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Fld1p, a functional homologue of human seipin, regulates the size of lipid droplets in yeast.

Fei W, Shui G, Gaeta B, Du X, Kuerschner L, Li P, Brown AJ, Wenk MR, Parton RG, Yang H - J. Cell Biol. (2008)

Bottom Line: Cells lacking FLD1 contain strikingly enlarged (supersized) LDs, and LDs from fld1Delta cells demonstrate significantly enhanced fusion activities both in vivo and in vitro.Interestingly, the expression of human seipin, whose mutant forms are associated with Berardinelli-Seip congenital lipodystrophy and motoneuron disorders, rescues LD-associated defects in fld1Delta cells.These results suggest that an evolutionally conserved function of seipin in phospholipid metabolism and LD formation may be functionally important in human adipogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, National University of Singapore, Singapore 117597, Republic of Singapore.

ABSTRACT
Lipid droplets (LDs) are emerging cellular organelles that are of crucial importance in cell biology and human diseases. In this study, we present our screen of approximately 4,700 Saccharomyces cerevisiae mutants for abnormalities in the number and morphology of LDs; we identify 17 fld (few LDs) and 116 mld (many LDs) mutants. One of the fld mutants (fld1) is caused by the deletion of YLR404W, a previously uncharacterized open reading frame. Cells lacking FLD1 contain strikingly enlarged (supersized) LDs, and LDs from fld1Delta cells demonstrate significantly enhanced fusion activities both in vivo and in vitro. Interestingly, the expression of human seipin, whose mutant forms are associated with Berardinelli-Seip congenital lipodystrophy and motoneuron disorders, rescues LD-associated defects in fld1Delta cells. Lipid profiling reveals alterations in acyl chain compositions of major phospholipids in fld1Delta cells. These results suggest that an evolutionally conserved function of seipin in phospholipid metabolism and LD formation may be functionally important in human adipogenesis.

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The fld1Δ (ylr404wΔ) cells synthesize morphologically distinct LDs. (A) Both wild-type and fld1Δ cells were grown in YPD medium until stationary phase or until log phase. Cells were stained with 20 μg/ml Nile red and immediately observed for LDs under a fluorescence microscope. Micrographs of the wild-type cells (a and a′) and fld1Δ cells (b–e, b′, and c′) are shown. Supersized LDs (b, c, and b′) are indicated by arrows, and aggregation of LDs (c, d, and c′) is indicated by arrowheads. DIC, differential interference contrast. (B) Conventional TEM of wild-type and fld1Δ cells. Cells were grown in YPD to stationary phase, fixed with 2.5% (vol/vol) glutaraldehyde and 2% (wt/vol) osmium tetroxide, and subjected to EM. LDs are seen as electron-transparent droplets. TEM of wild-type (a) and fld1Δ (b–i) cells. (C) Culture media affect LD morphology in fld1Δ cells. Wild-type and fld1Δ cells were grown until stationary phase in YPD medium (a and d), SC medium (b and e), YPO (oleate) medium (c and f), or YPDO medium (g). The preparation of media was described in Materials and methods. Nile red staining of wild-type (a–c) and fld1Δ (d–g) cells. Arrows indicate supersized LDs, and arrowheads indicate aggregated LDs. Bars: (A and C) 5 μm; (B) 1 μm.
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fig1: The fld1Δ (ylr404wΔ) cells synthesize morphologically distinct LDs. (A) Both wild-type and fld1Δ cells were grown in YPD medium until stationary phase or until log phase. Cells were stained with 20 μg/ml Nile red and immediately observed for LDs under a fluorescence microscope. Micrographs of the wild-type cells (a and a′) and fld1Δ cells (b–e, b′, and c′) are shown. Supersized LDs (b, c, and b′) are indicated by arrows, and aggregation of LDs (c, d, and c′) is indicated by arrowheads. DIC, differential interference contrast. (B) Conventional TEM of wild-type and fld1Δ cells. Cells were grown in YPD to stationary phase, fixed with 2.5% (vol/vol) glutaraldehyde and 2% (wt/vol) osmium tetroxide, and subjected to EM. LDs are seen as electron-transparent droplets. TEM of wild-type (a) and fld1Δ (b–i) cells. (C) Culture media affect LD morphology in fld1Δ cells. Wild-type and fld1Δ cells were grown until stationary phase in YPD medium (a and d), SC medium (b and e), YPO (oleate) medium (c and f), or YPDO medium (g). The preparation of media was described in Materials and methods. Nile red staining of wild-type (a–c) and fld1Δ (d–g) cells. Arrows indicate supersized LDs, and arrowheads indicate aggregated LDs. Bars: (A and C) 5 μm; (B) 1 μm.

