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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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Neutral lipid uptake does not require endocytosis.(A–B) Wing imaginal disc of Df(3R)lpr1/2 genotype expressing UAS-lpr2E in the posterior compartment driven by en-gal4. Neutral lipids were revealed by nile red staining (red). Lpr2E distribution was detected with α-HA antibody (green). Lipophorin distribution is shown in magenta to mark cell outlines. The wing imaginal disc is shown in a cross-section in (A), with the apical side at the top and the basal at the bottom. A basal section of the same disc is shown in (B). Note that Lpr2E expressing cells and two rows of adjacent non-expressing anterior cells (arrows) accumulate high levels of lipid droplets in a basal location. A line marks the limit of Lpr2E expression. Nile red staining is also shown in a separate panel (B'). (C–E) Detail of the somatic follicle epithelium (f) wrapping around the oocyte (o) in a stage 10 egg chamber dissected from a Df(3R)lpr1/2 female (C) and similar Df(3R)lpr1/2 mutant egg chambers overexpressing UASp-lpr2E in the germ-line driven by V32-gal4 (D) or UAS-lpr1J in the follicular epithelium driven by CY2-gal4 (E). Lpr2E and Lpr1J were detected with an α-HA antibody (green). The oocyte membrane was marked with a dotted line. Overexpressed proteins accumulated at the oocyte membrane (arrow in D) and at the apical region of the follicle cells (arrow in E). DAPI marks the cell nuclei (blue). Lipids are shown in grey as a separate channel (C', D' and E'). Note that expression of UASp-lpr2E in the nurse cells and oocyte induces an autonomous increase in lipid accumulation in these cells and also a non-autonomous rescue in the adjacent follicular epithelium (yellow arrows in D'). Expression of Lpr1J in the follicular epithelium rescues lipid accumulation in these cells to a similar extent (yellow arrows in E'). Note that the Df(3R)lpr1/2 egg chamber shown in (C) was dissected from young females in which a few egg chambers from each ovary escaped mid oogenesis degeneration. (F–G) Egg chambers stained with the lipophilic dye nile red (red) and DAPI (blue) to mark cell nuclei. (F) Wild-type genotype. Note the accumulation of lipids (arrow) in the nurse cells (n) and the auto-fluorescence of yolk proteins (Y) (in F') in the DAPI channel within the oocyte (o). (G) rab5 mutant egg chamber. Lipids accumulate in the nurse cells (arrow) similarly to the wild-type but no yolk proteins are present in the oocyte (G'), confirming that endocytosis is blocked in the rab5 genetic background. Scale bars: 10 µm (A–B), 20 µm (C–E), 100 µm (F–G).
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pgen-1001297-g006: Neutral lipid uptake does not require endocytosis.(A–B) Wing imaginal disc of Df(3R)lpr1/2 genotype expressing UAS-lpr2E in the posterior compartment driven by en-gal4. Neutral lipids were revealed by nile red staining (red). Lpr2E distribution was detected with α-HA antibody (green). Lipophorin distribution is shown in magenta to mark cell outlines. The wing imaginal disc is shown in a cross-section in (A), with the apical side at the top and the basal at the bottom. A basal section of the same disc is shown in (B). Note that Lpr2E expressing cells and two rows of adjacent non-expressing anterior cells (arrows) accumulate high levels of lipid droplets in a basal location. A line marks the limit of Lpr2E expression. Nile red staining is also shown in a separate panel (B'). (C–E) Detail of the somatic follicle epithelium (f) wrapping around the oocyte (o) in a stage 10 egg chamber dissected from a Df(3R)lpr1/2 female (C) and similar Df(3R)lpr1/2 mutant egg chambers overexpressing UASp-lpr2E in the germ-line driven by V32-gal4 (D) or UAS-lpr1J in the follicular epithelium driven by CY2-gal4 (E). Lpr2E and Lpr1J were detected with an α-HA antibody (green). The oocyte membrane was marked with a dotted line. Overexpressed proteins accumulated at the oocyte membrane (arrow in D) and at the apical region of the follicle cells (arrow in E). DAPI marks the cell nuclei (blue). Lipids are shown in grey as a separate channel (C', D' and E'). Note that expression of UASp-lpr2E in the nurse cells and oocyte induces an autonomous increase in lipid accumulation in these cells and also a non-autonomous rescue in the adjacent follicular epithelium (yellow arrows in D'). Expression of Lpr1J in the follicular epithelium rescues lipid accumulation in these cells to a similar extent (yellow arrows in E'). Note that the Df(3R)lpr1/2 egg chamber shown in (C) was dissected from young females in which a few egg chambers from each ovary escaped mid oogenesis degeneration. (F–G) Egg chambers stained with the lipophilic dye nile red (red) and DAPI (blue) to mark cell nuclei. (F) Wild-type genotype. Note the accumulation of lipids (arrow) in the nurse cells (n) and the auto-fluorescence of yolk proteins (Y) (in F') in the DAPI channel within the oocyte (o). (G) rab5 mutant egg chamber. Lipids accumulate in the nurse cells (arrow) similarly to the wild-type but no yolk proteins are present in the oocyte (G'), confirming that endocytosis is blocked in the rab5 genetic background. Scale bars: 10 µm (A–B), 20 µm (C–E), 100 µm (F–G).

