DOI: 10.5281/zenodo.15835847
title: Distinct Effects of Progesterone and Cholesterol on Lipid Membranes: Insights from Biophysical Experiments and Molecular Dynamics Simulations

description: Files legend:



Circularity_ploted – Circularity analysis of domains (So or Lo phases) in heated mica-supported lipid bilayers composed of an equimolar mixture of: DmOPC:SM:Chol, DmOPC:SM:Prog and DmOPC:SM. Each value represents the circularity coefficient averaged over individual domains and calculated using the equation (circularity = 4π area/perimeter^2), and averaged over at least two distinct samples per composition. The number of analysed domains per condition ranges from 116 to 353. Only domains clearly above the diffraction limit (i.e. with a diameter of over 3 μm) were included in the analysis.

Circularity_all_data – Circularity data used for obtaining the averaged values described above (File 1.) 

Laurdan_spectra_phase_separated_membranes – Changes in the fluorescence spectrum of Laurdan embedded in lipid bilayers in response to different phase-separated (the So/Lo and Ld phases) membrane compositions: 1:1:1 DmOPC:SM (mol:mol:mol), 1:1:1 DmOPC:SM:Prog (mol:mol:mol) and 1:1:1 DmOPC:SM:Chol (mol:mol:mol). The spectra for each composition were averaged from two independent samples and normalized. 

Laurdan_spectra_single_phase_membranes_DmOPC_SM – Changes in the fluorescence spectrum of Laurdan embedded in lipid bilayers in response to different membrane compositions and specific steroid content: 7:3 DmOPC:Prog (mol:mol) and 7:3 SM:Prog (mol:mol). The spectra for each composition were averaged from two independent samples and normalized. 

Laurdan_spectra_single_phase_membranes_DOPC – Changes in the fluorescence spectrum of Laurdan embedded in lipid bilayers in response to increasing amount percentage of progesterone and cholesterol, ranging from 0 mol% to 50 mol%. Intermediate amounts include 10 mol%, 20 mol%, 30 mol% and 40 mol%. The spectra for each steroid content were averaged from two independent samples and normalized. 

Laurdan_spectra_centre_of_mass – Centre of mass parameter calculated for Laurdan fluorescence spectra from File 5. 

Diffusion_coefficient_EXP_SIM_summary – Diffusion coefficients for mica-supported lipid bilayers composed of pure DOPC and binary mixtures with 25 mol% progesterone or 25 mol% cholesterol. The experimental data represent the average of at least 20 FRAP traces per composition, collected from 2 independent samples. The data were averaged from samples containing 20 and 30 mol% of progesterone or cholesterol, respectively, within the Ld phase labelled with Atto 633-DOPE. There are also values that correspond to the diffusion coefficients obtained from MD simulations – they were obtained from the mean square displacement (MSD) of the phosphate atoms, gathered over 100 ns and via performing a linear fitting of the MSD data on the time interval between 10 ns and 90 ns. Mean square of the translation distance of the particle is proportional to 4 × D × t. Both MSD and D were calculated by the program gmx msd.

DOPC_Prog_FRAP_ploted_averaged – Averaged diffusion coefficients for mica-supported lipid bilayers composed of pure DOPC and binary mixtures of DOPC and a steroid molecule at progesterone amounts ranging from 0 up to 50 mol% (the intermediate values are 10, 20, 30, and 40 mol%).

DOPC_Prog_FRAP_columns_all_data – Diffusion coefficients data used for obtaining the averaged values described above (File 8.)

DOPC_Prog_FRAP_traces – FRAP traces for mica-supported lipid bilayers composed of pure DOPC and binary mixtures of DOPC and a steroid molecule at progesterone amounts ranging from 0 up to 50 mol% (the intermediate values are 10, 20, 30, and 40 mol%).

DOPC_Chol_FRAP_ploted_averaged – Averaged diffusion coefficients for mica-supported lipid bilayers composed of pure DOPC and binary mixtures of DOPC and a steroid molecule at cholesterol amounts ranging from 0 up to 50 mol% (the intermediate values are 10, 20, 30, and 40 mol%).

DOPC_Chol_FRAP_columns_all_data – Diffusion coefficients data used for obtaining the averaged values described above (File 11.)

DOPC_Chol_FRAP_traces – FRAP traces for mica-supported lipid bilayers composed of pure DOPC and binary mixtures of DOPC and a steroid molecule at cholesterol amounts ranging from 0 up to 50 mol% (the intermediate values are 10, 20, 30, and 40 mol%).

DOPC_Prog_and_DOPC_Chol_FRAP_mobile_fraction_ploted_averaged – Mobile fractions extracted from the fits of the modified Soumpasis formula to the FRAP traces presented in Files 10 and 13 using additional equation (R_mobile = a/(1-b), where a and b were determined from the fitting procedure: a – the amplitude of the fitted recovery curve, b – the residual fluorescence after photobleaching). 

DOPC_Prog_and_DOPC_Chol_FRAP_mobile_fraction_all_data – Mobile fractions data used for obtaining the averaged values described above (File 14.)

MD_Mean_Square_Displacement – Mean squared displacement (MSD) of phosphorus atoms in the last 100 nm of the simulation for pure DOPC and binary mixtures containing 25 mol% progesterone or 25 mol% cholesterol. Further information can be found in the description of File 7.

MD_density_profiles – Steroid ring positions in systems containing DOPC and 25 mol% progesterone or 25 mol% cholesterol based on MD simulations – distribution of steroid ring centres along the membrane normal (Z-axis) relative to the bilayer midplane (Z=0). 

MD_angle_distribution – Steroid ring orientations in systems containing DOPC and 25 mol% progesterone (pink) or 25 mol% cholesterol (blue) based on MD simulations. (A) Distribution of steroid ring centres along the membrane normal (Z-axis) relative to the bilayer midplane (Z=0) – distribution of tilt angles (θ) between the long axis of the steroid core and the Z-axis for progesterone and cholesterol (the vector defined by two atoms located at the ends of steroid rings, we used the C3 and C16 atoms from Chol and Prog, according to the IUPAC numbering).

MD_heatmap_datasets – Steroid ring positions and orientations in systems containing DOPC and 25 mol% progesterone or 25 mol% cholesterol based on MD simulations – joint probability distributions for tilt angles and ring positions with respect to the Z-axis.

MD_order_parameter_relative_to_DOPC – Relative order parameters SCH with respect to pure DOPC for two binary mixtures: DOPC with 25 mol% of progesterone and DOPC with 25 mol% of cholesterol.

MD_order_parameter_sn1_sn2 – Order parameter for three different compositions: pure DOPC and two binary mixtures of DOPC with either 25 mol% of progesterone or 25 mol% of cholesterol, for the first and second chain of DOPC.

MD_APL – Area per lipid parameter for membranes composed of pure DOPC and two binary mixtures: DOPC with 25 mol% of progesterone or cholesterol. 

Figure 1. – Representative confocal fluorescence microscopy images of mica-supported lipid bilayers composed of an equimolar mixture of DmOPC:SM (A, D), DmOPC:SM:Prog (B, E), and DmOPC:SM:Chol (C, F) prepared under two different temperature conditions. The top row shows samples prepared at ambient temperature (21°C), while the bottom row shows samples prepared with the modifications as described in the Materials and methods section. The Ld phase is labeled with Atto 633-DOPE. Images for each temperature condition were acquired from distinct samples. The scale bar corresponds to 10 μm.