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Seipin performs dissectible functions in promoting lipid droplet biogenesis and regulating droplet morphology.

Cartwright BR, Binns DD, Hilton CL, Han S, Gao Q, Goodman JM - Mol. Biol. Cell (2014)

Bottom Line: Furthermore, we find that the normal rate of droplet initiation depends on 14 amino acids at the amino terminus of seipin, deletion of which results in fewer, larger droplets that are consistent with a delay in initiation but are otherwise normal in morphology.Importantly, other functions of seipin, namely vectorial budding and resistance to inositol, are retained in this mutant.We conclude that seipin has dissectible roles in both promoting early LD initiation and in regulating LD morphology, supporting its importance in LD biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75235-9041.

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Deletion of 14 amino acids from the seipin N-terminus results in an SLD phenotype. Seipin knockout cells (fld1∆) were complemented with plasmids overexpressing FLD1, fld1∆Nterm, or empty vector and cultured in rich oleate medium. (A) Representative bright-field or fluorescence microscopy projection images after staining with BODIPY to visualize LDs. Scale bar: 5 μm. (B) Number of FBs per total number of cells. (C) Percent of cells displaying one or more SLDs (defined as >1 μm diameter). For B and C, error bars represent SEMs from four independent experiments; for each, n = 100 cells from at least three fields. (D–F) Analysis of electron micrographs (sample images in Figure 4A); error bars represent SEMs from 100 cells. (D) Number of LDs per total number of cells. (E) Number of SLDs. (F) Number of LD clusters (defined as >5 adjacent droplets). (G) Phospholipid to NL ratios of isolated LD fractions, analyzed by TLC. (H) Phospholipid levels of whole-cell lysates by TLC, normalized to cell pellet wet weight. (I) Neutral lipid levels of whole-cell lysates as in H. (G–I) Error bars represent SEMs from four independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 by one-way analysis of variance (ANOVA) with correction for multiple comparisons.
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Figure 3: Deletion of 14 amino acids from the seipin N-terminus results in an SLD phenotype. Seipin knockout cells (fld1∆) were complemented with plasmids overexpressing FLD1, fld1∆Nterm, or empty vector and cultured in rich oleate medium. (A) Representative bright-field or fluorescence microscopy projection images after staining with BODIPY to visualize LDs. Scale bar: 5 μm. (B) Number of FBs per total number of cells. (C) Percent of cells displaying one or more SLDs (defined as >1 μm diameter). For B and C, error bars represent SEMs from four independent experiments; for each, n = 100 cells from at least three fields. (D–F) Analysis of electron micrographs (sample images in Figure 4A); error bars represent SEMs from 100 cells. (D) Number of LDs per total number of cells. (E) Number of SLDs. (F) Number of LD clusters (defined as >5 adjacent droplets). (G) Phospholipid to NL ratios of isolated LD fractions, analyzed by TLC. (H) Phospholipid levels of whole-cell lysates by TLC, normalized to cell pellet wet weight. (I) Neutral lipid levels of whole-cell lysates as in H. (G–I) Error bars represent SEMs from four independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 by one-way analysis of variance (ANOVA) with correction for multiple comparisons.

Mentions: To dissect the role of seipin in LD formation, we screened a small panel of seipin-deletion mutants for defects in steady-state LD morphology (unpublished data). Most remarkable of these was a deletion of 14 amino acids (MKINVSRPLQFLQW), comprising the amino terminal domain that faces the cytosol and precedes the first membrane span. While the seipin- strain is characterized by droplets of widely varying size, especially when cultured in medium containing oleic acid (Szymanski et al., 2007), cells expressing the N-terminal deletion mutant (fld1∆Nterm) from a plasmid in the background resulted in a remarkable prominence of SLDs (Figure 3A). This is consistent with a recently reported phenotype of fewer, larger droplets for a 10–amino acid N-terminal deletion of seipin, which was not characterized further (Wang et al., 2014). The fld1∆Nterm mutant produced fewer droplets per cell (Figure 3B) and more cells with SLDs (defined as LDs with >1-μm diameter by Yang; Fei et al., 2011a; Figure 3C) than both wild-type and seipin knockout cells. The overexpressed N-terminal deletion mutant protein both localized to the ER and formed high-molecular-weight complexes (Supplemental Figure S8), similar to wild-type. An SLD phenotype was also observed for endogenously expressed fld1∆Nterm integrated at the genomic seipin locus (Supplemental Figure S9A). Fluorescently tagged fld1∆Nterm forms several puncta similar to wild-type. Whereas wild-type seipin clearly localizes at or adjacent to the several droplets in cells, most puncta from fld1∆Nterm were dispersed in the cell, with at least one seipin spot associated with each of the few supersized droplets. (Supplemental Figure S9B).


