Physical chemistry of lipid bilayers

Lipids in the plasma membrane exhibit differential lateral diffusion as membrane components assemble in a selective manner to form different phases – liquid-ordered (Lo) and liquid-disordered (Ld). The Lo phase is introduced by the presence of cholesterol, which induce short-range order in unsaturated lipids while disrupting long-range order in saturated lipids simultaneously. This Lo phase is still considered to be fluid, as translational disorder exists where the lipids retain substantial mobility in the plane of the lipid bilayer, but a high order of lipid packing persists simultaneously. Notably, there is a marked difference in lateral lipid mobility between the Ld and Lo phases, where the Ld phase has a lateral diffusion coefficient (D) which is at least twice or thrice the value of the Lo phase.

In order to investigate this physical phenomenon in plasma membranes, we started by looking at the difference in diffusion coefficients in supported lipid bilayers (SLBs) of different compositions and at different temperatures using Imaging Total Internal Reflection-Fluorescence Correlation Spectroscopy (ITIR-FCS). As shown in Figure 1A, DOPC:DPPC:cholesterol mixtures exhibit lower D values than their counterparts with no cholesterol. This supports the proposition that adding cholesterol introduces greater order in lipids, hence restraining their mobility. In addition, higher Arrhenius activation energies (EArr) (Figures 1B, C) proved that there was indeed tighter packing in DOPC:DPPC:cholesterol mixtures where the lipids required more energy to overcome the diffusion energy barrier, supporting the existence of the Lo phase.

Figure 1: (A) Diffusion coefficients, D, of SLBs of different compositions across a temperature ramp from 298 to 313 K. (B) Arrhenius plots of SLBs – DLPC:DPPC (solid circle), DOPC:DPPC (solid square), DOPC:DPPC:Chol (5:5:2) (open circle), DOPC:DPPC:Chol (5:5:5) (open square), DOPC:DPPC:Chol (5:5:10) (open triangle), DOPC:DPPC:Chol (5:5:5) + mβCD (solid triangle), DOPC:DPPC + mβCD (solid diamond). (C) Arrhenius activation energies (EArr) of the SLBs. (Adapted from Bag et al., 2014)

The same investigation was extended to live cells, where lipid diffusion was measured using two lipid probes – DiI and GFP-GPI. Lipid diffusion slowed down when temperature fell, evident by the fall in D values (Figure 2A). Since it was proven that lipid diffusion is temperature-dependent, we can plot Arrhenius plots as shown in Figure 2B, where EArr was derived from the slope. From these results, it was postulated that different cell types have different physical properties and possibly membrane composition because they have different D (Figure 2A) and EArr (Figure 2C) values when the same lipid probe (DiI) was used. Notably, diffusion was much slower for transfected HeLa cells with GFP-GPI, a raft marker. This is expected as lipids in rafts, designated as the Lo phase, are packed in a tighter and more ordered manner than the Ld phase, which is highlighted by the DiI marker. This phenomenon is further supported by the larger EArr value of HeLa cells transfected with GFP-GPI (Figure 2C), which is around a factor of two larger than that of DiI-stained HeLa cells. Furthermore, using FCS diffusion law analysis, where a positive intercept suggests raft partitioning while zero intercept indicates free diffusion, we were able to probe the mode of diffusion in the plasma membrane. FCS diffusion law plots were obtained (Figure 3A) and it was found that the intercepts were positive for GFP-GPI and zero for DiI (Figure 3B). This proves that GFP-GPI and DiI are indeed markers for the raft (Lo) and non-raft (Ld) phase respectively.

Figure 2: (A) Diffusion coefficients, D, of different cell lines (HeLa, SH-SY5Y and Fibroblast) probed with different lipid markers (DiI and GFP-GPI) across a temperature ramp from 298 to 310 K. (B) Arrhenius plots of different cell lines probed with DiI and GFP-GPI, where error bars are only shown for DiI-stained fibroblast cells for clarity. (C) Comparison of Arrhenius activation energies (EArr) of different cell lines and lipid probes. (Adapted from Bag et al., 2014)

Figure 3: (A) FCS diffusion law plots of HeLa cells probed with DiI (Ld) and GFP-GPI (Lo) markers at two different temperatures – 298 K and 310 K. (B) Comparison of FCS diffusion law intercept values across different temperatures for HeLa cells probed with the two markers. (Adapted from Bag et al., 2014)

Reference:

Bag, N.; Yap, D. H. X.; Wohland, T., Temperature dependence of diffusion in model and live cell membranes characterized by imaging fluorescence correlation spectroscopy. Biochimica et Biophysica Acta (BBA) - Biomembranes 2014, 1838 (3), 802-813.

  • NUS
  • Biophysical Fluorescence Laboratory