Mentions: During the rescue experiments described in the previous sections, we realized that expression of UAS-lpr2E in the posterior compartment driven by en-gal4 not only autonomously rescued lipid uptake in that compartment but also promoted the formation of lipid droplets in a one to two cells wide region of anterior tissue abutting the anterior-posterior compartment border (Figure 6B, arrow). Analysis of confocal sections spanning the thickness of the imaginal disc confirmed that cells not expressing UAS-lpr2E but adjacent to expressing cells, accumulated higher levels of lipid droplets than more anteriorly located cells (Figure 6A, arrow). Similar results were obtained using a different Gal4 driver line to express UAS-lpr2E in the dorsal wing disc compartment (Figure S5C, S5D). To analyze whether non-autonomous lipid uptake could be detected in other tissues, we returned to the egg chamber because of its particular morphology. In the Drosophila egg chamber, the oocyte and nurse cells are surrounded by a closely associated somatic follicular epithelium. This allowed us to examine whether expression of the lipophorin receptors in the germ-line could non-autonomously direct lipid uptake in the follicular epithelium. Follicle cells of ovaries dissected from Df(3R)lpr1/2 females had very few lipid droplets (Figure 6C). However, after V32-gal4-mediated expression of UASp-lpr2E exclusively in the germ-line, we observed a remarkable increase in the number and size of lipid droplets in the follicular epithelium (Figure 6D). In fact, the rescue was similar to the one obtained by the expression of the lipid uptake-promoting UAS-lpr1J isoform directly in the follicle cells using the follicle cell driver CY2-gal4 (Figure 6E). These results indicated that Lpr2E can non-autonomously promote neutral lipid uptake in adjacent cells.


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)

Neutral lipid uptake does not require endocytosis.(A–B) Wing imaginal disc of Df(3R)lpr1/2 genotype expressing UAS-lpr2E in the posterior compartment driven by en-gal4. Neutral lipids were revealed by nile red staining (red). Lpr2E distribution was detected with α-HA antibody (green). Lipophorin distribution is shown in magenta to mark cell outlines. The wing imaginal disc is shown in a cross-section in (A), with the apical side at the top and the basal at the bottom. A basal section of the same disc is shown in (B). Note that Lpr2E expressing cells and two rows of adjacent non-expressing anterior cells (arrows) accumulate high levels of lipid droplets in a basal location. A line marks the limit of Lpr2E expression. Nile red staining is also shown in a separate panel (B'). (C–E) Detail of the somatic follicle epithelium (f) wrapping around the oocyte (o) in a stage 10 egg chamber dissected from a Df(3R)lpr1/2 female (C) and similar Df(3R)lpr1/2 mutant egg chambers overexpressing UASp-lpr2E in the germ-line driven by V32-gal4 (D) or UAS-lpr1J in the follicular epithelium driven by CY2-gal4 (E). Lpr2E and Lpr1J were detected with an α-HA antibody (green). The oocyte membrane was marked with a dotted line. Overexpressed proteins accumulated at the oocyte membrane (arrow in D) and at the apical region of the follicle cells (arrow in E). DAPI marks the cell nuclei (blue). Lipids are shown in grey as a separate channel (C', D' and E'). Note that expression of UASp-lpr2E in the nurse cells and oocyte induces an autonomous increase in lipid accumulation in these cells and also a non-autonomous rescue in the adjacent follicular epithelium (yellow arrows in D'). Expression of Lpr1J in the follicular epithelium rescues lipid accumulation in these cells to a similar extent (yellow arrows in E'). Note that the Df(3R)lpr1/2 egg chamber shown in (C) was dissected from young females in which a few egg chambers from each ovary escaped mid oogenesis degeneration. (F–G) Egg chambers stained with the lipophilic dye nile red (red) and DAPI (blue) to mark cell nuclei. (F) Wild-type genotype. Note the accumulation of lipids (arrow) in the nurse cells (n) and the auto-fluorescence of yolk proteins (Y) (in F') in the DAPI channel within the oocyte (o). (G) rab5 mutant egg chamber. Lipids accumulate in the nurse cells (arrow) similarly to the wild-type but no yolk proteins are present in the oocyte (G'), confirming that endocytosis is blocked in the rab5 genetic background. Scale bars: 10 µm (A–B), 20 µm (C–E), 100 µm (F–G).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037410&req=5

