Abstract
Membrane organization of lipid anchored proteins plays a fundamental role in proper cell signaling behavior. Several important signaling proteins, such as members of the Src family of kinases, are anchored to the membrane by covalent acyl, alkyl, and glycosylphosphatidylinositol (GPI) moieties. A convenient approach to studying this lipid anchor organization in cells is to image genetically encoded lipidation motifs of various signaling proteins fused to a fluorescent protein. However, previous efforts to reduce this lateral organization to a simple model often ignore the complexity inherent in cell membranes and have contributed to an inconsistent picture of membrane organization. We use two-color fluorescence fluctuation spectroscopy to study the spatial organization of fluorescently tagged lipid anchors in live cell membranes. In order to retain the complexity of this organization, we simultaneously observe several modes of lipid anchor dynamics by employing a multi-dimensional analysis of the time-resolved fluorescence data. Time-correlated single photon counting (TCSPC) data is gathered on a 3-detector system optimized for simultaneous polarization and two-color fluorescence correlation spectroscopy (FCS). The degree of dynamic colocalization of eGFP- and mCherry-fused lipid anchors can be quantified by pulse-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS), while single color FCS evaluates the translational mobility of the lipid anchors. The photon counting method also allows for time-resolved fluorescence anisotropy (TRFA) measurements, evaluating the rotational diffusion of the fluorescent protein, as well as fluorescence lifetime measurements to determine energy transfer. Taken together, this simultaneous multi-dimensional analysis of time-resolved fluorescence data offers a powerful approach towards determining the complex organizations of lipid anchors in live cell membranes.
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