Characterization of diffusion behavior of a novel extra-cellular sphingolipid associated peptide probe by fluorescence correlation spectroscopy and imaging total internal reflection fluorescence correlation spectroscopy

Abstract

Cell membrane is a very interesting and widely studied research area due to its physiological importance. Membrane heterogeneity also gained interest over the last few decades due to their relevance with different diseases. The heterogeneity arises due to some membrane proteins surrounded by some selective classes of lipids. The lipids of interest to this work belong to the sphingolipid family. Faulty intracellular trafficking or storage of sphingolipids and cholesterol can lead to an array of lipid storage diseases. Therefore studies of sub-cellular movements of sphingolipids and domains consist of sphingolipids have high level of importance. The major limitation associated with the field of sphingolipid trafficking is lack of commercially available reliable markers that can be used to trace lipid microdomains or sphingolipids in living cells. The easily synthesizable molecular fluorophore conjugated, 25 amino acid sequence of Amyloid beta peptide has been characterized in this study, to test the hypothesis that this peptide, the Sphingolipid Binding Domain (SBD), could mediate tagging of the sphingolipid rich domains found in the plasma membrane that constitute rafts. For the characterization of SBD?s diffusion behaviour on live cell surface, Fluorescence Correlation Spectroscopy, a widely used biophysical technique has been used in this study. Furthermore to visualize dynamic heterogeneous cell membrane organization traced by SBD, two new biophysical tool Imaging Total Internal Reflection-Fluorescence Correlation Spectroscopy (ITIR-FCS) and Imaging Total Internal Reflection-Fluorescence Cross Correlation Spectroscopy (ITIR-FCCS) has been introduced in this study.