Detection and quantitation of ferritinophagy using HaloTag tracing

Area of Science:

• Cell Biology
• Biochemistry
• Molecular Biology

Background:

• Iron is vital for cellular functions, requiring release from ferritin via ferritinophagy.
• Ferritinophagy, involving lysosomal degradation of ferritin, is implicated in various diseases but lacks specific detection methods.
• Nuclear receptor coactivator 4 mediates ferritin degradation during ferritinophagy.

Purpose of the Study:

• To develop and validate a novel assay for detecting and quantifying ferritinophagy.
• To provide a tool for studying cellular iron metabolism and its role in disease.

Main Methods:

• Generation of a Halo-tagged ferritin heavy chain 1 (FTH1) construct.
• Utilizing pulse-chase experiments with Halo-FTH1 and fluorescent Halo ligands.
• Quantification via in-gel fluorescence, immunoblotting, and microscopy.
• Validation by silencing nuclear receptor coactivator 4.

Main Results:

• Halo-FTH1 functions similarly to endogenous FTH1.
• The assay successfully detected and quantified lysosomal accumulation of Halo fragments during ferritinophagy.
• Silencing nuclear receptor coactivator 4 inhibited Halo fragment accumulation and FTH1 degradation, confirming assay specificity.

Conclusions:

• A HaloTag-FTH1 tool enables specific detection and quantification of ferritinophagy in mammalian cells.
• This assay is valuable for investigating cellular iron metabolism and associated diseases.
• The method provides a sensitive approach to study the dynamics of iron release from ferritin.