Examining the Role of Glycolipids in Integrin and B Cell Receptor Activation

Abstract

Glycolipids are an important component of the plasma membrane, and often play important roles in the spatial organization of membrane proteins. Our group is interested in the role of enzymes which alter the glycolipid composition of the plasma membrane in lymphocytes. Glycosyl hydrolase enzymes which catabolize membrane glycolipids have been proposed to regulate receptor signaling by altering membrane glycolipid composition. The human neuraminidase 3 (NEU3) is a membrane-associated enzyme that cleaves terminal neuraminic acid (also known as sialic acid) residues from membrane glycolipids, such as GM3. Cells treated with recombinant NEU3 show changes in their glycolipid composition, thus making NEU3 a powerful tool for probing the role of gangliosides in receptor biophysics. Using single dye tracking (SDT) of membrane receptors by total-internal reflection fluorescence (TIRF) microscopy, we have examined the influence of glycolipids on the lateral diffusion of membrane proteins on lymphocytes. We first examined the T cell integrin, LFA-1. SDT data show clear changes in LFA-1 diffusion, with an increased population of receptors with a large diffusion coefficient after NEU3 treatment. Imaging of LFA-1 by TIRF shows that NEU3 treatment results in co-localization of GM1 and LFA-1, which is distinct from its localization in cells activated with phorbol-12-myristate-13-acetate. To understand the generality of this observation, we examined the influence of NEU3 treatment on the B cell receptor (BCR) complex. NEU3 treatment again increased the proportion of mobile receptors in the B cell membrane relative to control. A similar increase in mobile receptors was observed when cells were activated with PMA. We will present our analysis of SDT and imaging data which support that altering the membrane composition of lymphocytes by NEU3 treatment results in changes to the lateral mobility and subcellular localization of specific receptors.