Limits...
13C- and 15N-Labeling Strategies Combined with Mass Spectrometry Comprehensively Quantify Phospholipid Dynamics in C. elegans.

Dancy BC, Chen SW, Drechsler R, Gafken PR, Olsen CP - PLoS ONE (2015)

Bottom Line: Using multiple measurements of phospholipid dynamics, we found that the phospholipid pools are replaced rapidly and at rates nearly double the turnover measured for neutral lipid populations.Furthermore, we found that stearoyl-CoA desaturases (SCDs), critical enzymes in polyunsaturated fatty acid production, play an unexpected role in influencing the overall rates of membrane maintenance as SCD depletion affected the turnover of nearly all membrane lipids.Additionally, the compromised membrane maintenance as defined by LC-MS/MS with SCD RNAi resulted in active phospholipid remodeling that we predict is critical to alleviate the impact of reduced membrane maintenance in these animals.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America.

ABSTRACT
Membranes define cellular and organelle boundaries, a function that is critical to all living systems. Like other biomolecules, membrane lipids are dynamically maintained, but current methods are extremely limited for monitoring lipid dynamics in living animals. We developed novel strategies in C. elegans combining 13C and 15N stable isotopes with mass spectrometry to directly quantify the replenishment rates of the individual fatty acids and intact phospholipids of the membrane. Using multiple measurements of phospholipid dynamics, we found that the phospholipid pools are replaced rapidly and at rates nearly double the turnover measured for neutral lipid populations. In fact, our analysis shows that the majority of membrane lipids are replaced each day. Furthermore, we found that stearoyl-CoA desaturases (SCDs), critical enzymes in polyunsaturated fatty acid production, play an unexpected role in influencing the overall rates of membrane maintenance as SCD depletion affected the turnover of nearly all membrane lipids. Additionally, the compromised membrane maintenance as defined by LC-MS/MS with SCD RNAi resulted in active phospholipid remodeling that we predict is critical to alleviate the impact of reduced membrane maintenance in these animals. Not only have these combined methodologies identified new facets of the impact of SCDs on the membrane, but they also have great potential to reveal many undiscovered regulators of phospholipid metabolism.

No MeSH data available.


Related in: MedlinePlus

Characterization of SCDs as Membrane Maintenance Regulators.(A) The major FAs in C. elegans are produced by the elongation and desaturation pathway shown here. An alternate pathway downstream of FA synthase (fasn-1) is used for the production of monomethyl-branched chain FAs, C15iso and C17iso. (B) The amount of new FA incorporated each hour was quantified in animals treated with adult-only RNAi against the FA synthesis, elongation and desaturation genes for 48 hours. The resulting data are summarized here with species with less replenishment than control RNAi colored light blue (>25% decrease) and dark blue (>50% decrease). See S1 Dataset for complete source data. (C) To determine the overall impact on membrane replenishment, we averaged the replacement rates for each FA species and normalized by the abundance of each species in the PL population. RNAi of pod-2, fat-5, fat-6 and fat-7 resulted in significant reduction in overall membrane maintenance. (D) The relative amount of synthesized FA is reduced with fat-7 RNAi treatment (blue) compared to control RNAi (black). Numbers shown represent the mean of at least three experiments ± SEM. Statistical significance was determined by two-tailed unpaired t-tests (*p<0.05, **p<0.01, ***p<0.001).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4631354&req=5

pone.0141850.g003: Characterization of SCDs as Membrane Maintenance Regulators.(A) The major FAs in C. elegans are produced by the elongation and desaturation pathway shown here. An alternate pathway downstream of FA synthase (fasn-1) is used for the production of monomethyl-branched chain FAs, C15iso and C17iso. (B) The amount of new FA incorporated each hour was quantified in animals treated with adult-only RNAi against the FA synthesis, elongation and desaturation genes for 48 hours. The resulting data are summarized here with species with less replenishment than control RNAi colored light blue (>25% decrease) and dark blue (>50% decrease). See S1 Dataset for complete source data. (C) To determine the overall impact on membrane replenishment, we averaged the replacement rates for each FA species and normalized by the abundance of each species in the PL population. RNAi of pod-2, fat-5, fat-6 and fat-7 resulted in significant reduction in overall membrane maintenance. (D) The relative amount of synthesized FA is reduced with fat-7 RNAi treatment (blue) compared to control RNAi (black). Numbers shown represent the mean of at least three experiments ± SEM. Statistical significance was determined by two-tailed unpaired t-tests (*p<0.05, **p<0.01, ***p<0.001).

Mentions: We sought to identify regulators of membrane maintenance and to exclusively look for genes and pathways that impact day-to-day membrane maintenance, we initiated an adult-only RNAi approach at day 1 of adulthood after membranes have been fully formed. To validate our strategy, we targeted FA synthesis with RNAi of the C. elegans acetyl-CoA carboxylase gene, pod-2. After 48 hours of treatment, there is significant reduction in de novo synthesis as measured by 13C labeling, indicating the efficacy of the adult-only RNAi treatment (S4 Fig). Then, we examined the total amount of new fat provided to the membrane in pod-2 treated adults as pod-2 depletions would limit the total amount of new fats available for the membrane. Indeed, we found a significant decrease in overall membrane maintenance in pod-2 RNAi-treated animals (Fig 3C). We looked for altered dynamics in a very limited and specific subset of FAs by disrupting mmBCFAs through RNAi of elo-5 and elo-6 (Fig 3A). Both elo-5 and elo-6 RNAi resulted in a significant decrease in new monomethyl-branched chain FAs, C15iso and C17iso, but not any other FA species, indicating that the 13C method allows for specific monitoring of individual FA pools (Fig 3B).


