Imaging Total Internal Reflection Fluorescence Correlation Spectroscopy (ITIR-FCS) Detects Multiple Lipid Domains on Live Cell Membranes

Abstract

The cell membrane consists of a large number of different lipid molecules, of membrane spanning and associated proteins and is mechanically linked to the cytoskeleton and the extracellular matrix. It exhibits a complicated, highly dynamic structure which reflects its large number of constituent components and which is essential for signalling and trafficking in live cells. This structure is thought to have different characteristic length scales which can range from tens to hundreds of nanometers. Thus new methods are needed which can provide information on membrane structures at these different length scales with high temporal resolution. Imaging Total Internal Reflection Fluorescence Correlation Spectroscopy (ITIR-FCS) provides fluorescence correlation measurements on each point of a whole cell membranes with diffraction limited resolution and a better than millisecond time resolution. In particular the differences between spatial cross-correlation measurements, so-called ΔCCF, allows to infer changes in membrane organization and in combination with the diffusion coefficients provides information about membrane dynamics, organization and their changes during drug treatment. Here, in a combination of simulations, measurements on pure and mixed lipid supported bilayers, and live cell membranes stained with domain markers, we show that cell membrane composition is a crucial determinant of the type of domains existing at any point in time. By using markers for liquid ordered (sphingolipid binding domain, SBD) and disordered (DiIC18) lipid phases and a range of membrane composition- and cytoskeleton-influencing agents (methyl-β-cyclodextrin, latrunculin A, NB-DNJ, fumonisin B1, sphingomyelinase) we demonstrate that we have at least two different, ceramide and sphingolipid dependent, domain types, and that membrane fluidity and organization are not necessarily correlated and exist over different length scales.