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 (). 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
) mutants and strains accumulating more than seven LDs as mld
) 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
Figure 1. 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 (more ...)
We focused on FLD1, which corresponds to a previously uncharacterized ORF, YLR404W, because its deletion not only affected the number of LDs but also gave rise to strikingly enlarged or aggregated LDs (). When grown in rich medium until stationary phase, wild-type cells usually display three to seven LDs under the microscope. The LDs were between 0.2 and 0.4 μm in diameter and were almost spherical in shape (). In contrast, LDs observed in fld1Δ cells were very irregular in terms of quantity, shape, and size. Up to 30% of the total population of fld1Δ cells contained one or a few supersized LDs that were spherical in shape and were about 0.5–1.5 μm in diameter (; arrow), which means that the volume of the largest LD of the fld1Δ cells was about 50 times that of the largest LD found in wild-type cells. About 60% of the fld1Δ population contained an amorphous aggregation of neutral lipids in addition to several small LDs (; arrowheads). The remaining ~10% of the fld1Δ cells contained scattered and weakly stained LDs, which had diameters of <0.1 μm (). The phenotypic characteristics of fld1Δ were also observed in log-phase cells. When grown to log phase (OD600 = 0.8), most of the wild-type cells contained two or three LDs that were slightly smaller than those of stationary-phase cells (). The LDs in fld1Δ at log phase show similar morphology to cells grown to stationary phase except that the supersized LDs and the aggregation of neutral lipids were smaller and more weakly stained (; arrow and arrowhead, respectively).
To examine the ultrastructure of fld1Δ cells, we performed transmission EM (TEM) of the wild-type and fld1Δ strains. Cells were grown in rich medium until stationary phase and were subjected to TEM analysis. One typical cross section of a wild-type cell contained five LD profiles, which were round and about 0.2–0.4 μm in diameter (). The cross sections of fld1Δ mutant cells again showed three classes of LDs with distinct morphologies. Up to 30% of cells displayed one or a few supersized LDs per section, which were either round or oval (). Consistent with the result of fluorescence microscopy, the diameters of some LDs were up to 1.5 μm (). Aggregated LDs were found in ~60% of the mutant population (). These aggregations were reminiscent of the amorphous neutral lipid clump observed under fluorescence microscopy (). The rest of the fld1Δ cells (~10% of the total population) contained many tiny LDs, most of which had a diameter of <0.1 μm and were loosely scattered ().
Yeast cells undergo marked proliferation of LDs when they are grown in defined medium (synthetic complete [SC]) or in an oleate-based medium (YPO [YP plus oleate, no glucose]; Binns et al., 2006
). As shown in , a great increase in the number of LDs was observed in wild-type cells grown in SC or YPO media. When fld1Δ
cells were grown in YPD, the majority (~70%) of the cells showed amorphous aggregations of many intermediate-sized LDs (). In contrast, >70% of the fld1Δ
cells displayed only one or two supersized LDs when cultured in SC media (). Moreover, amorphous aggregation of many small LDs that were common in cells cultured in YPD media were only observed in about 10% of the cells grown in SC. Interestingly, when fld1Δ
cells were cultured in YPO medium, >95% (191/200 cells examined) of the cells displayed amorphous aggregations of LDs without the supersized LDs (). However, when fld1Δ
cells were cultured in YPDO medium (YPD + oleate), the large LDs appeared again together with the aggregation of smaller LDs (). Thus, we identified a commonly used growth medium (SC) that allowed us to easily distinguish wild-type and fld1Δ
cells based on the presence or absence of supersized LDs (, compare b with e).
We measured the steady-state levels of TAG and SE as well as the rate of oleate incorporation into TAG and SE (Oelkers et al., 2002
). For cells grown in YPD to log phase, the deletion of FLD1
caused about a doubling in the steady-state levels of both TAG and SE (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200711136/DC1
). The rate of SE synthesis was also upregulated by 70% in fld1Δ
deletion cells, but little difference in the rate of oleate incorporation into TAG was observed (Fig. S1 B). Similar patterns of changes were detected for cells grown in YPD to stationary phase or in SC to either log or stationary phase (unpublished data).