Seipin performs dissectible functions in promoting lipid droplet biogenesis and regulating droplet morphology.

Cartwright BR, Binns DD, Hilton CL, Han S, Gao Q, Goodman JM - Mol. Biol. Cell (2014)

Deletion of 14 amino acids from the seipin N-terminus results in an SLD phenotype. Seipin knockout cells (fld1∆) were complemented with plasmids overexpressing FLD1, fld1∆Nterm, or empty vector and cultured in rich oleate medium. (A) Representative bright-field or fluorescence microscopy projection images after staining with BODIPY to visualize LDs. Scale bar: 5 μm. (B) Number of FBs per total number of cells. (C) Percent of cells displaying one or more SLDs (defined as >1 μm diameter). For B and C, error bars represent SEMs from four independent experiments; for each, n = 100 cells from at least three fields. (D–F) Analysis of electron micrographs (sample images in Figure 4A); error bars represent SEMs from 100 cells. (D) Number of LDs per total number of cells. (E) Number of SLDs. (F) Number of LD clusters (defined as >5 adjacent droplets). (G) Phospholipid to NL ratios of isolated LD fractions, analyzed by TLC. (H) Phospholipid levels of whole-cell lysates by TLC, normalized to cell pellet wet weight. (I) Neutral lipid levels of whole-cell lysates as in H. (G–I) Error bars represent SEMs from four independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 by one-way analysis of variance (ANOVA) with correction for multiple comparisons.
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Figure 3: Deletion of 14 amino acids from the seipin N-terminus results in an SLD phenotype. Seipin knockout cells (fld1∆) were complemented with plasmids overexpressing FLD1, fld1∆Nterm, or empty vector and cultured in rich oleate medium. (A) Representative bright-field or fluorescence microscopy projection images after staining with BODIPY to visualize LDs. Scale bar: 5 μm. (B) Number of FBs per total number of cells. (C) Percent of cells displaying one or more SLDs (defined as >1 μm diameter). For B and C, error bars represent SEMs from four independent experiments; for each, n = 100 cells from at least three fields. (D–F) Analysis of electron micrographs (sample images in Figure 4A); error bars represent SEMs from 100 cells. (D) Number of LDs per total number of cells. (E) Number of SLDs. (F) Number of LD clusters (defined as >5 adjacent droplets). (G) Phospholipid to NL ratios of isolated LD fractions, analyzed by TLC. (H) Phospholipid levels of whole-cell lysates by TLC, normalized to cell pellet wet weight. (I) Neutral lipid levels of whole-cell lysates as in H. (G–I) Error bars represent SEMs from four independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 by one-way analysis of variance (ANOVA) with correction for multiple comparisons.
Mentions: To dissect the role of seipin in LD formation, we screened a small panel of seipin-deletion mutants for defects in steady-state LD morphology (unpublished data). Most remarkable of these was a deletion of 14 amino acids (MKINVSRPLQFLQW), comprising the amino terminal domain that faces the cytosol and precedes the first membrane span. While the seipin- strain is characterized by droplets of widely varying size, especially when cultured in medium containing oleic acid (Szymanski et al., 2007), cells expressing the N-terminal deletion mutant (fld1∆Nterm) from a plasmid in the background resulted in a remarkable prominence of SLDs (Figure 3A). This is consistent with a recently reported phenotype of fewer, larger droplets for a 10–amino acid N-terminal deletion of seipin, which was not characterized further (Wang et al., 2014). The fld1∆Nterm mutant produced fewer droplets per cell (Figure 3B) and more cells with SLDs (defined as LDs with >1-μm diameter by Yang; Fei et al., 2011a; Figure 3C) than both wild-type and seipin knockout cells. The overexpressed N-terminal deletion mutant protein both localized to the ER and formed high-molecular-weight complexes (Supplemental Figure S8), similar to wild-type. An SLD phenotype was also observed for endogenously expressed fld1∆Nterm integrated at the genomic seipin locus (Supplemental Figure S9A). Fluorescently tagged fld1∆Nterm forms several puncta similar to wild-type. Whereas wild-type seipin clearly localizes at or adjacent to the several droplets in cells, most puncta from fld1∆Nterm were dispersed in the cell, with at least one seipin spot associated with each of the few supersized droplets. (Supplemental Figure S9B).

Bottom Line: Furthermore, we find that the normal rate of droplet initiation depends on 14 amino acids at the amino terminus of seipin, deletion of which results in fewer, larger droplets that are consistent with a delay in initiation but are otherwise normal in morphology.Importantly, other functions of seipin, namely vectorial budding and resistance to inositol, are retained in this mutant.We conclude that seipin has dissectible roles in both promoting early LD initiation and in regulating LD morphology, supporting its importance in LD biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75235-9041.

Show MeSH
Related in: MedlinePlus