Mitochondria’s division of labor sheds light on how cancer cells survive harsh conditions

In a recent study published in Nature, researchers from the United States of America investigated how cells separate competing metabolic pathways within mitochondria, namely oxidative phosphorylation (OXPHOS) and reductive synthesis of proline and ornithine.

They found that mitochondrial fusion and fission enable cells to balance competing metabolic demands by creating two specialized mitochondrial subpopulations: one subset containing P5CS (pyrroline-5-carboxylate synthase), which lacks cristae and adenosine triphosphate (ATP) synthase, and the other dedicated to OXPHOS.

Background

Mammalian cells contain 50 to 1,000 mitochondria that constantly undergo fusion and fission to maintain their functions, eliminate defects, and adjust to cellular needs. Known mainly for producing ATP through OXPHOS, mitochondria also play a key role in making essential molecules needed for cell growth. When nutrients are abundant, mitochondria can use excess resources to support these biosynthetic functions. However, during nutrient scarcity, it’s unclear how mitochondria balance their energy production with the need to synthesize crucial molecules for cell maintenance.

While the pathways for OXPHOS and biosynthesis (like amino acid and one-carbon metabolism) have each been extensively studied individually, how mitochondria manage these processes together— especially under bioenergetic and nutrient stress— remains poorly understood. As understanding this balance is crucial for insights into cellular growth and survival, researchers in the present study examined how these competing processes are balanced within the mitochondria to meet the metabolic needs of the cell.

About the study

The researchers used a STRING protein-protein interaction (PPI) analysis to identify mitochondrial enzyme clusters based on functional roles. Mouse embryonic fibroblasts (MEFs) were cultured in glucose-deficient or galactose medium to rely on OXPHOS for ATP production. [U-¹³C] glutamine tracing was employed to study metabolic pathways of glutamate into the tricarboxylic acid cycle (TCA) cycle and reductive biosynthesis.

Mitochondrial activity was manipulated with various treatments, and a P5CS knockout was developed via gene editing. The filament formation of P5CS was assessed using imaging under various nutrient conditions and proliferative states. Mutant P5CS forms were expressed to test filament dynamics in proline synthesis, with proline and ornithine supplementation to assess their impact.

To examine P5CS clustering in vivo, tissue sections from human pancreatic ductal adenocarcinoma (PDAC) were analyzed for P5CS filaments in mitochondria. These tumors are reported to struggle to supply sufficient energy to their cells as they grow due to limitations in blood supply and nutrient availability. High-resolution microscopy revealed P5CS segregation from ATP synthase. Interactions between P5CS and ATP synthase complexes were confirmed, and mitochondrial membrane potential was assessed.

Ultrastructural features of P5CS-containing mitochondria were analyzed using correlative light and electron microscopy (CLEM). OPA1-knockout MEFs were studied for cristae formation and proline biosynthesis. Mitochondrial dynamics in fusion-deficient Mfn1/2−/− and fission-deficient Drp1−/− MEFs were assessed for mitochondrial morphology, OXPHOS activity, and proline synthesis.

Results

Mitochondrial enzymes were classified into three functional clusters: TCA cycle (cluster 1), amino acid biosynthesis (cluster 2), and one-carbon metabolism (cluster 3), with P5CS bridging all three pathways. Proline synthesis was maintained when cells relied on OXPHOS, suggesting a balance between oxidative and reductive metabolism.

Imaging revealed that P5CS formed filaments in mitochondria, particularly under OXPHOS-dependent conditions or nutrient stress. Mutant P5CS that could not form filaments resulted in reduced proline synthesis, confirming the necessity of filament formation. Adding proline or ornithine reversed P5CS filament formation, indicating that metabolic demand regulates this process.

In vivo, P5CS clustering was observed in a subset of mitochondria in pancreatic tumor cells, while adjacent normal tissues lacked this clustering. In the tumor cells, mitochondria containing P5CS lacked ATP synthase components, whereas those enriched in ATP synthase did not contain P5CS. P5CS was found to be less associated with ATP synthase when mitochondria were segregated, even though total protein levels remained unchanged.

P5CS-containing mitochondria showed higher membrane potential, suggesting that they engage in reductive metabolism for proline and ornithine synthesis, while ATP synthase-enriched mitochondria are less involved in this process. Furthermore, reduced nicotinamide adenine dinucleotide (NADH) levels compromised proline synthesis, confirming that a reductive mitochondrial environment is essential for proline production.

Further, P5CS-containing mitochondria showed a near-complete loss of cristae, replaced by stacks of protein filaments, while ATP synthase-enriched mitochondria were found to maintain cristae. P5CS-containing mitochondria lacked the MICOS complex component MIC60 and ATP synthase subunit ATP5I. Lack of OPA1 disrupted the cristae but did not prevent proline biosynthesis.

Live-cell imaging showed that P5CS-containing mitochondria fused into larger networks in galactose medium. Fusion-deficient cells failed to separate P5CS from ATP synthase, showing impaired respiratory activity but maintained proline synthesis. Fission-deficient cells showed elongated mitochondria, failed to separate P5CS, and showed reduced proline synthesis, impairing collagen production. Reintroducing DRP1 restored proline and collagen synthesis, linking mitochondrial fission to proline biosynthesis.

Conclusion

In conclusion, while mitochondrial fusion and fission help maintain similarity among mitochondria, they also help create and maintain specialized groups of mitochondria within a cell, including pancreatic cancer cells, such that each group focuses on different tasks. The ability of mitochondria to adapt to nutrient scarcity by presenting in two distinct forms could potentially be a key survival strategy even for cancer cells. This discovery offers a promising therapeutic target to potentially inhibit tumor growth by disrupting their metabolic adaptability.

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