Tissue-Specific Fluorescent Protein Turnover in Free-Moving Flies.

Area of Science:

• * Molecular Biology
• * Genetics
• * Biochemistry

Background:

• * Protein turnover is crucial for cellular function and is tightly regulated.
• * Existing methods for studying protein degradation in vivo are limited.
• * Development of novel assays is needed to understand protein dynamics.

Purpose of the Study:

• * To establish a robust in vivo assay for measuring protein turnover in adult Drosophila melanogaster.
• * To determine the half-lives of various fluorescent proteins in vivo.
• * To investigate factors influencing protein degradation, including inhibitors, protein type, tissue, age, and sex.

Main Methods:

• * Generation of conditional transgenic Drosophila melanogaster expressing fluorescent proteins.
• * In vivo measurement of fluorescent protein decay over time using video tracking.
• * Calculation of protein half-life based on fluorescence decay.
• * Application of proteasome and protein synthesis inhibitors to assess degradation pathways.
• * Analysis of protein half-life across different tissues, sexes, and ages.

Main Results:

• * The proteasome inhibitor bortezomib increased eGFP half-life, confirming proteasomal degradation.
• * Cycloheximide decreased eGFP accumulation without altering half-life, indicating reduced synthesis.
• * Half-lives varied significantly among fluorescent proteins (e.g., DsRED > eGFP > mCherry).
• * Tissue-specific differences in half-life were observed (e.g., higher eGFP half-life in muscle).
• * Age-dependent changes in half-life were detected for DsRED but not eGFP.

Conclusions:

• * The developed in vivo assay is a promising tool for studying protein turnover in Drosophila.
• * Protein degradation is influenced by intrinsic protein properties, cellular localization, and physiological state.
• * This assay facilitates research into small molecule effects on protein degradation pathways.