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A new fluorescence-based method identifies protein phosphatases regulating lipid droplet metabolism.

Bozaquel-Morais BL, Madeira JB, Maya-Monteiro CM, Masuda CA, Montero-Lomeli M - PLoS ONE (2010)

Bottom Line: Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4), type 2A phosphatase and its related regulator (pph21 and sap185), type 2C protein phosphatases (ptc1, ptc4, ptc7) and dual phosphatases (pps1, msg5) were catalogued as high-lipid droplet content strains.We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive) in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity.In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Bioquímica Médica, Programa de Biologia Molecular e Biotecnologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil.

ABSTRACT
In virtually every cell, neutral lipids are stored in cytoplasmic structures called lipid droplets (LDs) and also referred to as lipid bodies or lipid particles. We developed a rapid high-throughput assay based on the recovery of quenched BODIPY-fluorescence that allows to quantify lipid droplets. The method was validated by monitoring lipid droplet turnover during growth of a yeast culture and by screening a group of strains deleted in genes known to be involved in lipid metabolism. In both tests, the fluorimetric assay showed high sensitivity and good agreement with previously reported data using microscopy. We used this method for high-throughput identification of protein phosphatases involved in lipid droplet metabolism. From 65 yeast knockout strains encoding protein phosphatases and its regulatory subunits, 13 strains revealed to have abnormal levels of lipid droplets, 10 of them having high lipid droplet content. Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4), type 2A phosphatase and its related regulator (pph21 and sap185), type 2C protein phosphatases (ptc1, ptc4, ptc7) and dual phosphatases (pps1, msg5) were catalogued as high-lipid droplet content strains. Only reg1, a targeting subunit of the type 1 phosphatase Glc7p, and members of the nutrient-sensitive TOR pathway (sit4 and the regulatory subunit sap190) were catalogued as low-lipid droplet content strains, which were studied further. We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive) in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity. In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis.

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The liquid fluorescence recovery (LFR) assay can detect LD dynamics.A. Stationary yeast cells pre-grown for 48 hours, were inoculated into fresh YPD medium and cellular growth was recorded by measuring absorbance at 600 nm over the course of 48 hours (n = 3± S.D.). B. Aliquots of cells were withdrawn during growth and fixed in formaldehyde. The LD index was determined (fluorescence/OD cells) using the LFR assay (n = 3± S.D.) C. The triacylglycerol content was measured during growth. D and E. LD content was determined by fluorescence microscopy. Cells were grown for 0 (corresponding to 48 h pre-grown cells), 6 and 24 hours in YPD, incubated with BODIPY and photographed by fluorescence microscopy. The total fluorescence area/cell (white bars) were determined and expressed in pixels/cell (white bars). LDs per cell were quantified using the same images (gray bars). Data are for at least 50 individual cells. *p<0.05, ** p<0.01, ***p<0.001, in comparison to WT values.
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pone-0013692-g004: The liquid fluorescence recovery (LFR) assay can detect LD dynamics.A. Stationary yeast cells pre-grown for 48 hours, were inoculated into fresh YPD medium and cellular growth was recorded by measuring absorbance at 600 nm over the course of 48 hours (n = 3± S.D.). B. Aliquots of cells were withdrawn during growth and fixed in formaldehyde. The LD index was determined (fluorescence/OD cells) using the LFR assay (n = 3± S.D.) C. The triacylglycerol content was measured during growth. D and E. LD content was determined by fluorescence microscopy. Cells were grown for 0 (corresponding to 48 h pre-grown cells), 6 and 24 hours in YPD, incubated with BODIPY and photographed by fluorescence microscopy. The total fluorescence area/cell (white bars) were determined and expressed in pixels/cell (white bars). LDs per cell were quantified using the same images (gray bars). Data are for at least 50 individual cells. *p<0.05, ** p<0.01, ***p<0.001, in comparison to WT values.

Mentions: To assess whether our method was able to detect small fluctuations in LD levels, we quantified this parameter during yeast growth as LDs are actively metabolized during growth [23], [40] (Figure 4A, B). Results obtained with the LFR assay (Figure 4B) were similar to those obtained by quantifying the TAG content (Figure 4C) or through microscopic analysis, from which we calculated the total fluorescence area/cell. Furthermore it did not reflect the number of LDs/cell that does not vary as much as the size of LDs along the growth curve (Figure 4D, E). The results were also in agreement with previous results obtained by microscopic analysis [23] and biochemical quantification of cellular TAG [40], which indicated that yeast cells consume LDs when diluted in fresh medium. After six hours, LDs are re-synthesized reaching highest levels at stationary phase [23], [40]. This result demonstrates that our assay was able to detect small physiological lipid stores fluctuations during yeast growth without the need for time-consuming image analysis.


