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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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The N-terminal seipin-deletion phenotype is resistant to manipulation by inositol. Seipin knockout cells (fld1∆) complemented with plasmids overexpressing empty vector, FLD1, or fld1∆Nterm, and the ER marker CFP-HDEL were grown in minimal glucose medium with or without the indicated PL precursor supplements. (A) Representative fluorescence microscopy projection images after staining with BODIPY. Scale bar: 5 μm. CFP-HDEL is false-colored red in the merged images. Arrowhead indicates an LD–ER tangle, defined as an irregular BODIPY body colocalized with a similarly shaped ER density. (B and D) Percent of cells displaying SLDs. (C and E) Percent of cells displaying LD–ER tangles. Error bars for B–E represent SEMs from three independent experiments; for each, n = 100 cells from at least three fields. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 by two-way ANOVA with correction for multiple comparisons.
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Figure 5: The N-terminal seipin-deletion phenotype is resistant to manipulation by inositol. Seipin knockout cells (fld1∆) complemented with plasmids overexpressing empty vector, FLD1, or fld1∆Nterm, and the ER marker CFP-HDEL were grown in minimal glucose medium with or without the indicated PL precursor supplements. (A) Representative fluorescence microscopy projection images after staining with BODIPY. Scale bar: 5 μm. CFP-HDEL is false-colored red in the merged images. Arrowhead indicates an LD–ER tangle, defined as an irregular BODIPY body colocalized with a similarly shaped ER density. (B and D) Percent of cells displaying SLDs. (C and E) Percent of cells displaying LD–ER tangles. Error bars for B–E represent SEMs from three independent experiments; for each, n = 100 cells from at least three fields. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 by two-way ANOVA with correction for multiple comparisons.

Mentions: We further analyzed the supersized droplet phenotype of fld1∆Nterm from oleate-grown cells by electron microscopy (Figure 3, D–F; sample images are in Figure 4A), finding fewer droplets in fld1∆Nterm with an increased number of supersized droplets, consistent with the phenotype from fluorescence microscopy. Additionally, we detected a concomitant decrease in the prevalence of LD clusters (defined as at least five droplets adjacent to one another) compared with fld1∆ cells. This was consistent with a decrease in LD–ER tangles later observed by fluorescence microscopy in minimal medium (Figure 5, C and E). The N-terminal deletion therefore appears to display an extreme supersized phenotype but is otherwise normal in droplet morphology, without the droplet clustering or size heterogeneity of the knockout strain.


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)

The N-terminal seipin-deletion phenotype is resistant to manipulation by inositol. Seipin knockout cells (fld1∆) complemented with plasmids overexpressing empty vector, FLD1, or fld1∆Nterm, and the ER marker CFP-HDEL were grown in minimal glucose medium with or without the indicated PL precursor supplements. (A) Representative fluorescence microscopy projection images after staining with BODIPY. Scale bar: 5 μm. CFP-HDEL is false-colored red in the merged images. Arrowhead indicates an LD–ER tangle, defined as an irregular BODIPY body colocalized with a similarly shaped ER density. (B and D) Percent of cells displaying SLDs. (C and E) Percent of cells displaying LD–ER tangles. Error bars for B–E represent SEMs from three independent experiments; for each, n = 100 cells from at least three fields. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 by two-way ANOVA with correction for multiple comparisons.
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Figure 5: The N-terminal seipin-deletion phenotype is resistant to manipulation by inositol. Seipin knockout cells (fld1∆) complemented with plasmids overexpressing empty vector, FLD1, or fld1∆Nterm, and the ER marker CFP-HDEL were grown in minimal glucose medium with or without the indicated PL precursor supplements. (A) Representative fluorescence microscopy projection images after staining with BODIPY. Scale bar: 5 μm. CFP-HDEL is false-colored red in the merged images. Arrowhead indicates an LD–ER tangle, defined as an irregular BODIPY body colocalized with a similarly shaped ER density. (B and D) Percent of cells displaying SLDs. (C and E) Percent of cells displaying LD–ER tangles. Error bars for B–E represent SEMs from three independent experiments; for each, n = 100 cells from at least three fields. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 by two-way ANOVA with correction for multiple comparisons.
Mentions: We further analyzed the supersized droplet phenotype of fld1∆Nterm from oleate-grown cells by electron microscopy (Figure 3, D–F; sample images are in Figure 4A), finding fewer droplets in fld1∆Nterm with an increased number of supersized droplets, consistent with the phenotype from fluorescence microscopy. Additionally, we detected a concomitant decrease in the prevalence of LD clusters (defined as at least five droplets adjacent to one another) compared with fld1∆ cells. This was consistent with a decrease in LD–ER tangles later observed by fluorescence microscopy in minimal medium (Figure 5, C and E). The N-terminal deletion therefore appears to display an extreme supersized phenotype but is otherwise normal in droplet morphology, without the droplet clustering or size heterogeneity of the knockout strain.

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