Fluorescence correlation methods for imaging cellular behavior of sphingolipid-interacting probes.

For cell biologists interested in the properties of cell membranes, their composition, and dynamics, the realization that sphingolipids and cholesterol have the capacity to self-organize into ordered domains has given rise to a need to visualize these lipids in actual living cell membranes. In order to find out how various classes of lipids distribute in the membrane and what their behaviors are, it is extremely useful to apply fluorescent probes that either interact with these lipids, or that themselves behave like naturally occurring lipids. At the same time, imaging modalities to observe their behaviors require the appropriate spatial and temporal resolution, on the milli- or microsecond timescale. Knowledge of membrane organization and how it changes during processes like cell signaling and invasion by pathogens will undoubtedly be relevant to our understanding of the action of infectious diseases, bacterial toxins, and even disease pathologies like prion and Alzheimer's disease, where glycosphingolipids (GSL) and sphingolipid-rich domains act as membrane receptors or docking sites. In such cases, membrane composition and dynamics will clearly play a role in infectivity. The present challenge is in coming up with high-resolution and non-invasive approaches to observing these dynamic and structural features of sphingolipid-containing membrane domains. This chapter will discuss several variants and applications of fluorescence correlation spectroscopy as well as probes that can be used to study sphingolipid dynamics.