tpHusion: An efficient tool for clonal pH determination in Drosophila

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.