Concentration Dependent Membrane Anchor Colocalization Study by Fluorescence Cross-Correlation Spectroscopy in Live Cells

Abstract

Membrane anchors such as protein lipidations and glypiations have been proposed to play essential roles in the sorting and organization of plasma membrane-associated proteins, especially those involved in cell signaling. Here, we investigate the concentration dependence and variability of anchor colocalization in live cells by transfecting various cell types with pairs of fusion proteins created by replacing all but short tails of natively lipidated proteins with either red or green fluorescent proteins. These fusion proteins remove any native protein-protein interactions while fluorescently tagging membrane anchors in live cells. To observe sub-cellular organization, we use Fluorescence Cross-Correlation Spectroscopy (FCCS) to quantify the dynamic colocalization between green- and red-labeled anchors. FCCS allows observations of dynamic colocalization in live cells at a greater range of separation distances than is allowed by FRET, and because it is a dynamic measurement FCCS avoids ambiguous or false positive colocalization that can result from static studies. Fusion protein expression level, as determined by overall intensity of cell fluorescence, naturally varies in a population of transiently transfected cells. Using this to our advantage, we are able to observe cells within a wide range of protein expression and explore trends between concentration and fusion protein colocalization. We also analyze variation in the amount of colocalization and observe a difference between the variability from cell-to-cell and the variability from spot-to-spot within one cell across several anchor types and different cell lines.