Monitoring Association of Membrane Proteins with Micro-Domains and Cytoskeleton in Live Cells During Signaling and Perturbation

Abstract

A central challenge to understanding cell signaling is to quantify how the membrane ultra-structure modulates signaling dynamics and how vice-versa signaling influences the membrane ultra-structure. Spatial membrane domains, such as created by lipid rafts and the membrane cytoskeleton, influence membrane protein mobility and hence membrane bound processes. Not only are these domains modified in a range of pathologies but various drugs are also thought to influence them. However, direct visualization of these lipid domains in intact cells is challenging because of their small dimension and dynamics.We present a non-destructive a camera based FCS method for quantifying the membrane protein association with lipid domains in intact cells and with membrane cytoskeleton domains. Most importantly, our method provides continuous monitoring of changes in the protein domain interactions over time. We investigated the modulation of GPI-anchored GFP with lipid domains over time in response to cross linking, Ethanol, Cholesterol Sulfate or Cholera toxin addition, as well as cholesterol extraction. As an example for signaling induced changes, we studied the changes in the interaction of temperature sensitive ion channel, TRPV2, with lipid domains and membrane cytoskeleton upon activation by 2-APB.