Quantitative FRET Microscopy Reveals a Crucial Role of Cytoskeleton in Promoting PI(4,5)P2 Confinement
Abstract
:1. Introduction
2. Results
2.1. 2D FRET between Non-Compartmentalized Proteins Shows a Linear Dependence with Acceptor Concentration in the Low FRET Range
2.2. Bystander FRET in the Absence of Compartmentalization
2.3. Clustering of PI(4,5)P2 in HEK293T Cells
2.4. Clustering of PI(4,5)P2 in HeLa Cells
2.5. Determinants of PI-(4,5)P2 Clustering in HeLa Cells
3. Discussion
4. Materials and Methods
4.1. Cell Culture and Transfection
4.2. pDNA Constructs
4.3. Fluorescence Linear Dichroism Imaging
4.4. Three-Filter Cube FRET Microscopy
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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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
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 StyleSarmento, 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