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Drosophila lipophorin receptors mediate the uptake of neutral lipids in oocytes and imaginal disc cells by an endocytosis-independent mechanism.

Parra-Peralbo E, Culi J - PLoS Genet. (2011)

Bottom Line: Furthermore, our data indicate that endocytosis of the lipophorin receptors is not required to mediate the uptake of neutral lipids.These findings suggest a model where lipophorin receptors promote the extracellular lipolysis of lipophorins.This model is reminiscent of the lipolytic processing of triglyceride-rich lipoproteins that occurs at the mammalian capillary endothelium, suggesting an ancient role for LDLR-like proteins in this process.

View Article: PubMed Central - PubMed

Affiliation: Centro Andaluz de Biología del Desarrollo (CSIC-UPO), Universidad Pablo de Olavide, Sevilla, Spain.

ABSTRACT
Lipids are constantly shuttled through the body to redistribute energy and metabolites between sites of absorption, storage, and catabolism in a complex homeostatic equilibrium. In Drosophila, lipids are transported through the hemolymph in the form of lipoprotein particles, known as lipophorins. The mechanisms by which cells interact with circulating lipophorins and acquire their lipidic cargo are poorly understood. We have found that lipophorin receptor 1 and 2 (lpr1 and lpr2), two partially redundant genes belonging to the Low Density Lipoprotein Receptor (LDLR) family, are essential for the efficient uptake and accumulation of neutral lipids by oocytes and cells of the imaginal discs. Females lacking the lpr2 gene lay eggs with low lipid content and have reduced fertility, revealing a central role for lpr2 in mediating Drosophila vitellogenesis. lpr1 and lpr2 are transcribed into multiple isoforms. Interestingly, only a subset of these isoforms containing a particular LDLR type A module mediate neutral lipid uptake. Expression of these isoforms induces the extracellular stabilization of lipophorins. Furthermore, our data indicate that endocytosis of the lipophorin receptors is not required to mediate the uptake of neutral lipids. These findings suggest a model where lipophorin receptors promote the extracellular lipolysis of lipophorins. This model is reminiscent of the lipolytic processing of triglyceride-rich lipoproteins that occurs at the mammalian capillary endothelium, suggesting an ancient role for LDLR-like proteins in this process.

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Germ-line expression of Lpr1J or Lpr2E rescues the oogenesis phenotypes of lpr1-, lpr2− double mutant females.(A–F) Df(3R)lpr1/2 egg chambers overexpressing the lipid uptake-defective UASp-lpr2F isoform (A–B) or the lipid uptake-promoting UASp-lpr2E (C–D) or UASp-lpr1J (E–F) isoforms in the germ-line driven by V32-gal4. (A,C,E) Lpr2F (A), Lpr2E (C) and Lpr1J (E) proteins were detected in the nurse cells (n) and oocyte (o) membranes using an α-HA antibody (arrowheads, shown in green). Egg chambers were stained with the lipophilic nile red dye (red) and DAPI (blue) to mark nuclei. (B, D, F) Ovarioles containing multiple egg chambers stained with DAPI. Several degenerating egg chambers are labeled with a bracket in (B). Note that germ-line specific expression of UASp-lpr2E and UASp-lpr1J but not of UASp-lpr2F induced accumulation of neutral lipids in nurse cells (arrows in C and E) and rescued mid-oogenesis degeneration (D and F, compare also with the wild-type and Df(3R)lpr1/2 egg chambers in Figure 2). (G) Semiquantitative RT-PCR showing the relative abundance of lpr1 and lpr2 transcripts in several tissues, as indicated (wing imaginal discs, third instar larva fat body, ovary, adult fat body, adult brain and whole female fly). The oligo pairs used discriminated between isoforms transcribed from the proximal promoter (PP) and from distal promoter (DP). Analysis of ribosomal protein rp49 transcription was included as a control. Scale bars: (A, C and E) 50 µm, (B, D and F) 100 µm.
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pgen-1001297-g005: Germ-line expression of Lpr1J or Lpr2E rescues the oogenesis phenotypes of lpr1-, lpr2− double mutant females.(A–F) Df(3R)lpr1/2 egg chambers overexpressing the lipid uptake-defective UASp-lpr2F isoform (A–B) or the lipid uptake-promoting UASp-lpr2E (C–D) or UASp-lpr1J (E–F) isoforms in the germ-line driven by V32-gal4. (A,C,E) Lpr2F (A), Lpr2E (C) and Lpr1J (E) proteins were detected in the nurse cells (n) and oocyte (o) membranes using an α-HA antibody (arrowheads, shown in green). Egg chambers were stained with the lipophilic nile red dye (red) and DAPI (blue) to mark nuclei. (B, D, F) Ovarioles containing multiple egg chambers stained with DAPI. Several degenerating egg chambers are labeled with a bracket in (B). Note that germ-line specific expression of UASp-lpr2E and UASp-lpr1J but not of UASp-lpr2F induced accumulation of neutral lipids in nurse cells (arrows in C and E) and rescued mid-oogenesis degeneration (D and F, compare also with the wild-type and Df(3R)lpr1/2 egg chambers in Figure 2). (G) Semiquantitative RT-PCR showing the relative abundance of lpr1 and lpr2 transcripts in several tissues, as indicated (wing imaginal discs, third instar larva fat body, ovary, adult fat body, adult brain and whole female fly). The oligo pairs used discriminated between isoforms transcribed from the proximal promoter (PP) and from distal promoter (DP). Analysis of ribosomal protein rp49 transcription was included as a control. Scale bars: (A, C and E) 50 µm, (B, D and F) 100 µm.

