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...
Guardado en:
| Autores principales: | , , , , , , , |
|---|---|
| Formato: | article |
| Lenguaje: | EN |
| Publicado: |
MDPI AG
2021
|
| Materias: | |
| Acceso en línea: | https://doaj.org/article/2e18d906e1f346aa964b02c4c62d8240 |
| Etiquetas: |
Agregar Etiqueta
Sin Etiquetas, Sea el primero en etiquetar este registro!
|
| Sumario: | 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. |
|---|