Measuring the Lateral Diffusion of Plasma Membrane Receptors Using Raster Image Correlation Spectroscopy.

Raster image correlation spectroscopy (RICS) enables detecting and quantifying diffusion in live cells using standard commercial laser scanning confocal microscopes. Here, we describe a protocol based on RICS for measuring the lateral diffusion of two immunoreceptors within the plasma membrane of the macrophage cell line RAW 264.7. The sample images and measurements presented in this chapter were obtained from RICS analysis of Toll-like receptor 2 (TLR2) and cluster of differentiation 14 (CD14), which are transmembrane and membrane-anchored receptors, respectively. A step-by-step guideline is provided to acquire raster-scanned images and to extract the diffusion coefficients using RICS analysis.

Lateral diffusion of CD14 and TLR2 in macrophage plasma membrane assessed by raster image correlation spectroscopy and single particle tracking.

The diffusion of membrane receptors is central to many biological processes, such as signal transduction, molecule translocation, and ion transport, among others; consequently, several advanced fluorescence microscopy techniques have been developed to measure membrane receptor mobility within live cells. The membrane-anchored receptor cluster of differentiation 14 (CD14) and the transmembrane toll-like receptor 2 (TLR2) are important receptors in the plasma membrane of macrophages that activate the intracellular signaling cascade in response to pathogenic stimuli. The aim of the present work was to compare the diffusion coefficients of CD14 and TLR2 on the apical and basal membranes of macrophages using two fluorescence-based methods: raster image correlation spectroscopy (RICS) and single particle tracking (SPT). In the basal membrane, the diffusion coefficients obtained from SPT and RICS were found to be comparable and revealed significantly faster diffusion of CD14 compared with TLR2. In addition, RICS showed that the diffusion of both receptors was significantly faster in the apical membrane than in the basal membrane, suggesting diffusion hindrance by the adhesion of the cells to the substrate. This finding highlights the importance of selecting the appropriate membrane (i.e., basal or apical) and corresponding method when measuring receptor diffusion in live cells. Accurately knowing the diffusion coefficient of two macrophage receptors involved in the response to pathogen insults will facilitate the study of changes that occur in signaling in these cells as a result of aging and disease.