PMF-seq: a highly scalable screening strategy for linking genetics to mitochondrial bioenergetics
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The paper introduces a new high-throughput technique called PMF-seq (permeabilized-cell mitochondrial function sequencing) that combines the power of classical mitochondrial bioenergetics studies with highly parallel genetic screening using CRISPR technology. The key invention is interrogating mitochondrial function in pools of mutagenized cells where the plasma membrane has been selectively permeabilized, leaving the mitochondria intact and accessible to mitochondrial substrates and inhibitors.
Classical mitochondrial bioenergetics studies have provided deep insights by allowing detailed monitoring of parameters like oxygen consumption, membrane potential, and substrate oxidation in suspensions of isolated mitochondria treated with specific substrates and inhibitors. However, these studies are inherently low-throughput. In contrast, recent genetic screening approaches using pooled CRISPR libraries have enabled systematic mapping of genes required for mitochondrial respiration, but provide only coarse-grained information about mitochondrial physiology.
PMF-seq combines the strengths of both approaches. The authors CRISPR-mutagenized cultured human cells with a custom single guide RNA library ("MitoPlus") targeting 1,864 genes involved in mitochondrial function and metabolism. The mutagenized cells were permeabilized using perfringolysin O, which selectively permeabilizes the cholesterol-rich plasma membrane while leaving mitochondrial membranes intact. In the permeabilized cells, the authors could energize the mitochondria using cell-impermeable substrates and monitor the mitochondrial membrane potential (Δψm) using the fluorescent dye TMRM.
By flow cytometry, the authors separated the cell population into those with high or low Δψm for a given substrate condition. Sequencing the guide RNA abundances in each population revealed genes required for polarizing or depolarizing the mitochondria under that specific substrate. This enabled linking individual genes to detailed mitochondrial bioenergetic phenotypes at scale.
The authors established PMF-seq by recapitulating known aspects of mitochondrial physiology:
1) They recovered genes encoding specific respiratory chain complexes only when using substrates feeding into those complexes, clearly delineating the branched topology of the respiratory chain.
2) When using ATP as a substrate, they observed a strong requirement for complex V (ATP synthase) subunits and assembly factors, validating the reversibility of this complex to generate a membrane potential.
3) Using D-lactate as a substrate, they identified a genetic requirement for the D-lactate dehydrogenase LDHD and confirmed its direct role in transferring electrons from D-lactate to cytochrome c through biochemical assays.
Generating new biological insights - the complex V assembly factor ATPAF2 emerged as a genetic vulnerability that sensitizes cells to acute mitochondrial damage by the pro-apoptotic protein tBID. Follow-up studies revealed striking mitochondrial morphology defects in ATPAF2 knockout cells treated with tBID. PMF-seq also uncovered dependencies on respiratory chain complexes III and IV when membrane potential was generated by complex V operating in reverse, suggesting these complexes may play a role in this process.
In essence, PMF-seq provides a powerful experimental platform to systematically map the genetic basis of mitochondrial physiology and organelle function at high resolution. A new way to link genetic perturbations to detailed bioenergetic phenotypes. Using the method to dissect genetics of mitochondrial ETC branching and acute tBID action. By carefully controlling the substrate milieu, PMF-seq can force cells to utilize specific mitochondrial pathways and identify the underlying genetic requirements.
The authors anticipate PMF-seq will enable a variety of future applications, such as investigating genes regulating NADH or calcium dynamics within mitochondria, studying other pro-apoptotic proteins like BAX/BAK, or even extending the approach to analyze other organelles like lysosomes.