13C- and 15N-Labeling Strategies Combined with Mass Spectrometry Comprehensively Quantify Phospholipid Dynamics in C. elegans.

Dancy BC, Chen SW, Drechsler R, Gafken PR, Olsen CP - PLoS ONE (2015)

Characterization of SCDs as Membrane Maintenance Regulators.(A) The major FAs in C. elegans are produced by the elongation and desaturation pathway shown here. An alternate pathway downstream of FA synthase (fasn-1) is used for the production of monomethyl-branched chain FAs, C15iso and C17iso. (B) The amount of new FA incorporated each hour was quantified in animals treated with adult-only RNAi against the FA synthesis, elongation and desaturation genes for 48 hours. The resulting data are summarized here with species with less replenishment than control RNAi colored light blue (>25% decrease) and dark blue (>50% decrease). See S1 Dataset for complete source data. (C) To determine the overall impact on membrane replenishment, we averaged the replacement rates for each FA species and normalized by the abundance of each species in the PL population. RNAi of pod-2, fat-5, fat-6 and fat-7 resulted in significant reduction in overall membrane maintenance. (D) The relative amount of synthesized FA is reduced with fat-7 RNAi treatment (blue) compared to control RNAi (black). Numbers shown represent the mean of at least three experiments ± SEM. Statistical significance was determined by two-tailed unpaired t-tests (*p<0.05, **p<0.01, ***p<0.001).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4631354&req=5

pone.0141850.g003: Characterization of SCDs as Membrane Maintenance Regulators.(A) The major FAs in C. elegans are produced by the elongation and desaturation pathway shown here. An alternate pathway downstream of FA synthase (fasn-1) is used for the production of monomethyl-branched chain FAs, C15iso and C17iso. (B) The amount of new FA incorporated each hour was quantified in animals treated with adult-only RNAi against the FA synthesis, elongation and desaturation genes for 48 hours. The resulting data are summarized here with species with less replenishment than control RNAi colored light blue (>25% decrease) and dark blue (>50% decrease). See S1 Dataset for complete source data. (C) To determine the overall impact on membrane replenishment, we averaged the replacement rates for each FA species and normalized by the abundance of each species in the PL population. RNAi of pod-2, fat-5, fat-6 and fat-7 resulted in significant reduction in overall membrane maintenance. (D) The relative amount of synthesized FA is reduced with fat-7 RNAi treatment (blue) compared to control RNAi (black). Numbers shown represent the mean of at least three experiments ± SEM. Statistical significance was determined by two-tailed unpaired t-tests (*p<0.05, **p<0.01, ***p<0.001).
Mentions: We sought to identify regulators of membrane maintenance and to exclusively look for genes and pathways that impact day-to-day membrane maintenance, we initiated an adult-only RNAi approach at day 1 of adulthood after membranes have been fully formed. To validate our strategy, we targeted FA synthesis with RNAi of the C. elegans acetyl-CoA carboxylase gene, pod-2. After 48 hours of treatment, there is significant reduction in de novo synthesis as measured by 13C labeling, indicating the efficacy of the adult-only RNAi treatment (S4 Fig). Then, we examined the total amount of new fat provided to the membrane in pod-2 treated adults as pod-2 depletions would limit the total amount of new fats available for the membrane. Indeed, we found a significant decrease in overall membrane maintenance in pod-2 RNAi-treated animals (Fig 3C). We looked for altered dynamics in a very limited and specific subset of FAs by disrupting mmBCFAs through RNAi of elo-5 and elo-6 (Fig 3A). Both elo-5 and elo-6 RNAi resulted in a significant decrease in new monomethyl-branched chain FAs, C15iso and C17iso, but not any other FA species, indicating that the 13C method allows for specific monitoring of individual FA pools (Fig 3B).

Bottom Line: Using multiple measurements of phospholipid dynamics, we found that the phospholipid pools are replaced rapidly and at rates nearly double the turnover measured for neutral lipid populations.Furthermore, we found that stearoyl-CoA desaturases (SCDs), critical enzymes in polyunsaturated fatty acid production, play an unexpected role in influencing the overall rates of membrane maintenance as SCD depletion affected the turnover of nearly all membrane lipids.Additionally, the compromised membrane maintenance as defined by LC-MS/MS with SCD RNAi resulted in active phospholipid remodeling that we predict is critical to alleviate the impact of reduced membrane maintenance in these animals.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America.

ABSTRACT
Membranes define cellular and organelle boundaries, a function that is critical to all living systems. Like other biomolecules, membrane lipids are dynamically maintained, but current methods are extremely limited for monitoring lipid dynamics in living animals. We developed novel strategies in C. elegans combining 13C and 15N stable isotopes with mass spectrometry to directly quantify the replenishment rates of the individual fatty acids and intact phospholipids of the membrane. Using multiple measurements of phospholipid dynamics, we found that the phospholipid pools are replaced rapidly and at rates nearly double the turnover measured for neutral lipid populations. In fact, our analysis shows that the majority of membrane lipids are replaced each day. Furthermore, we found that stearoyl-CoA desaturases (SCDs), critical enzymes in polyunsaturated fatty acid production, play an unexpected role in influencing the overall rates of membrane maintenance as SCD depletion affected the turnover of nearly all membrane lipids. Additionally, the compromised membrane maintenance as defined by LC-MS/MS with SCD RNAi resulted in active phospholipid remodeling that we predict is critical to alleviate the impact of reduced membrane maintenance in these animals. Not only have these combined methodologies identified new facets of the impact of SCDs on the membrane, but they also have great potential to reveal many undiscovered regulators of phospholipid metabolism.

No MeSH data available.


Related in: MedlinePlus