Biophysical Society Thematic Meeting - October 13-15, 2015

Biophysics of Proteins at Surfaces: Assembly, Activation, Signaling

Wednesday Speaker Abstracts

FRET Analysis of the Nanoscale Organization of PI(4,5)P 2 in Living Cells Maria J. Sarmento 1 , Ana Coutinho 1,2 , Manuel Prieto 1 , Fabio Fernandes 1 . 1 Instituto Superior Técnico, Lisbon, Portugal, 2 Faculdade de Ciências, Universidade de Lisboa, Lisbon, Portugal. Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ) is a phospholipid concentrated in the inner leaflet of the plasma membrane, to which it recruits proteins involved in several cellular functions, many of which are abrogated in the absence of PI(4,5)P 2 , illustrating the importance of this lipid. Protein regulation by PI(4,5)P 2 occurs as a result of spatially and temporally localized fluctuations of its concentration in the plasma membrane. In fact, the distribution of this lipid in the plasma membrane has been proposed to be heterogeneous, and PI(4,5)P 2 clustering is detected on model membranes under specific conditions. Domains highly enriched in PI(4,5)P 2 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)P 2 segregation/clustering in the plasma membrane. Here, we aimed to characterize the distribution of PI(4,5)P 2 in the plasma membrane of cells where no heterogeneity in PI(4,5)P 2 lateral distribution had been previously detected. To this end, FRET microscopy measurements with pleckstrin homology (PH) domains tagged with different fluorescent proteins were carried out. FRET microscopy data is evaluated through comparison with the theoretical expectation for FRET in the case of a homogeneous distribution of PH domains, and evidence for the formation of PI(4,5)P 2 enriched nanodomains is obtained. Results confirm that distinct PI(4,5)P 2 local densities are found in different cellular models, suggesting that PI(4,5)P 2 organization varies significantly between eukaryotic cells. In HeLa cells, disruption of the cytoskeleton decreased the compartmentalization of PI(4,5)P 2 , proving that the organization of at least a pool of PI(4,5)P 2 molecules depends on the presence of membrane-cytoskeleton interactions. 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.

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