Membrane Anchor Dependent Colocalization in Cellular Membranes Observed by Fluorescence Cross-Correlation Spectroscopy

Abstract

Membrane anchors exist on many proteins in a variety of combinations of enzymatically attached fatty acids and glypiations. These anchors play a part in protein trafficking within cells and in associating proteins with cell membranes. They are also frequently found on well-known signaling proteins. Given the variety of anchor composition, we question whether these anchors play a more significant role in the lateral sorting or dynamic colocalization of proteins within cell membranes. To observe this in vivo, we create fusion proteins of red and green fluorescent proteins with the consensus protein lipidation motif of various signaling proteins and express both red and green constructs in HEK293T cells. The dynamic colocalization of red and green fluorescent proteins, and therefore the dynamic colocalization of membrane anchors, can be directly observed using Fluorescence Cross-Correlation Spectroscopy (FCCS). FCCS allows us to observe dynamic colocalization on the nanometer length scale. Unlike FRET, FCCS can detect positive colocalization regardless of orientation and at lengths larger than 10nm. Recent results will be discussed.