Quantitative FRET Microscopy Reveals a Crucial Role of Cytoskeleton in Promoting PI(4,5)P<sub>2</sub> Confinement

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P<sub>2</sub>) 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 o...

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Autores principales:	Maria J. Sarmento, Luís Borges-Araújo, Sandra N. Pinto, Nuno Bernardes, Joana C. Ricardo, Ana Coutinho, Manuel Prieto, Fábio Fernandes
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	PI(4,5)P<sub>2</sub> PH domains membrane organization membrane domains FRET microscopy Biology (General) QH301-705.5 Chemistry QD1-999
Acceso en línea:	https://doaj.org/article/2e18d906e1f346aa964b02c4c62d8240
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spelling	oai:doaj.org-article:2e18d906e1f346aa964b02c4c62d82402021-11-11T17:11:06ZQuantitative FRET Microscopy Reveals a Crucial Role of Cytoskeleton in Promoting PI(4,5)P<sub>2</sub> Confinement10.3390/ijms2221117271422-00671661-6596https://doaj.org/article/2e18d906e1f346aa964b02c4c62d82402021-10-01T00:00:00Zhttps://www.mdpi.com/1422-0067/22/21/11727https://doaj.org/toc/1661-6596https://doaj.org/toc/1422-0067Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P<sub>2</sub>) 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)P<sub>2</sub> within the membrane. Here, we use a Förster resonance energy transfer (FRET) approach to quantitatively assess the extent of PI(4,5)P<sub>2</sub> confinement within the plasma membrane. This methodology relies on the rigorous evaluation of the dependence of absolute FRET efficiencies between pleckstrin homology domains (PH<sub>PLCδ</sub>) fused with fluorescent proteins and their average fluorescence intensity at the membrane. PI(4,5)P<sub>2</sub> 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)P<sub>2</sub> is almost entirely eliminated, showing that the cytoskeleton network is the critical component responsible for the formation of nanoscale PI(4,5)P<sub>2</sub> domains in HeLa cells.Maria J. SarmentoLuís Borges-AraújoSandra N. PintoNuno BernardesJoana C. RicardoAna CoutinhoManuel PrietoFábio FernandesMDPI AGarticlePI(4,5)P<sub>2</sub>PH domainsmembrane organizationmembrane domainsFRET microscopyBiology (General)QH301-705.5ChemistryQD1-999ENInternational Journal of Molecular Sciences, Vol 22, Iss 11727, p 11727 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	PI(4,5)P<sub>2</sub> PH domains membrane organization membrane domains FRET microscopy Biology (General) QH301-705.5 Chemistry QD1-999
spellingShingle	PI(4,5)P<sub>2</sub> PH domains membrane organization membrane domains FRET microscopy Biology (General) QH301-705.5 Chemistry QD1-999 Maria J. Sarmento Luís Borges-Araújo Sandra N. Pinto Nuno Bernardes Joana C. Ricardo Ana Coutinho Manuel Prieto Fábio Fernandes Quantitative FRET Microscopy Reveals a Crucial Role of Cytoskeleton in Promoting PI(4,5)P<sub>2</sub> Confinement
description	Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P<sub>2</sub>) 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)P<sub>2</sub> within the membrane. Here, we use a Förster resonance energy transfer (FRET) approach to quantitatively assess the extent of PI(4,5)P<sub>2</sub> confinement within the plasma membrane. This methodology relies on the rigorous evaluation of the dependence of absolute FRET efficiencies between pleckstrin homology domains (PH<sub>PLCδ</sub>) fused with fluorescent proteins and their average fluorescence intensity at the membrane. PI(4,5)P<sub>2</sub> 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)P<sub>2</sub> is almost entirely eliminated, showing that the cytoskeleton network is the critical component responsible for the formation of nanoscale PI(4,5)P<sub>2</sub> domains in HeLa cells.
format	article
author	Maria J. Sarmento Luís Borges-Araújo Sandra N. Pinto Nuno Bernardes Joana C. Ricardo Ana Coutinho Manuel Prieto Fábio Fernandes
author_facet	Maria J. Sarmento Luís Borges-Araújo Sandra N. Pinto Nuno Bernardes Joana C. Ricardo Ana Coutinho Manuel Prieto Fábio Fernandes
author_sort	Maria J. Sarmento
title	Quantitative FRET Microscopy Reveals a Crucial Role of Cytoskeleton in Promoting PI(4,5)P<sub>2</sub> Confinement
title_short	Quantitative FRET Microscopy Reveals a Crucial Role of Cytoskeleton in Promoting PI(4,5)P<sub>2</sub> Confinement
title_full	Quantitative FRET Microscopy Reveals a Crucial Role of Cytoskeleton in Promoting PI(4,5)P<sub>2</sub> Confinement
title_fullStr	Quantitative FRET Microscopy Reveals a Crucial Role of Cytoskeleton in Promoting PI(4,5)P<sub>2</sub> Confinement
title_full_unstemmed	Quantitative FRET Microscopy Reveals a Crucial Role of Cytoskeleton in Promoting PI(4,5)P<sub>2</sub> Confinement
title_sort	quantitative fret microscopy reveals a crucial role of cytoskeleton in promoting pi(4,5)p<sub>2</sub> confinement
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/2e18d906e1f346aa964b02c4c62d8240
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Quantitative FRET Microscopy Reveals a Crucial Role of Cytoskeleton in Promoting PI(4,5)P<sub>2</sub> Confinement

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