Abstract
Cholesterol constitutes ∼30–40% of the mammalian plasma membrane, a larger fraction than of any other single component. It is a major player in numerous signaling processes as well as in shaping molecular membrane architecture. However, our knowledge of the dynamics of cholesterol in the plasma membrane is limited, restricting our understanding of the mechanisms regulating its involvement in cell signaling. Here, we applied advanced fluorescence imaging and spectroscopy approaches on in vitro (model membranes) and in vivo (live cells and embryos) membranes as well as in silico analysis to systematically study the nanoscale dynamics of cholesterol in biological membranes. Our results indicate that cholesterol diffuses faster than phospholipids in live membranes, but not in model membranes. Interestingly, a detailed statistical diffusion analysis suggested two-component diffusion for cholesterol in the plasma membrane of live cells. One of these components was similar to a freely diffusing phospholipid analogue, whereas the other one was significantly faster. When a cholesterol analogue was localized to the outer leaflet only, the fast diffusion of cholesterol disappeared, and it diffused similarly to phospholipids. Overall, our results suggest that cholesterol diffusion in the cell membrane is heterogeneous and that this diffusional heterogeneity is due to cholesterol's nanoscale interactions and localization in the membrane.
Highlights
Cholesterol constitutes ϳ30 – 40% of the mammalian plasma membrane, a larger fraction than of any other single component
We investigated the diffusion of fluorescent cholesterol analogues in both model and cellular membranes using advanced imaging and spectroscopy tools as well as molecular
We found that cholesterol moves only slightly faster (Ϸ1.2 times) than phospholipids and sphingolipids in model membranes that are thermodynamically in equilibrium
Summary
To understand cholesterol dynamics at the nanoscale in biomembranes, we first compared the diffusion dynamics between various cholesterol analogues and phospholipid analogues (Fig. 1). We used TF-Chol attached to a saturated (16:0) and an unsaturated (18:2) acyl chain to study whether the acyl chains make cholesterol behave similar to phospholipids in the membrane. Diffusion of cholesterol analogues compared with phospholipid analogues in model membranes Previous reports suggested fast diffusion of TF-Chol (Ϸ3 m2/s) in the plasma membrane of live cells [18, 19]. We compared whether an acyl chain attachment changes the cholesterol mobility To this end, we compared the diffusion of TF-Chol with TF-Chol bearing either saturated or unsaturated acyl chains (16:0 TF-Chol and 18:2 TF-Chol, respectively) (Fig. 1). In GUVs, acyl-chain carrying cholesterol analogues did not show notably different diffusion (D16:0 ϭ 10.0 Ϯ 0.9 m2/s; D18:2 ϭ 10.3 Ϯ 0.8 m2/s) compared with TF-Chol (DTF-Chol ϭ 10.5 Ϯ 1.2 m2/s) (Fig. 2C).
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