Mentions: In an effort to identify novel gene products that may play a role in LD formation, Nile red, a vital dye specific for intracellular LDs, was used to visually screen the entire collection of viable single-gene deletion mutants of the budding yeast Saccharomyces cerevisiae for abnormalities in the number and morphology of LDs (Greenspan et al., 1985). Because the number and morphology of LDs may vary depending on growth phases, wild-type cells and all mutants were grown overnight to stationary phase (OD600 = ∼5) in this study immediately followed by Nile red staining and fluorescence microcopy. Wild-type cells at stationary phase showed 5.16 ± 2.18 LDs per cell on average (±SD; n = 200), and ∼80% of the cells displayed three to seven LDs (Fig. 1 A, a). To simplify the screening process, we arbitrarily categorized deletion strains with the majority (>80%) of cells accumulating on average less than three LDs as fld (few LDs) mutants and strains accumulating more than seven LDs as mld (many LDs) mutants. Among the mld mutants, strains containing >11 LDs were classified as strong mld mutants. We isolated 17 fld mutants and 116 mld mutants (Tables S1 and S2, available at http://www.jcb.org/cgi/content/full/jcb.200711136/DC1).


Fld1p, a functional homologue of human seipin, regulates the size of lipid droplets in yeast.

Fei W, Shui G, Gaeta B, Du X, Kuerschner L, Li P, Brown AJ, Wenk MR, Parton RG, Yang H - J. Cell Biol. (2008)

The fld1Δ (ylr404wΔ) cells synthesize morphologically distinct LDs. (A) Both wild-type and fld1Δ cells were grown in YPD medium until stationary phase or until log phase. Cells were stained with 20 μg/ml Nile red and immediately observed for LDs under a fluorescence microscope. Micrographs of the wild-type cells (a and a′) and fld1Δ cells (b–e, b′, and c′) are shown. Supersized LDs (b, c, and b′) are indicated by arrows, and aggregation of LDs (c, d, and c′) is indicated by arrowheads. DIC, differential interference contrast. (B) Conventional TEM of wild-type and fld1Δ cells. Cells were grown in YPD to stationary phase, fixed with 2.5% (vol/vol) glutaraldehyde and 2% (wt/vol) osmium tetroxide, and subjected to EM. LDs are seen as electron-transparent droplets. TEM of wild-type (a) and fld1Δ (b–i) cells. (C) Culture media affect LD morphology in fld1Δ cells. Wild-type and fld1Δ cells were grown until stationary phase in YPD medium (a and d), SC medium (b and e), YPO (oleate) medium (c and f), or YPDO medium (g). The preparation of media was described in Materials and methods. Nile red staining of wild-type (a–c) and fld1Δ (d–g) cells. Arrows indicate supersized LDs, and arrowheads indicate aggregated LDs. Bars: (A and C) 5 μm; (B) 1 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2234226&req=5

