Protocol for real-time imaging of membrane fission by mitofissin

Area of Science:

• Cell Biology
• Mitochondrial Dynamics
• Autophagy

Background:

• Yeast mitofissin Atg44 is a mitochondrial intermembrane space protein.
• Atg44 is essential for membrane fission during mitophagy.
• Understanding Atg44's function requires methods to observe its membrane fission activity.

Purpose of the Study:

• To present a detailed protocol for observing Atg44-mediated membrane fission.
• To provide a method for studying mitochondrial dynamics and mitophagy.
• To enable the investigation of other membrane fission proteins.

Main Methods:

• Recombinant Atg44 protein purification.
• Preparation of lipid nanotubes as model membranes.
• Real-time observation of Atg44-mediated membrane fission using confocal microscopy.
• Tube radius estimation using confocal microscopy.

Main Results:

• The protocol successfully enables observation of Atg44-mediated membrane fission.
• The method allows for quantitative analysis of membrane fission events.
• The protocol is adaptable for studying other fission proteins.

Conclusions:

• This protocol provides a valuable tool for studying Atg44 function in mitophagy.
• The developed method facilitates research into mitochondrial dynamics.
• The protocol's adaptability broadens its utility in membrane fission research.