Mentions: We used a Lpr2-specific antibody to examine the distribution of Lpr2 in wild-type egg chambers, detecting Lpr2 protein at the membranes of nurse cells and oocytes in vitellogenic egg chambers (Figure 2C–2E). This expression was low at the beginning of vitellogenesis (stage 8, Figure 2C asterisk) and increased as the egg chamber matured, being maximal at stage 11 (Figure 2E). In situ hybridization detected a similar expression pattern for lpr2 transcripts (Figure 2B). Together, these results demonstrate that Lpr2 is the major receptor involved in the uptake of neutral lipids by nurse cells and oocytes. In contrast, our results indicate that lpr1 is not essential for this process since egg chambers from Df(3R)lpr1 females had normal amounts of neutral lipids (Figure 2L, Figure S3A). However, lpr1 appears to play some role in oogenesis since Df(3R)lpr1/2 females, which lack both receptors, exhibited stronger and qualitatively distinct phenotypes compared to Df(3R)lpr2 mutants. Ovaries from Df(3R)lpr1/2 females were severely reduced in size and contained abundant cellular debris. DAPI staining revealed condensation and fragmentation of the nurse cell nuclei at stages 9–10, indicating cell degeneration (Figure 2O, compare to Figure 2N. Figure S3B), a phenotype not observed in Df(3R)lpr2 females, explaining the egg laying phenotype described for these mutants earlier. Starvation and other adverse stimuli are known to activate an oogenesis checkpoint that results in the apoptosis of egg chambers at mid-oogenesis [24]. Thus, the degeneration of Df(3R)lpr1/2 ovaries probably results from activation of this checkpoint. As expected, the few non-degenerating stage 10 egg chambers that can be found in young Df(3R)lpr1/2 females had extremely low lipid content (Figure 2K). These results indicate that lpr1 has a partially redundant function during oogenesis, which was only revealed in the absence of lpr2. Accordingly, we detected lpr1 expression in nurse cells and follicle cells by in situ hybridization and RT-PCR (Figure 2A, Figure 5G) but not by antibody staining (not shown), suggesting that Lrp1 protein levels might be low. Oogenesis is a complex process regulated by hormonal signals that relay information about the nutritional status of the female and other stimuli [25]. Thus, it is conceivable that mutations in lpr1 and lpr2 could affect oogenesis, at least in part, by altering the hormonal or nutritional status of the female. To address this point, we eliminated lpr1 and lpr2 exclusively in the oocyte and nurse cells by generating Df(3R)lpr1/2 germ-line clones. The resulting females were sterile but laid abundant non-viable eggs, the egg chambers had low lipid content (Figure 2M) and no signs of degeneration were observed at mid oogenesis (not shown). Taken together, these experiments indicated that lpr2 and to a minor extent lpr1, are autonomously required in the oocyte and nurse cells to mediate lipid uptake during vitellogenesis. In addition, they suggest that somatic expression of the lipophorin receptors contributes to the regulation of the mid-oogenesis checkpoint.


