Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)

Summary

A technique to probe the lipid raft partitioning of fluorescent proteins at the plasma membrane of living cells is described. It takes advantage of the disparity in diffusion times of proteins located inside or outside of lipid rafts. Acquisition can be performed dynamically in control conditions or after drug addition.

Abstract

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. They play a role in cellular physiological processes such as signalling, and trafficking^1,2 but are also thought to be key players in several diseases including viral or bacterial infections and neurodegenerative diseases³.

Yet their existence is still a matter of controversy^4,5. Indeed, lipid raft size has been estimated to be around 20 nm⁶, far under the resolution limit of conventional microscopy (around 200 nm), thus precluding their direct imaging. Up to now, the main techniques used to assess the partition of proteins of interest inside lipid rafts were Detergent Resistant Membranes (DRMs) isolation and co-patching with antibodies. Though widely used because of their rather easy implementation, these techniques were prone to artefacts and thus criticized^7,8. Technical improvements were therefore necessary to overcome these artefacts and to be able to probe lipid rafts partition in living cells.

Here we present a method for the sensitive analysis of lipid rafts partition of fluorescently-tagged proteins or lipids in the plasma membrane of living cells. This method, termed Fluorescence Correlation Spectroscopy (FCS), relies on the disparity in diffusion times of fluorescent probes located inside or outside of lipid rafts. In fact, as evidenced in both artificial membranes and cell cultures, probes would diffuse much faster outside than inside dense lipid rafts^9,10. To determine diffusion times, minute fluorescence fluctuations are measured as a function of time in a focal volume (approximately 1 femtoliter), located at the plasma membrane of cells with a confocal microscope (Fig. 1). The auto-correlation curves can then be drawn from these fluctuations and fitted with appropriate mathematical diffusion models¹¹.

FCS can be used to determine the lipid raft partitioning of various probes, as long as they are fluorescently tagged. Fluorescent tagging can be achieved by expression of fluorescent fusion proteins or by binding of fluorescent ligands. Moreover, FCS can be used not only in artificial membranes and cell lines but also in primary cultures, as described recently¹². It can also be used to follow the dynamics of lipid raft partitioning after drug addition or membrane lipid composition change¹².

Protocol

1. Calibration of the FCS Setup

1. Start the confocal microscope, lasers, computers, incubator for temperature and CO₂ control.
2. Make sure the SPAD (Single Photon Avalanche Diode) is on and the fluorescence filter inside the SPAD is well suited to your sample. Check that the SPAD is synchronized in time. Beware to only start your FCS software once your SPAD settings are ready for acquisition.
3. Prepare a fresh solution of cholera toxin-Alexa488 diluted in PBS to reach a concentration of 1 μg/ml (17.5 nM).
4. Optimize confocal imaging of the solution using internal detection of the microscope.
5. Switch to point scanning mo....

Discussion

The FCS method presented here enables a sensitive and rapid analysis of the lipid raft partitioning of fluorescent probes of interest in living cells. FCS combines the accuracy of localization of confocal microscopy with the sensitivity of single photon counting. The main difference between FCS and standard biochemical techniques is that FCS enables the absolute determination of the lipid rafts partition of the target and not the relative partition as is the case for DRMs isolation or co-patching.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cholera toxin subunit B-Alexa 488	Invitrogen	C-34775	MW (pentamer) = 57 kg/mol
Confocal microscope	Leica Microsystems	SP5
Incubator for temperature and CO2 control	Life imaging services	The Cube and the Box
SPAD (Single Photon Avalanche Diode)	MPD (Micro Photon Devices)	PDM serie (100 µm sensitive area)
High pass 488 nm filter	Semrock	488 nm blocking edge BrightLine long-pass filter Part # FF01-488/LP-25
FCS detection unit	Picoquant	Picoharp 300 module
Acquisition and auto-correlation software	Picoquant	SymPhoTime
Fitting software	OriginLab	OriginPro8