Effect of Lipid Raft Disruptors on Cell Membrane Fluidity Studied by Fluorescence Spectroscopy

Abstract

Lipid rafts are specialized microdomains in cell membranes, rich in cholesterol and sphingolipids, and play an integrative role in several physiological and pathophysiological processes. The integrity of rafts can be disrupted via their cholesterol content-with methyl-β-cyclodextrin (MCD) or with our own carboxamido-steroid compound (C1)-or via their sphingolipid content-with sphingomyelinase (SMase) or with myriocin (Myr). We previously proved by the fluorescent spectroscopy method with LAURDAN that treatment with lipid raft disruptors led to a change in cell membrane polarity. In this study, we focused on the alteration of parameters describing membrane fluidity, such as generalized polarization (GP), characteristic time of the GP values change-Center of Gravity (τ_CoG)-and rotational mobility (τ_rot) of LAURDAN molecules. Myr caused a blue shift of the LAURDAN spectrum (higher GP value), while other agents lowered GP values (red shift). MCD decreased the CoG values, while other compounds increased it, so MCD lowered membrane stiffness. In the case of τ_rot, only Myr lowered the rotation of LAURDAN, while the other compounds increased the speed of τ_rot, which indicated a more disordered membrane structure. Overall, MCD appeared to increase the fluidity of the membranes, while treatment with the other compounds resulted in decreased fluidity and increased stiffness of the membranes.

Keywords: LAURDAN; cholesterol; fluorescens spectroscopy; lipid raft; methyl-beta-cyclodextrin; myriocin; sphingomyelinase.

Figure 1

LAURDAN emission decays at 420 nm (violet), 460 nm (blue), and 530 nm (green). LAURDAN in cell suspension, treated with Smase. λ_ex = 369 nm.

Figure 2

CoG function of LAURDAN. Black line is the CoG function of LAURDAN and red dashed line is a mono-exponential fit giving excellent cover with the measured CoG function.

Figure 3

TRANES of LAURDAN. (A), Laurdan in cell suspension; (B), Laurdan in non-cell solution. λ_ex = 369 nm.

Figure 4

Anisotropy decay of LAURDAN. λ = 450 nm. Black—Anisotropy data. Red—1-exponential fit.

Figure 5

Structure of C1 compound and cholesterol.

Figure 6

Calculated steady-state spectra of LAURDAN. λ_ex = 369 nm.