A new fluorescence-based method identifies protein phosphatases regulating lipid droplet metabolism.

Bozaquel-Morais BL, Madeira JB, Maya-Monteiro CM, Masuda CA, Montero-Lomeli M - PLoS ONE (2010)

The liquid fluorescence recovery (LFR) assay can detect LD dynamics.A. Stationary yeast cells pre-grown for 48 hours, were inoculated into fresh YPD medium and cellular growth was recorded by measuring absorbance at 600 nm over the course of 48 hours (n = 3± S.D.). B. Aliquots of cells were withdrawn during growth and fixed in formaldehyde. The LD index was determined (fluorescence/OD cells) using the LFR assay (n = 3± S.D.) C. The triacylglycerol content was measured during growth. D and E. LD content was determined by fluorescence microscopy. Cells were grown for 0 (corresponding to 48 h pre-grown cells), 6 and 24 hours in YPD, incubated with BODIPY and photographed by fluorescence microscopy. The total fluorescence area/cell (white bars) were determined and expressed in pixels/cell (white bars). LDs per cell were quantified using the same images (gray bars). Data are for at least 50 individual cells. *p<0.05, ** p<0.01, ***p<0.001, in comparison to WT values.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2965658&req=5

pone-0013692-g004: The liquid fluorescence recovery (LFR) assay can detect LD dynamics.A. Stationary yeast cells pre-grown for 48 hours, were inoculated into fresh YPD medium and cellular growth was recorded by measuring absorbance at 600 nm over the course of 48 hours (n = 3± S.D.). B. Aliquots of cells were withdrawn during growth and fixed in formaldehyde. The LD index was determined (fluorescence/OD cells) using the LFR assay (n = 3± S.D.) C. The triacylglycerol content was measured during growth. D and E. LD content was determined by fluorescence microscopy. Cells were grown for 0 (corresponding to 48 h pre-grown cells), 6 and 24 hours in YPD, incubated with BODIPY and photographed by fluorescence microscopy. The total fluorescence area/cell (white bars) were determined and expressed in pixels/cell (white bars). LDs per cell were quantified using the same images (gray bars). Data are for at least 50 individual cells. *p<0.05, ** p<0.01, ***p<0.001, in comparison to WT values.
Mentions: To assess whether our method was able to detect small fluctuations in LD levels, we quantified this parameter during yeast growth as LDs are actively metabolized during growth [23], [40] (Figure 4A, B). Results obtained with the LFR assay (Figure 4B) were similar to those obtained by quantifying the TAG content (Figure 4C) or through microscopic analysis, from which we calculated the total fluorescence area/cell. Furthermore it did not reflect the number of LDs/cell that does not vary as much as the size of LDs along the growth curve (Figure 4D, E). The results were also in agreement with previous results obtained by microscopic analysis [23] and biochemical quantification of cellular TAG [40], which indicated that yeast cells consume LDs when diluted in fresh medium. After six hours, LDs are re-synthesized reaching highest levels at stationary phase [23], [40]. This result demonstrates that our assay was able to detect small physiological lipid stores fluctuations during yeast growth without the need for time-consuming image analysis.

Bottom Line: Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4), type 2A phosphatase and its related regulator (pph21 and sap185), type 2C protein phosphatases (ptc1, ptc4, ptc7) and dual phosphatases (pps1, msg5) were catalogued as high-lipid droplet content strains.We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive) in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity.In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Bioquímica Médica, Programa de Biologia Molecular e Biotecnologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil.

ABSTRACT
In virtually every cell, neutral lipids are stored in cytoplasmic structures called lipid droplets (LDs) and also referred to as lipid bodies or lipid particles. We developed a rapid high-throughput assay based on the recovery of quenched BODIPY-fluorescence that allows to quantify lipid droplets. The method was validated by monitoring lipid droplet turnover during growth of a yeast culture and by screening a group of strains deleted in genes known to be involved in lipid metabolism. In both tests, the fluorimetric assay showed high sensitivity and good agreement with previously reported data using microscopy. We used this method for high-throughput identification of protein phosphatases involved in lipid droplet metabolism. From 65 yeast knockout strains encoding protein phosphatases and its regulatory subunits, 13 strains revealed to have abnormal levels of lipid droplets, 10 of them having high lipid droplet content. Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4), type 2A phosphatase and its related regulator (pph21 and sap185), type 2C protein phosphatases (ptc1, ptc4, ptc7) and dual phosphatases (pps1, msg5) were catalogued as high-lipid droplet content strains. Only reg1, a targeting subunit of the type 1 phosphatase Glc7p, and members of the nutrient-sensitive TOR pathway (sit4 and the regulatory subunit sap190) were catalogued as low-lipid droplet content strains, which were studied further. We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive) in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity. In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis.

Show MeSH