The expression of FLD1-GFP in fld1Δ cells restored the normal morphology of LDs (). We performed a series of subcellular fractionation experiments to analyze the cellular distribution of Fld1p. Cell extracts prepared from the fld1Δ strain expressing Fld1-GFP were fractionated by centrifugation at 13,000 g for 10 min, resulting in P13 pellet and S13 supernatant fractions that were probed with antibodies against GFP and Dpm1p, an ER marker. Both Fld1p and Dpm1p were found in the P13 fraction, which contains large membranous structures such as the vacuole, ER, and plasma membrane (). The same cell extracts were subjected to continuous sucrose density gradient analysis. 13 fractions were collected from top to bottom (1–13) and were probed for the presence of GFP and Dpm1p by immunoblotting. Dpm1p and Fld1p appeared to exist in the same density fractions (). Localization of Fld1-GFP was also examined in live cells by fluorescent microscopy, and Fld1-GFP was found in both perinuclear and peripheral ER (). Finally, immuno-EM was used to pinpoint the exact location of Fld1p. Fld1p-GFP was found to be associated with the cortical ER and the nuclear envelope, which is consistent with the putative ER localization observed by light microscopy. In addition, labeling was observed throughout the ER, including regions in contact with LDs ().
Figure 2. Fld1p localizes predominantly to the ER. (A) The expression of FLD1-GFP complements the fld1Δ phenotype. Cells were transformed either with YCplac111 vector alone or with YCplac111-FLD1-GFP and grown in SC media without leucine. (B) fld1Δ (more ...)
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 , 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.
Figure 3. 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 (more ...)
To further demonstrate enhanced LD fusion in fld1Δ
cells, we isolated LDs from wild-type and mutant cells both carrying Tgl3p-GFP. Tgl3p is a triglyceride lipase that localizes to the surface of LDs (; Athenstaedt and Daum, 2003
). Purified LDs from both wild-type and fld1Δ
cells were left in PBS buffer and examined by microscopy before and after 60 min. Whereas LDs from wild-type cells remained scattered and unchanged in size, LDs from fld1Δ
cells formed aggregates or fused into huge lipid inclusions reminiscent of the supersized LDs observed in live fld1Δ
cells (). The lipid inclusions observed in vitro are much larger than the supersized LDs in live cells (~5 μm vs. ~1 μm in diameter), suggesting that additional fusion events occurred in vitro.
Remote homology detection techniques were used to look for a mammalian homologue to Fld1p. Human seipin, a protein associated with Berardinelli-Seip congenital lipodystrophy (BSCL) and motorneuron disorders, was identified (Agarwal and Garg, 2004
). Seipin shows weak sequence conservations to Fld1p, but they both contain two predicted transmembrane domains, and their predicted secondary structures are also very similar (). Human seipin is encoded by the BSCL2
gene, which gives rise to at least three different mRNAs and two peptides with 398 or 462 amino acids (Lundin et al., 2006
). The region that covers the first 280 amino acids of human seipin is 88% identical among rat, mouse, chimpanzee, and human homologues (Agarwal and Garg, 2004
). This is interesting because all of the sequence conservations between yeast Fld1p (285 amino acids) and seipin fall within this region. To test whether seipin is a functional homologue of Fld1p, full-length human and mouse seipin homologues were expressed in fld1Δ
cells grown in SC media. The average diameter of the LDs is 1.27 ± 0.19 μm (n
= 117) without human seipin and 0.43 ± 0.05 μm (n
= 106) with seipin (). We also examined the effects of point mutations in seipin that are implicated in lipodystrophy (A212P) and motoneuron disorders (N88S and S90L). The expression of N88S and S90L but not A212P rescued the defects in LD morphology (). This is not surprising because A212P is considered a loss-of-function mutation, whereas N88S and S90L may represent gain-of-function mutations (Windpassinger et al., 2004
). Lastly, expression of the highly conserved 280–amino acid region of seipin rescued the defects in LD morphology (). Together, these results strongly suggest that human seipin represents the functional homologue of Fld1p.