Drosophila lipophorin receptors mediate the uptake of neutral lipids in oocytes and imaginal disc cells by an endocytosis-independent mechanism.

Parra-Peralbo E, Culi J - PLoS Genet. (2011)

Germ-line expression of Lpr1J or Lpr2E rescues the oogenesis phenotypes of lpr1-, lpr2− double mutant females.(A–F) Df(3R)lpr1/2 egg chambers overexpressing the lipid uptake-defective UASp-lpr2F isoform (A–B) or the lipid uptake-promoting UASp-lpr2E (C–D) or UASp-lpr1J (E–F) isoforms in the germ-line driven by V32-gal4. (A,C,E) Lpr2F (A), Lpr2E (C) and Lpr1J (E) proteins were detected in the nurse cells (n) and oocyte (o) membranes using an α-HA antibody (arrowheads, shown in green). Egg chambers were stained with the lipophilic nile red dye (red) and DAPI (blue) to mark nuclei. (B, D, F) Ovarioles containing multiple egg chambers stained with DAPI. Several degenerating egg chambers are labeled with a bracket in (B). Note that germ-line specific expression of UASp-lpr2E and UASp-lpr1J but not of UASp-lpr2F induced accumulation of neutral lipids in nurse cells (arrows in C and E) and rescued mid-oogenesis degeneration (D and F, compare also with the wild-type and Df(3R)lpr1/2 egg chambers in Figure 2). (G) Semiquantitative RT-PCR showing the relative abundance of lpr1 and lpr2 transcripts in several tissues, as indicated (wing imaginal discs, third instar larva fat body, ovary, adult fat body, adult brain and whole female fly). The oligo pairs used discriminated between isoforms transcribed from the proximal promoter (PP) and from distal promoter (DP). Analysis of ribosomal protein rp49 transcription was included as a control. Scale bars: (A, C and E) 50 µm, (B, D and F) 100 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3037410&req=5

pgen-1001297-g005: Germ-line expression of Lpr1J or Lpr2E rescues the oogenesis phenotypes of lpr1-, lpr2− double mutant females.(A–F) Df(3R)lpr1/2 egg chambers overexpressing the lipid uptake-defective UASp-lpr2F isoform (A–B) or the lipid uptake-promoting UASp-lpr2E (C–D) or UASp-lpr1J (E–F) isoforms in the germ-line driven by V32-gal4. (A,C,E) Lpr2F (A), Lpr2E (C) and Lpr1J (E) proteins were detected in the nurse cells (n) and oocyte (o) membranes using an α-HA antibody (arrowheads, shown in green). Egg chambers were stained with the lipophilic nile red dye (red) and DAPI (blue) to mark nuclei. (B, D, F) Ovarioles containing multiple egg chambers stained with DAPI. Several degenerating egg chambers are labeled with a bracket in (B). Note that germ-line specific expression of UASp-lpr2E and UASp-lpr1J but not of UASp-lpr2F induced accumulation of neutral lipids in nurse cells (arrows in C and E) and rescued mid-oogenesis degeneration (D and F, compare also with the wild-type and Df(3R)lpr1/2 egg chambers in Figure 2). (G) Semiquantitative RT-PCR showing the relative abundance of lpr1 and lpr2 transcripts in several tissues, as indicated (wing imaginal discs, third instar larva fat body, ovary, adult fat body, adult brain and whole female fly). The oligo pairs used discriminated between isoforms transcribed from the proximal promoter (PP) and from distal promoter (DP). Analysis of ribosomal protein rp49 transcription was included as a control. Scale bars: (A, C and E) 50 µm, (B, D and F) 100 µm.
Mentions: We used a Lpr2-specific antibody to examine the distribution of Lpr2 in wild-type egg chambers, detecting Lpr2 protein at the membranes of nurse cells and oocytes in vitellogenic egg chambers (Figure 2C–2E). This expression was low at the beginning of vitellogenesis (stage 8, Figure 2C asterisk) and increased as the egg chamber matured, being maximal at stage 11 (Figure 2E). In situ hybridization detected a similar expression pattern for lpr2 transcripts (Figure 2B). Together, these results demonstrate that Lpr2 is the major receptor involved in the uptake of neutral lipids by nurse cells and oocytes. In contrast, our results indicate that lpr1 is not essential for this process since egg chambers from Df(3R)lpr1 females had normal amounts of neutral lipids (Figure 2L, Figure S3A). However, lpr1 appears to play some role in oogenesis since Df(3R)lpr1/2 females, which lack both receptors, exhibited stronger and qualitatively distinct phenotypes compared to Df(3R)lpr2 mutants. Ovaries from Df(3R)lpr1/2 females were severely reduced in size and contained abundant cellular debris. DAPI staining revealed condensation and fragmentation of the nurse cell nuclei at stages 9–10, indicating cell degeneration (Figure 2O, compare to Figure 2N. Figure S3B), a phenotype not observed in Df(3R)lpr2 females, explaining the egg laying phenotype described for these mutants earlier. Starvation and other adverse stimuli are known to activate an oogenesis checkpoint that results in the apoptosis of egg chambers at mid-oogenesis [24]. Thus, the degeneration of Df(3R)lpr1/2 ovaries probably results from activation of this checkpoint. As expected, the few non-degenerating stage 10 egg chambers that can be found in young Df(3R)lpr1/2 females had extremely low lipid content (Figure 2K). These results indicate that lpr1 has a partially redundant function during oogenesis, which was only revealed in the absence of lpr2. Accordingly, we detected lpr1 expression in nurse cells and follicle cells by in situ hybridization and RT-PCR (Figure 2A, Figure 5G) but not by antibody staining (not shown), suggesting that Lrp1 protein levels might be low. Oogenesis is a complex process regulated by hormonal signals that relay information about the nutritional status of the female and other stimuli [25]. Thus, it is conceivable that mutations in lpr1 and lpr2 could affect oogenesis, at least in part, by altering the hormonal or nutritional status of the female. To address this point, we eliminated lpr1 and lpr2 exclusively in the oocyte and nurse cells by generating Df(3R)lpr1/2 germ-line clones. The resulting females were sterile but laid abundant non-viable eggs, the egg chambers had low lipid content (Figure 2M) and no signs of degeneration were observed at mid oogenesis (not shown). Taken together, these experiments indicated that lpr2 and to a minor extent lpr1, are autonomously required in the oocyte and nurse cells to mediate lipid uptake during vitellogenesis. In addition, they suggest that somatic expression of the lipophorin receptors contributes to the regulation of the mid-oogenesis checkpoint.

