Limits...
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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LDs from fld1Δ cells demonstrate enhanced fusion activity in vivo and in vitro. (A) fld1Δ cells were grown in SC medium until midlog phase (OD600 = ∼1) and were stained with Nile red. Cells in which two or several LDs lay close together were targeted. Images were collected at 2-s intervals. (B) Tgl3p-GFP localizes to LDs both in wild-type (WT) and fld1Δ strains. DIC, differential interference contrast. (C) Tgl3p-GFP–tagged LDs were isolated from fld1Δ cells grown in SC media, resuspended in PBS, and left for gentle shaking at 30°C for 1 h. The images were taken before (0 min) and after incubation (60 min; a–c). (a–c) Three typical results. (D) LDs isolated from wild-type cells grown in SC media do not fuse under the same conditions as described in C. Bars, 5 μm.
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fig3: LDs from fld1Δ cells demonstrate enhanced fusion activity in vivo and in vitro. (A) fld1Δ cells were grown in SC medium until midlog phase (OD600 = ∼1) and were stained with Nile red. Cells in which two or several LDs lay close together were targeted. Images were collected at 2-s intervals. (B) Tgl3p-GFP localizes to LDs both in wild-type (WT) and fld1Δ strains. DIC, differential interference contrast. (C) Tgl3p-GFP–tagged LDs were isolated from fld1Δ cells grown in SC media, resuspended in PBS, and left for gentle shaking at 30°C for 1 h. The images were taken before (0 min) and after incubation (60 min; a–c). (a–c) Three typical results. (D) LDs isolated from wild-type cells grown in SC media do not fuse under the same conditions as described in C. Bars, 5 μm.

Mentions: The existence of morphologically distinct LDs within fld1Δ suggests enhanced fusion activities of LDs: the small, discrete LDs may represent the newly synthesized LDs, which tend to aggregate before eventually fusing into a supersized LD. To test this hypothesis, wild-type and fld1Δ cells were cultured in SC medium until midlog phase (OD600 = ∼1.0), stained with Nile red, and observed for the fusion of LDs by fluorescent microscopy. Cells in which two or several LDs lay close together were targeted. We examined 200 cases of adjacent LDs each for mutant and wild-type cells and monitored every case for 1 min. The criteria we used to define fusion were described previously (Bostrom et al., 2007). No fusion events were observed in wild-type cells, but fusion was detectable for about 10% (19/200) of all mutant cases. As shown in Fig. 3 A, two closely positioned LDs appeared to completely fuse within a span of 2 s. The size of the newly formed LD appeared to be the combined size of the two parent LDs.


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)

LDs from fld1Δ cells demonstrate enhanced fusion activity in vivo and in vitro. (A) fld1Δ cells were grown in SC medium until midlog phase (OD600 = ∼1) and were stained with Nile red. Cells in which two or several LDs lay close together were targeted. Images were collected at 2-s intervals. (B) Tgl3p-GFP localizes to LDs both in wild-type (WT) and fld1Δ strains. DIC, differential interference contrast. (C) Tgl3p-GFP–tagged LDs were isolated from fld1Δ cells grown in SC media, resuspended in PBS, and left for gentle shaking at 30°C for 1 h. The images were taken before (0 min) and after incubation (60 min; a–c). (a–c) Three typical results. (D) LDs isolated from wild-type cells grown in SC media do not fuse under the same conditions as described in C. Bars, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2234226&req=5

fig3: LDs from fld1Δ cells demonstrate enhanced fusion activity in vivo and in vitro. (A) fld1Δ cells were grown in SC medium until midlog phase (OD600 = ∼1) and were stained with Nile red. Cells in which two or several LDs lay close together were targeted. Images were collected at 2-s intervals. (B) Tgl3p-GFP localizes to LDs both in wild-type (WT) and fld1Δ strains. DIC, differential interference contrast. (C) Tgl3p-GFP–tagged LDs were isolated from fld1Δ cells grown in SC media, resuspended in PBS, and left for gentle shaking at 30°C for 1 h. The images were taken before (0 min) and after incubation (60 min; a–c). (a–c) Three typical results. (D) LDs isolated from wild-type cells grown in SC media do not fuse under the same conditions as described in C. Bars, 5 μm.
Mentions: The existence of morphologically distinct LDs within fld1Δ suggests enhanced fusion activities of LDs: the small, discrete LDs may represent the newly synthesized LDs, which tend to aggregate before eventually fusing into a supersized LD. To test this hypothesis, wild-type and fld1Δ cells were cultured in SC medium until midlog phase (OD600 = ∼1.0), stained with Nile red, and observed for the fusion of LDs by fluorescent microscopy. Cells in which two or several LDs lay close together were targeted. We examined 200 cases of adjacent LDs each for mutant and wild-type cells and monitored every case for 1 min. The criteria we used to define fusion were described previously (Bostrom et al., 2007). No fusion events were observed in wild-type cells, but fusion was detectable for about 10% (19/200) of all mutant cases. As shown in Fig. 3 A, two closely positioned LDs appeared to completely fuse within a span of 2 s. The size of the newly formed LD appeared to be the combined size of the two parent LDs.

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