Diffusion of Receptors on Macrophage Plasma Membranes

Abstract

Among the central constituents of the innate immune system are macrophages, which are known for phagocytosis or ‘eating’ foreign particles or pathogens. Macrophages express several cell-surface proteins including transmembrane and membrane-anchored receptors, which play a vital role in their response to pathogenic stimuli. The plasma membrane is a highly fluid and dynamic environment, which facilitates the diffusion of lipids and proteins within the plane of the membrane. This study aims to measure the lateral diffusion of two types of plasma membrane receptors on macrophages, toll-like receptor II (TLR2) and cluster of differentiation 14 (CD14), to answer three main research questions: 1) Which type of fluorescence-based microscopy techniques is best suited for measuring the lateral diffusion of TLR2 and CD14 on macrophage plasma membrane? 2) Does culturing macrophages on different surface topographies impact the diffusion of TLR2 in the plasma membrane and its pro-inflammatory response, along with morphological changes? 3) Does aging alter the lateral diffusion of TLR2 in the plasma membrane of macrophages? To date, a variety of fluorescence-based methods have been developed to study the dynamics of cell membrane constituents. These techniques are based on either ensemble or single particle measurements. We have used single particle tracking methods to track the mobility of fluorescently labeled membrane receptors on murine bone marrow-derived macrophages. Total internal reflection fluorescence microscopy (TIRF) was used to visualize and capture the dynamics in live cells. Using a custom routine algorithm we detected, localized, and tracked the particles to calculate their diffusion coefficient, extracted from the mean-squared displacement as the most common measure of diffusion. We also measured the diffusion coefficient using an ensemble-based technique known as Raster Image Correlation Spectroscopy (RICS) with a confocal laser-scanning microscope. The use of confocal eliminates the out-of-focus signal and enables measurements that are confined to a narrow plane in the cell. Also, the ability of RICS to separate the slow and immobile fractions of particles makes it possible to detect heterogeneities in diffusion. To our knowledge, this is the first study that has utilized both SPT and RICS to directly compare receptors’ diffusion in different membrane sections. Moreover, this is the first study that has examined the diffusion of receptors on macrophages adhered to different surface topographies, and the first that has investigated the receptors’ diffusion in young and old macrophages.