Bottom Line: Furthermore, our data indicate that endocytosis of the lipophorin receptors is not required to mediate the uptake of neutral lipids.These findings suggest a model where lipophorin receptors promote the extracellular lipolysis of lipophorins.This model is reminiscent of the lipolytic processing of triglyceride-rich lipoproteins that occurs at the mammalian capillary endothelium, suggesting an ancient role for LDLR-like proteins in this process.

View Article: PubMed Central - PubMed

Affiliation: Centro Andaluz de Biología del Desarrollo (CSIC-UPO), Universidad Pablo de Olavide, Sevilla, Spain.

ABSTRACT
Lipids are constantly shuttled through the body to redistribute energy and metabolites between sites of absorption, storage, and catabolism in a complex homeostatic equilibrium. In Drosophila, lipids are transported through the hemolymph in the form of lipoprotein particles, known as lipophorins. The mechanisms by which cells interact with circulating lipophorins and acquire their lipidic cargo are poorly understood. We have found that lipophorin receptor 1 and 2 (lpr1 and lpr2), two partially redundant genes belonging to the Low Density Lipoprotein Receptor (LDLR) family, are essential for the efficient uptake and accumulation of neutral lipids by oocytes and cells of the imaginal discs. Females lacking the lpr2 gene lay eggs with low lipid content and have reduced fertility, revealing a central role for lpr2 in mediating Drosophila vitellogenesis. lpr1 and lpr2 are transcribed into multiple isoforms. Interestingly, only a subset of these isoforms containing a particular LDLR type A module mediate neutral lipid uptake. Expression of these isoforms induces the extracellular stabilization of lipophorins. Furthermore, our data indicate that endocytosis of the lipophorin receptors is not required to mediate the uptake of neutral lipids. These findings suggest a model where lipophorin receptors promote the extracellular lipolysis of lipophorins. This model is reminiscent of the lipolytic processing of triglyceride-rich lipoproteins that occurs at the mammalian capillary endothelium, suggesting an ancient role for LDLR-like proteins in this process.

Show MeSH
Related in: MedlinePlus