fig1: The fld1Δ (ylr404wΔ) cells synthesize morphologically distinct LDs. (A) Both wild-type and fld1Δ cells were grown in YPD medium until stationary phase or until log phase. Cells were stained with 20 μg/ml Nile red and immediately observed for LDs under a fluorescence microscope. Micrographs of the wild-type cells (a and a′) and fld1Δ cells (b–e, b′, and c′) are shown. Supersized LDs (b, c, and b′) are indicated by arrows, and aggregation of LDs (c, d, and c′) is indicated by arrowheads. DIC, differential interference contrast. (B) Conventional TEM of wild-type and fld1Δ cells. Cells were grown in YPD to stationary phase, fixed with 2.5% (vol/vol) glutaraldehyde and 2% (wt/vol) osmium tetroxide, and subjected to EM. LDs are seen as electron-transparent droplets. TEM of wild-type (a) and fld1Δ (b–i) cells. (C) Culture media affect LD morphology in fld1Δ cells. Wild-type and fld1Δ cells were grown until stationary phase in YPD medium (a and d), SC medium (b and e), YPO (oleate) medium (c and f), or YPDO medium (g). The preparation of media was described in Materials and methods. Nile red staining of wild-type (a–c) and fld1Δ (d–g) cells. Arrows indicate supersized LDs, and arrowheads indicate aggregated LDs. Bars: (A and C) 5 μm; (B) 1 μm.
Mentions: In an effort to identify novel gene products that may play a role in LD formation, Nile red, a vital dye specific for intracellular LDs, was used to visually screen the entire collection of viable single-gene deletion mutants of the budding yeast Saccharomyces cerevisiae for abnormalities in the number and morphology of LDs (Greenspan et al., 1985). Because the number and morphology of LDs may vary depending on growth phases, wild-type cells and all mutants were grown overnight to stationary phase (OD600 = ∼5) in this study immediately followed by Nile red staining and fluorescence microcopy. Wild-type cells at stationary phase showed 5.16 ± 2.18 LDs per cell on average (±SD; n = 200), and ∼80% of the cells displayed three to seven LDs (Fig. 1 A, a). To simplify the screening process, we arbitrarily categorized deletion strains with the majority (>80%) of cells accumulating on average less than three LDs as fld (few LDs) mutants and strains accumulating more than seven LDs as mld (many LDs) mutants. Among the mld mutants, strains containing >11 LDs were classified as strong mld mutants. We isolated 17 fld mutants and 116 mld mutants (Tables S1 and S2, available at http://www.jcb.org/cgi/content/full/jcb.200711136/DC1).

Bottom Line: Cells lacking FLD1 contain strikingly enlarged (supersized) LDs, and LDs from fld1Delta cells demonstrate significantly enhanced fusion activities both in vivo and in vitro.Interestingly, the expression of human seipin, whose mutant forms are associated with Berardinelli-Seip congenital lipodystrophy and motoneuron disorders, rescues LD-associated defects in fld1Delta cells.These results suggest that an evolutionally conserved function of seipin in phospholipid metabolism and LD formation may be functionally important in human adipogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, National University of Singapore, Singapore 117597, Republic of Singapore.

ABSTRACT
Lipid droplets (LDs) are emerging cellular organelles that are of crucial importance in cell biology and human diseases. In this study, we present our screen of approximately 4,700 Saccharomyces cerevisiae mutants for abnormalities in the number and morphology of LDs; we identify 17 fld (few LDs) and 116 mld (many LDs) mutants. One of the fld mutants (fld1) is caused by the deletion of YLR404W, a previously uncharacterized open reading frame. Cells lacking FLD1 contain strikingly enlarged (supersized) LDs, and LDs from fld1Delta cells demonstrate significantly enhanced fusion activities both in vivo and in vitro. Interestingly, the expression of human seipin, whose mutant forms are associated with Berardinelli-Seip congenital lipodystrophy and motoneuron disorders, rescues LD-associated defects in fld1Delta cells. Lipid profiling reveals alterations in acyl chain compositions of major phospholipids in fld1Delta cells. These results suggest that an evolutionally conserved function of seipin in phospholipid metabolism and LD formation may be functionally important in human adipogenesis.

Show MeSH
Related in: MedlinePlus