Analysis of PI(4,5)P2 Lateral Organization at the Plasma Membrane of Living Cells Through FRET

Abstract

Phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) is an important component of the inner leaflet of the plasma membrane of eukaryotic cells. Despite the fact that it only comprises approximately 1 mol% of the total membrane phospholipids, this phosphoinosite has been associated with many different cells functions including membrane trafficking, actin cytoskeleton remodeling and cell motility, among others. The physiological functions of this lipid seem to depend on localized concentration fluctuations within the plasma membrane. In fact, the distribution of this lipid in the plasma membrane has been proposed to be heterogeneous, and PI(4,5)P2 clustering is detected on model membranes under specific conditions. Domains highly enriched in PI(4,5)P2 were also reported at the plasma membrane of specific cell types. However, for most cellular models, scarce evidence has been found for PI(4,5)P2 segregation/clustering in the plasma membrane.In this context, our main goal was to study the distribution of PI(4,5)P2 molecules in cells lines where no heterogeneity in PI(4,5)P2 lateral distribution had been previously observed. The distribution of PI(4,5)P2 was assessed from FRET microscopy measurements with pleckstrin homology (PH) domains tagged with different fluorescent proteins. We applied a FRET methodology capable of discriminating between FRET from aggregates/clusters from non-interacting molecules within the plasma membrane of living cells. Our results clearly show distinct PI(4,5)P2 local densities in different cellular models, suggesting different patterns of PI(4,5)P2 lateral distributions within the plasma membrane. In addition, the effect of cholesterol removal on PI(4,5)P2 lateral organization is significantly different in distinct cell lines, suggesting that the role of cholesterol in the formation of PI(4,5)P2 enriched domains varies considerably.This work was supported by FCT - Foundation of Science and Technology (PTDC/QUI-BIQ/119494/2010 and RECI/CTM-POL/0342/2012). M.J.S. and F.F. acknowledge research grants (SFRH/BD/80575/2011 and SFRH/BPD/64320/2009) from FCT.