Abstract
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.
Highlights
The diffusion of membrane receptors is central to many biological processes, such as signal transduction, molecule translocation, and ion transport, among others; several advanced fluorescence microscopy techniques have been developed to measure membrane receptor mobility within live cells
Raster image correlation spectroscopy (RICS)[10,11] is in principle an extended version of fluorescence correlation spectroscopy (FCS), with the addition of a spatial component using confocal laser-scanning microscopy (CLSM), which uses the raster scanning to capture the fluctuations in the intensity caused by the movement of fluorescent molecules
Single particle tracking (SPT) is typically performed on images acquired with a total internal reflection fluorescence (TIRF) microscope, where the acquisition of the intensities of all the pixels in each frame is done simultaneously and isolated particles are followed for relatively long periods of time, with capture frame rates of up to 40 k Hz2
Summary
The diffusion of membrane receptors is central to many biological processes, such as signal transduction, molecule translocation, and ion transport, among others; several advanced fluorescence microscopy techniques have been developed to measure membrane receptor mobility within live cells. 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). Several powerful fluorescence-based methods have been used to study the dynamics of membrane components, such as the diffusion and association of immunoreceptors[2,3,4,5] These techniques are based on either ensemble measurements, where the diffusion coefficient is obtained from the average motion of groups of molecules, or single particle tracking where the mobility of individual particles is analyzed. The images are analyzed to precisely locate each fluorescent particle within the image and the trajectories are built by linking the positions of particles in consecutive frames
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