Seipin performs dissectible functions in promoting lipid droplet biogenesis and regulating droplet morphology.
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.
Affiliation: Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75235-9041.Show MeSH
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Mentions: 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.
Affiliation: Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75235-9041.