependency of the S-value order of POPC’s first acyl-chain bonds on the distance from the nearest PI(4,5)P2 molecule, in the presence and absence of calcium.
Fig. 6
Fig. 6. Calcium-induced PI(4,5)P2 clustering induces negative membrane curvature, as seen by CG MD simulations.
Curvature analysis of snapshots from asymmetric membrane simulations containing 10 mol% PI(4,5)P2 in the inner leaflet. Snapshots are shown for the three acyl-chain compositions studied, both in the presence and absence of calcium. For each system, a top and side view are presented, as well as a top view colored by local mean curvature value. PI(4,5)P2 phosphodiester (PO4) beads are represented by the black circles. In the molecular representations, PI(4,5)P2 headgroups are depicted in gray, with the phosphates discriminated in orange. PI(4,5)P2 acyl chains are colored according to the corresponding color code. Ca2+ ions are represented in blue. The bulk POPC lipids are represented by the translucent gray surface.
Fig. 7
Fig. 7. CG MD study of (16:0)2 PI(4,5)P2 gel-phase nanodomains.
a Final snapshot of a gel-forming simulation containing 50% mol PI(4,5)P2 at 280 K. PI(4,5)P2 gel crystals (b) were obtained from this simulation and used for the crystal scaffold simulations (c), which were used to characterize gel-phase properties. d Average system-wide acyl-chain hexagonality dependency with temperature for each of the gel-phase crystal scaffold systems studied. e Average system-wide S-value order parameter, calculated on the first acyl-chain bead of every lipid acyl chain, for each of the gel-phase crystal scaffold systems studied. f S-value order parameter for the first acyl-chain bead of every gel-forming lipid acyl chain for each of the systems studied. Error bars for all measurements represent the SD from N = 3 independent simulation experiments.
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