A high-throughput assay for mitochondrial membrane potential in permeabilized yeast cells

Area of Science:

• Biochemistry
• Cell Biology
• Biophysics

Background:

• Mitochondrial membrane potential is crucial for cellular energy production and function.
• Accurate measurement of mitochondrial membrane potential is essential for understanding cellular health and disease.
• Existing methods for assessing mitochondrial membrane potential can be limited in throughput and applicability.

Purpose of the Study:

• To develop a novel fluorometric assay for measuring mitochondrial membrane potential in permeabilized yeast cells.
• To validate the assay's sensitivity and specificity using known mitochondrial modulators.
• To adapt the assay for high-throughput screening applications.

Main Methods:

• Developed a fluorometric assay using 3,3'-dipropylthiadicarbocyanine iodide (DiSC(3)(5)) probe.
• Permeabilized yeast cell plasma membranes to isolate mitochondrial measurements.
• Utilized chemical uncouplers and Uncoupling Protein 1 (UCP1) for assay validation.

Main Results:

• DiSC(3)(5) fluorescence decreased in energized mitochondria and increased significantly upon membrane potential dissipation.
• Assay demonstrated sensitivity to specific modulators in UCP1-expressing cells.
• Confirmed abolition of plasma membrane potential post-permeabilization.

Conclusions:

• The developed fluorometric assay accurately measures mitochondrial membrane potential in permeabilized yeast.
• This method is suitable for high-throughput screening of mitochondrial membrane potential modulators.
• The assay provides a valuable tool for studying mitochondrial function and identifying novel therapeutic targets.