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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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N-terminal seipin deletion results in an initiation defect during de novo LD formation. Genomic knock-ins were generated at the seipin locus in the 3KO(GALDGA1) background for FLD1, fld1∆, and fld1∆Nterm. Cells were switched from raffinose to galactose media at t = 0 to induce droplet formation and stained with BODIPY. (A) Representative fluorescence microscopy projection images at indicated time points after galactose induction. Scale bars: 5 µm. (B) Histograms of FB size, given as area in pixels on a maximum-intensity projection image. (C) Percent of cells containing at least one FB. (D) Number of distinct FBs per cells that have at least one. Error bars in C and D represent SEMs from three independent experiments, each N = 100 cells from at least three fields. *, p < 0.05; #, p < 0.01; , p < 0.0001 by one-way ANOVA with correction for multiple comparisons. (E) Time-lapse fluorescence microscopy of cells embedded into agar after 1 h of galactose induction in liquid culture at 27–29°C. Images taken in 10-min increments (see Supplemental Videos S1–S3); representative projections of 30-min increment montages are shown. Note that cells were precultured at high density to prevent division during imaging. (F) Percent of cells that displayed at least one FB over the course of the time lapse. Error bars represent SEMs from three independent experiments. ***, p < 0.001 by one-way ANOVA with correction for multiple comparisons. (G) Histogram of time to first appearance for each droplet. (H) Average intensity curves for FBs during the time lapse. Time 0 defined as the frame before first appearance of the droplet. Error bars represent SEMs from average droplet intensity values per time point over three independent experiments. Absolute intensity values are likely underestimated due to bleaching. Scale bars: 5 µm
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Figure 6: N-terminal seipin deletion results in an initiation defect during de novo LD formation. Genomic knock-ins were generated at the seipin locus in the 3KO(GALDGA1) background for FLD1, fld1∆, and fld1∆Nterm. Cells were switched from raffinose to galactose media at t = 0 to induce droplet formation and stained with BODIPY. (A) Representative fluorescence microscopy projection images at indicated time points after galactose induction. Scale bars: 5 µm. (B) Histograms of FB size, given as area in pixels on a maximum-intensity projection image. (C) Percent of cells containing at least one FB. (D) Number of distinct FBs per cells that have at least one. Error bars in C and D represent SEMs from three independent experiments, each N = 100 cells from at least three fields. *, p < 0.05; #, p < 0.01; , p < 0.0001 by one-way ANOVA with correction for multiple comparisons. (E) Time-lapse fluorescence microscopy of cells embedded into agar after 1 h of galactose induction in liquid culture at 27–29°C. Images taken in 10-min increments (see Supplemental Videos S1–S3); representative projections of 30-min increment montages are shown. Note that cells were precultured at high density to prevent division during imaging. (F) Percent of cells that displayed at least one FB over the course of the time lapse. Error bars represent SEMs from three independent experiments. ***, p < 0.001 by one-way ANOVA with correction for multiple comparisons. (G) Histogram of time to first appearance for each droplet. (H) Average intensity curves for FBs during the time lapse. Time 0 defined as the frame before first appearance of the droplet. Error bars represent SEMs from average droplet intensity values per time point over three independent experiments. Absolute intensity values are likely underestimated due to bleaching. Scale bars: 5 µm

Mentions: After characterizing the effects of fld1∆Nterm on LD morphology and determining that the supersized phenotype was independent of inositol and its effect on PL synthesis, we then asked whether it could be explained by a defect in the rate of droplet initiation. We integrated fld1∆Nterm into the genomic seipin locus of the 3KO(GALDGA1) strain and observed LD formation after galactose induction as in Figure 1, with additional early time points at 1 and 2 h (Figure 6A). After 9 h, the “supersized” morphology of fld1∆Nterm was generally recapitulated, with the fld1∆Nterm droplet population displaying significantly larger sizes than FLD1 or fld1∆ (average area on projection image: FLD1, 55.02 pixels2; fld1∆, 80.43 pixels2; fld1∆Nterm, 116.6 pixels2), obviously more spherical shape, and a size distribution that exhibited less skewness than fld1∆ (average skewness of area distributions: FLD1, 1.094; fld1∆, 2.189; fld1∆Nterm, 1.406), indicating a more normally distributed droplet population than the knockout (Figure 6B).