Figure 4. Expression of human and mouse BSCL2 (seipin) rescues defects in LD morphology in fld1Δ cells. (A) Sequence alignment of seipin homologues with gene identification numbers. The alignment was created using the PRALINE server (Simossis and Heringa, (more ...)
Congenital generalized lipodystrophy (CGL; or BSCL) is an autosomal recessive disorder that is characterized by the almost complete absence of adipose tissue and severe insulin resistance (Agarwal and Garg, 2004
). Genome-wide linkage analysis identified two loci for CGL: CGL type 1 (CGL1) is caused by mutations in the AGPAT2 (1-acylglycerol-3-phosphate-O
-acyl transferase 2) gene, and CGL2 is caused by mutations in BSCL2
, which encodes seipin (Magre et al., 2001
; Agarwal et al., 2002
). AGPAT2 catalyzes the formation of phosphatidic acid (PA), but knocking down AGPAT2 led to elevated levels of several phospholipid species, including PA, and to a delay in the activation of key transcription factors for adipogenesis such as C/EBPβ and PPARγ (Gale et al., 2006
). Therefore, AGPAT2 controls adipogenesis through modulation of the synthesis of phospholipids. In contrast, little is known about the role of seipin in adipogenesis and lipodystrophy. Because mutations in AGPAT2 and BSCL2 cause similar clinical manifestations, we wondered whether aberrant phospholipid metabolism may underlie the cellular defects for both conditions. Lipid species from wild-type and fld1Δ
whole cell extracts were analyzed by electrospray ionization tandem mass spectrometry. The level of PA increased slightly in fld1Δ
cells (unpublished data). Interestingly, there is a shift from long-chain (18:1) to medium/short-chain (16:0, 14:0, and 12:0) fatty acid incorporation into all major phospholipids as a result of the deletion of FLD1
(nd Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200711136/DC1
Figure 5. Mass spectrometry analysis of glycerophospholipids. (A and B) Wild-type and fld1Δ cells were grown to stationary phase in SC media. Total lipids were extracted and analyzed by high performance liquid chromatography/mass spectrometry. The results (more ...)
We identified seipin (Fld1p) as a novel regulator of the cellular dynamics of LDs. The deletion of FLD1
causes increased levels of neutral lipids, clustering of LDs, and formation of enlarged (supersized) LDs. Increased neutral lipids often lead to an increase in the number but not the size of LDs ( and Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200711136/DC1
). Therefore, the appearance of supersized LDs in fld1Δ
cells is unlikely to be caused by an increase in neutral lipids. Rather, the physical property of the surface of LDs might have been altered in fld1Δ
cells as a result of changes in phospholipids, which may facilitate the clustering and fusion of LDs. In support of this, LDs isolated from fld1Δ
cells can aggregate and fuse without the supply of ATP and cytoplasmic proteins ().
What is the molecular function of Fld1p or seipin? We favor a role of Fld1p in phospholipid/fatty acid metabolism for the following reasons: (1) a shift from long to medium/short acyl chains was detected in fld1Δ
cells (); (2) LDs isolated from fld1Δ
cells can aggregate and fuse without the supply of ATP and cytoplasmic proteins, suggesting a role for phospholipids (); (3) the other gene that is associated with CGL is APGAT2, a major enzyme in phospholipid metabolism; and (4) lipin, another well-known protein that is associated with lipodystrophy in mice, is a phosphatidate phosphatase (Han et al., 2006
). Deletion of SPO7
, two genes involved in the activation of lipin, also caused aberrant LD morphology (Table S1 and our unpublished data).
Results described herein show for the first time that seipin and its homologues modulate the formation and especially fusion of the LDs. Our data also suggest that changes in phospholipid metabolism may be the unifying theme for both CGL1 and CGL2. Understanding the molecular function of seipin will provide mechanistic insights into LD formation and adipogenesis.