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tpHusion: An efficient tool for clonal pH determination in Drosophila.

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Summary

We developed a new genetically encoded pH indicator (GEpHI) for Drosophila, called tpHusion, that works independently of the Gal4/UAS system. This tool enables robust investigation of intracellular pH dynamics across various tissues and developmental stages.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Developmental Biology

Background:

  • Genetically encoded pH indicators (GEpHIs) are crucial for studying intracellular pH (pHi) dynamics.
  • Existing GEpHIs in Drosophila predominantly rely on the Gal4/UAS binary expression system, limiting their application.
  • A need exists for GEpHIs that offer ubiquitous expression and Gal4/UAS independence.

Purpose of the Study:

  • To generate and validate a novel, ubiquitously-expressed GEpHI for Drosophila.
  • To establish a tool for studying pHi dynamics independent of the Gal4/UAS system.
  • To demonstrate the utility of the new GEpHI in various Drosophila tissues and experimental contexts.

Main Methods:

  • Cloning of a super ecliptic pHluorin and FusionRed fusion protein under the tubulin promoter to create tpHusion.
  • Validation of tpHusion function in diverse Drosophila tissues and developmental stages.
  • Application of tpHusion for comparative pHi analysis in manipulated and surrounding epithelial cells.

Main Results:

  • Successful generation of a ubiquitously-expressed GEpHI (tpHusion) independent of the Gal4/UAS system.
  • Demonstrated functionality of tpHusion across various tissues and developmental stages in Drosophila.
  • Accurate indication of pHi differences in fixed tissues and comparative analysis of manipulated clones.

Conclusions:

  • tpHusion is a robust and versatile tool for studying intracellular pH dynamics in Drosophila.
  • The Gal4/UAS-independent nature of tpHusion expands the possibilities for pHi research in Drosophila.
  • This new GEpHI facilitates comparative analysis of pHi in cellular contexts within epithelial tissues.