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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.