PMF-seq: a highly scalable screening strategy for linking genetics to mitochondrial bioenergetics
Tsz-Leung To1,2,3, Jason G McCoy1,2,3, Naomi K Ostriker1,2,3
1Howard Hughes Medical Institute and Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA.
Nature Metabolism
|February 27, 2024
Summary
We developed permeabilized-cell mitochondrial function sequencing (PMF-seq) to link genes with specific mitochondrial functions. This method reveals new genes involved in mitochondrial respiration and apoptosis, advancing our understanding of organelle physiology.
Area of Science:
- Cell Biology
- Genetics
- Biochemistry
Background:
- Mitochondrial function is studied using isolated organelles or genetic screens.
- Existing methods have limitations in connecting genes to detailed bioenergetic phenotypes.
- A need exists for a technique combining high-throughput genetic analysis with precise bioenergetic measurements.
Purpose of the Study:
- To introduce permeabilized-cell mitochondrial function sequencing (PMF-seq).
- To bridge the gap between genetic screens and detailed bioenergetic measurements of mitochondria.
- To identify genes critical for mitochondrial physiology and response to apoptotic stimuli.
Main Methods:
- PMF-seq involves gentle permeabilization of CRISPR mutagenized cells.
- Mitochondrial bioenergetics are probed in situ using flow cytometry.
- Selected cells are analyzed via next-generation sequencing to identify genetic determinants.
Main Results:
- PMF-seq successfully identified genes affecting mitochondrial respiratory chain branching and reversibility.
- Human D-lactate dehydrogenase was shown to transfer electrons to cytochrome c, maintaining mitochondrial membrane potential.
- The study identified ATPAF2 as a genetic sensitizer of tBID-induced apoptosis.
Conclusions:
- PMF-seq is a powerful new method for dissecting mitochondrial function at a genetic level.
- The findings provide novel insights into mitochondrial respiratory pathways and apoptosis regulation.
- PMF-seq is expected to accelerate the discovery of genes impacting organelle physiology.
