Differential Clustering of Membrane Anchors Observed by FCCS in Live Cells

Abstract

Proper lateral organization of protein signaling elements in the cell membrane is crucial for proper cell behavior. Several important signaling proteins, such as some members of the Src family of kinases, are anchored to the membrane by covalent acyl, alkyl, and glycosylphosphatidylinositol (GPI) moieties. It has been proposed that these membrane anchors help to laterally sort proteins into signaling nanoclusters of unknown sizes. We set out to understand the role of these membrane anchors in the lateral sorting of proteins in the plasma membrane of a live cell. In order to observe this, we genetically encode lipidation motifs from a number of different cell signaling proteins fused to either a monomeric red or green fluorescent protein in living cells. Using Fluorescence Cross-Correlation Spectroscopy (FCCS) we are able to quantify the amount of dynamic colocalization of red and green fluorescent proteins anchored in the cell membrane. By only illuminating an area of the membrane with the cross-section of a focused laser beam, FCCS allows us to observe dynamic colocalization on the nanometer length scale, and unlike Forster Resonance Energy Transfer (FRET), FCCS can detect positive colocalization regardless of orientation and at lengths beyond the Forster radius of FRET pairs. We seek to understand if our fluorescent membrane anchors alone can dynamically colocalize in domains and if these domains can discriminate between different anchors. We have performed pairwise comparison of green-labeled and red-labeled membrane anchors in live cells and have seen different levels of cross-correlation for different anchor pairs.