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)

N-terminal seipin deletion results in an initiation defect during de novo LD formation. Genomic knock-ins were generated at the seipin locus in the 3KO(GALDGA1) background for FLD1, fld1∆, and fld1∆Nterm. Cells were switched from raffinose to galactose media at t = 0 to induce droplet formation and stained with BODIPY. (A) Representative fluorescence microscopy projection images at indicated time points after galactose induction. Scale bars: 5 µm. (B) Histograms of FB size, given as area in pixels on a maximum-intensity projection image. (C) Percent of cells containing at least one FB. (D) Number of distinct FBs per cells that have at least one. Error bars in C and D represent SEMs from three independent experiments, each N = 100 cells from at least three fields. *, p < 0.05; #, p < 0.01; , p < 0.0001 by one-way ANOVA with correction for multiple comparisons. (E) Time-lapse fluorescence microscopy of cells embedded into agar after 1 h of galactose induction in liquid culture at 27–29°C. Images taken in 10-min increments (see Supplemental Videos S1–S3); representative projections of 30-min increment montages are shown. Note that cells were precultured at high density to prevent division during imaging. (F) Percent of cells that displayed at least one FB over the course of the time lapse. Error bars represent SEMs from three independent experiments. ***, p < 0.001 by one-way ANOVA with correction for multiple comparisons. (G) Histogram of time to first appearance for each droplet. (H) Average intensity curves for FBs during the time lapse. Time 0 defined as the frame before first appearance of the droplet. Error bars represent SEMs from average droplet intensity values per time point over three independent experiments. Absolute intensity values are likely underestimated due to bleaching. Scale bars: 5 µm
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Figure 6: N-terminal seipin deletion results in an initiation defect during de novo LD formation. Genomic knock-ins were generated at the seipin locus in the 3KO(GALDGA1) background for FLD1, fld1∆, and fld1∆Nterm. Cells were switched from raffinose to galactose media at t = 0 to induce droplet formation and stained with BODIPY. (A) Representative fluorescence microscopy projection images at indicated time points after galactose induction. Scale bars: 5 µm. (B) Histograms of FB size, given as area in pixels on a maximum-intensity projection image. (C) Percent of cells containing at least one FB. (D) Number of distinct FBs per cells that have at least one. Error bars in C and D represent SEMs from three independent experiments, each N = 100 cells from at least three fields. *, p < 0.05; #, p < 0.01; , p < 0.0001 by one-way ANOVA with correction for multiple comparisons. (E) Time-lapse fluorescence microscopy of cells embedded into agar after 1 h of galactose induction in liquid culture at 27–29°C. Images taken in 10-min increments (see Supplemental Videos S1–S3); representative projections of 30-min increment montages are shown. Note that cells were precultured at high density to prevent division during imaging. (F) Percent of cells that displayed at least one FB over the course of the time lapse. Error bars represent SEMs from three independent experiments. ***, p < 0.001 by one-way ANOVA with correction for multiple comparisons. (G) Histogram of time to first appearance for each droplet. (H) Average intensity curves for FBs during the time lapse. Time 0 defined as the frame before first appearance of the droplet. Error bars represent SEMs from average droplet intensity values per time point over three independent experiments. Absolute intensity values are likely underestimated due to bleaching. Scale bars: 5 µm
Mentions: After characterizing the effects of fld1∆Nterm on LD morphology and determining that the supersized phenotype was independent of inositol and its effect on PL synthesis, we then asked whether it could be explained by a defect in the rate of droplet initiation. We integrated fld1∆Nterm into the genomic seipin locus of the 3KO(GALDGA1) strain and observed LD formation after galactose induction as in Figure 1, with additional early time points at 1 and 2 h (Figure 6A). After 9 h, the “supersized” morphology of fld1∆Nterm was generally recapitulated, with the fld1∆Nterm droplet population displaying significantly larger sizes than FLD1 or fld1∆ (average area on projection image: FLD1, 55.02 pixels2; fld1∆, 80.43 pixels2; fld1∆Nterm, 116.6 pixels2), obviously more spherical shape, and a size distribution that exhibited less skewness than fld1∆ (average skewness of area distributions: FLD1, 1.094; fld1∆, 2.189; fld1∆Nterm, 1.406), indicating a more normally distributed droplet population than the knockout (Figure 6B).

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