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Article

Quantitative FRET Microscopy Reveals a Crucial Role of Cytoskeleton in Promoting PI(4,5)P2 Confinement

1
Centro de Química-Física Molecular and Institute of Nanoscience and Nanotechnology, Instituto Superior Técnico, University of Lisbon, 1049-001 Lisbon, Portugal
2
IBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, University of Lisbon, 1049-001 Lisbon, Portugal
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Associate Laboratory i4HB—Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisbon, Portugal
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Departamento de Química e Bioquímica, Faculty of Sciences, University of Lisbon, 1749-016 Lisbon, Portugal
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Department of Bioengineering, Instituto Superior Técnico, University of Lisbon, 1049-001 Lisbon, Portugal
*
Authors to whom correspondence should be addressed.
Current address: Instituto de Medicina Molecular, Faculty of Medicine, University of Lisbon, Av. Prof. Egas Moniz, 1649-028 Lisbon, Portugal.
Academic Editor: Jean-Pierre Jaffrézou
Int. J. Mol. Sci. 2021, 22(21), 11727; https://doi.org/10.3390/ijms222111727
Received: 1 September 2021 / Revised: 24 October 2021 / Accepted: 26 October 2021 / Published: 29 October 2021
(This article belongs to the Special Issue Raft Microdomains and Cell Signaling)
Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is an essential plasma membrane component involved in several cellular functions, including membrane trafficking and cytoskeleton organization. This function multiplicity is partially achieved through a dynamic spatiotemporal organization of PI(4,5)P2 within the membrane. Here, we use a Förster resonance energy transfer (FRET) approach to quantitatively assess the extent of PI(4,5)P2 confinement within the plasma membrane. This methodology relies on the rigorous evaluation of the dependence of absolute FRET efficiencies between pleckstrin homology domains (PHPLCδ) fused with fluorescent proteins and their average fluorescence intensity at the membrane. PI(4,5)P2 is found to be significantly compartmentalized at the plasma membrane of HeLa cells, and these clusters are not cholesterol-dependent, suggesting that membrane rafts are not involved in the formation of these nanodomains. On the other hand, upon inhibition of actin polymerization, compartmentalization of PI(4,5)P2 is almost entirely eliminated, showing that the cytoskeleton network is the critical component responsible for the formation of nanoscale PI(4,5)P2 domains in HeLa cells. View Full-Text
Keywords: PI(4,5)P2; PH domains; membrane organization; membrane domains; FRET microscopy PI(4,5)P2; PH domains; membrane organization; membrane domains; FRET microscopy
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MDPI and ACS Style

Sarmento, M.J.; Borges-Araújo, L.; Pinto, S.N.; Bernardes, N.; Ricardo, J.C.; Coutinho, A.; Prieto, M.; Fernandes, F. Quantitative FRET Microscopy Reveals a Crucial Role of Cytoskeleton in Promoting PI(4,5)P2 Confinement. Int. J. Mol. Sci. 2021, 22, 11727. https://doi.org/10.3390/ijms222111727

AMA Style

Sarmento MJ, Borges-Araújo L, Pinto SN, Bernardes N, Ricardo JC, Coutinho A, Prieto M, Fernandes F. Quantitative FRET Microscopy Reveals a Crucial Role of Cytoskeleton in Promoting PI(4,5)P2 Confinement. International Journal of Molecular Sciences. 2021; 22(21):11727. https://doi.org/10.3390/ijms222111727

Chicago/Turabian Style

Sarmento, Maria J., Luís Borges-Araújo, Sandra N. Pinto, Nuno Bernardes, Joana C. Ricardo, Ana Coutinho, Manuel Prieto, and Fábio Fernandes. 2021. "Quantitative FRET Microscopy Reveals a Crucial Role of Cytoskeleton in Promoting PI(4,5)P2 Confinement" International Journal of Molecular Sciences 22, no. 21: 11727. https://doi.org/10.3390/ijms222111727

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