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Related Experiment Video

Updated: Jul 9, 2025

Mapping the Cellular Distribution of an Optogenetic Protein Using a Light-Stimulation Grid
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Protocol for screening cellular outputs activated by optogenetically controlled temporal PI3K signaling activation

Yoshibumi Ueda1, Shohei Matsushita2, Mitsugu Suzuki3

  • 1Department of Chemistry, School of Science, The University of Tokyo, Tokyo, Japan; Department of Quality Assurance and Radiological Protection, National Institute of Infectious Diseases, Tokyo, Japan.

STAR Protocols
|December 7, 2023
PubMed
Summary

This study introduces a new protocol for screening cellular responses to controlled Phosphoinositide 3-kinase (PI3K) signaling patterns. This method aids in understanding PI3K signaling and its effects on cancer drug efficacy.

Keywords:
CancerCell BiologyCell Membrane

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Area of Science:

  • Cellular Biology
  • Molecular Signaling
  • Biotechnology

Background:

  • Phosphoinositide 3-kinase (PI3K) signaling pathways generate diverse cellular outputs based on stimulation patterns.
  • Understanding these distinct outputs is crucial for deciphering cellular responses and developing targeted therapies.

Purpose of the Study:

  • To present a novel protocol for screening cellular outputs triggered by optogenetically controlled temporal PI3K signaling activation.
  • To enable high-throughput analysis of PI3K signaling dynamics in response to various stimulation patterns.

Main Methods:

  • Development of a protocol using stable cell lines (PPAP2) for PI3K signaling.
  • Integration of optogenetic control for precise temporal activation of PI3K signaling.
  • Establishment of procedures for probe expression, blue light irradiation, and analysis of translation activity in 96-well plates.

Main Results:

  • A comprehensive protocol for screening PI3K-activated cellular outputs was successfully established.
  • The protocol allows for the systematic investigation of how different temporal PI3K signaling patterns influence cellular responses.
  • Demonstrated feasibility for analyzing translation activity as a key cellular output.

Conclusions:

  • The presented protocol offers a robust platform for dissecting PI3K signaling dynamics and cellular responses.
  • This method can be applied to investigate the impact of PI3K signaling on various cellular processes, including the efficacy of anticancer drugs.
  • Facilitates high-throughput screening for a deeper understanding of PI3K pathway regulation.