pgen-1001297-g006: Neutral lipid uptake does not require endocytosis.(A–B) Wing imaginal disc of Df(3R)lpr1/2 genotype expressing UAS-lpr2E in the posterior compartment driven by en-gal4. Neutral lipids were revealed by nile red staining (red). Lpr2E distribution was detected with α-HA antibody (green). Lipophorin distribution is shown in magenta to mark cell outlines. The wing imaginal disc is shown in a cross-section in (A), with the apical side at the top and the basal at the bottom. A basal section of the same disc is shown in (B). Note that Lpr2E expressing cells and two rows of adjacent non-expressing anterior cells (arrows) accumulate high levels of lipid droplets in a basal location. A line marks the limit of Lpr2E expression. Nile red staining is also shown in a separate panel (B'). (C–E) Detail of the somatic follicle epithelium (f) wrapping around the oocyte (o) in a stage 10 egg chamber dissected from a Df(3R)lpr1/2 female (C) and similar Df(3R)lpr1/2 mutant egg chambers overexpressing UASp-lpr2E in the germ-line driven by V32-gal4 (D) or UAS-lpr1J in the follicular epithelium driven by CY2-gal4 (E). Lpr2E and Lpr1J were detected with an α-HA antibody (green). The oocyte membrane was marked with a dotted line. Overexpressed proteins accumulated at the oocyte membrane (arrow in D) and at the apical region of the follicle cells (arrow in E). DAPI marks the cell nuclei (blue). Lipids are shown in grey as a separate channel (C', D' and E'). Note that expression of UASp-lpr2E in the nurse cells and oocyte induces an autonomous increase in lipid accumulation in these cells and also a non-autonomous rescue in the adjacent follicular epithelium (yellow arrows in D'). Expression of Lpr1J in the follicular epithelium rescues lipid accumulation in these cells to a similar extent (yellow arrows in E'). Note that the Df(3R)lpr1/2 egg chamber shown in (C) was dissected from young females in which a few egg chambers from each ovary escaped mid oogenesis degeneration. (F–G) Egg chambers stained with the lipophilic dye nile red (red) and DAPI (blue) to mark cell nuclei. (F) Wild-type genotype. Note the accumulation of lipids (arrow) in the nurse cells (n) and the auto-fluorescence of yolk proteins (Y) (in F') in the DAPI channel within the oocyte (o). (G) rab5 mutant egg chamber. Lipids accumulate in the nurse cells (arrow) similarly to the wild-type but no yolk proteins are present in the oocyte (G'), confirming that endocytosis is blocked in the rab5 genetic background. Scale bars: 10 µm (A–B), 20 µm (C–E), 100 µm (F–G).
Mentions: During the rescue experiments described in the previous sections, we realized that expression of UAS-lpr2E in the posterior compartment driven by en-gal4 not only autonomously rescued lipid uptake in that compartment but also promoted the formation of lipid droplets in a one to two cells wide region of anterior tissue abutting the anterior-posterior compartment border (Figure 6B, arrow). Analysis of confocal sections spanning the thickness of the imaginal disc confirmed that cells not expressing UAS-lpr2E but adjacent to expressing cells, accumulated higher levels of lipid droplets than more anteriorly located cells (Figure 6A, arrow). Similar results were obtained using a different Gal4 driver line to express UAS-lpr2E in the dorsal wing disc compartment (Figure S5C, S5D). To analyze whether non-autonomous lipid uptake could be detected in other tissues, we returned to the egg chamber because of its particular morphology. In the Drosophila egg chamber, the oocyte and nurse cells are surrounded by a closely associated somatic follicular epithelium. This allowed us to examine whether expression of the lipophorin receptors in the germ-line could non-autonomously direct lipid uptake in the follicular epithelium. Follicle cells of ovaries dissected from Df(3R)lpr1/2 females had very few lipid droplets (Figure 6C). However, after V32-gal4-mediated expression of UASp-lpr2E exclusively in the germ-line, we observed a remarkable increase in the number and size of lipid droplets in the follicular epithelium (Figure 6D). In fact, the rescue was similar to the one obtained by the expression of the lipid uptake-promoting UAS-lpr1J isoform directly in the follicle cells using the follicle cell driver CY2-gal4 (Figure 6E). These results indicated that Lpr2E can non-autonomously promote neutral lipid uptake in adjacent cells.

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