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Related Experiment Video

Updated: Mar 6, 2026

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation
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Fluorescent FYVE Chimeras to Quantify PtdIns3P Synthesis During Autophagy.

S M S Yakhine-Diop1, G Martínez-Chacón1, R A González-Polo1

  • 1Centro de Investigación Biomédica en Red en Enfermedades Neurodegenerativas (CIBERNED), Universidad de Extremadura, Cáceres, Spain; Facultad de Enfermería y Terapia Ocupacional, Universidad de Extremadura, Cáceres, Spain.

Methods in Enzymology
|March 4, 2017
PubMed
Summary

This study quantifies phosphatidylinositol 3-phosphate (PtdIns3P) synthesis, a key molecule in autophagy. The method uses cells expressing FYVE-dsRed to measure PtdIns3P production, aiding autophagy research.

Keywords:
3-MethyladenineAutophagyFYVE domainFluorescence microscopyPtdIns3KPtdIns3PStarvation

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Area of Science:

  • Cellular Biology
  • Molecular Biology

Background:

  • Autophagy is a critical cellular degradation process regulated by signaling pathways.
  • Phosphatidylinositol 3-kinases (PtdIns3K) class III (Vps34) and class I modulate autophagy positively and negatively, respectively.
  • Both PtdIns3K classes synthesize phosphatidylinositol 3-phosphate (PtdIns3P), vital for autophagosome biogenesis and membrane traffic.

Purpose of the Study:

  • To present a method for quantifying phosphatidylinositol 3-phosphate (PtdIns3P) synthesis.
  • To utilize FYVE domain-containing proteins for PtdIns3P detection and measurement.

Main Methods:

  • Employing cells transiently transfected with FYVE-dsRed.
  • Utilizing cells stably expressing FYVE-dsRed.
  • Quantifying PtdIns3P synthesis through these cellular models.

Main Results:

  • Demonstrated a method for quantifying PtdIns3P synthesis.
  • Successfully used FYVE-dsRed expressing cells to measure PtdIns3P production.

Conclusions:

  • The presented method allows for the quantification of PtdIns3P synthesis.
  • This technique can be applied to study the role of PtdIns3P in cellular processes like autophagy.