Limits...
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.

Show MeSH

Related in: MedlinePlus

Lpr2 is required for the uptake of neutral lipids during Drosophila vitellogenesis.(A–B) lpr1 (A) and lpr2 (B) expression in wild-type ovarioles detected by in situ hybridization. Transcripts of both genes were first visible in the nurse cells (n) of stage 8 egg chambers (asterisk) and their levels increased thereafter. lrp1 transcripts were also detected in the follicle cells of mature egg chambers (A, arrow in inset). (C–E) Immunostaining showing Lpr2 protein localization during oogenesis. Lpr2 is first detected at low levels in stage 8 egg chambers, coinciding with the start of vitellogenesis (asterisks in C). (D) Magnification of a stage 10 egg chamber showing expression at the nurse cells (n) and oocyte (o) membranes. Maximal expression was detected at stage 11 egg chambers (E and E', two focal planes). (F–M) Nile red staining of egg chambers (F–H and J–M) and one blastoderm stage embryos (I) to reveal lipid droplets (yellow, nile red dye fluorescence was captured in the green and red channels). (F–I) Wild-type (wt) genotype. Neutral lipids start to accumulate at vitellogenic stages and reach a maximum in blastoderm embryos. Note that near the end of vitellogenesis, nurse cells degenerate and dump their content into the oocyte. (F) stage 9 (asterisk), (G) stage 10 and (H) stage 11 egg chambers. (J–M) stage 10 egg chambers of the indicated genotypes. Accumulation of neutral lipids is reduced in Df(3R)lpr2 (J), Df(3R)lpr1/2 (K, egg chamber outlined) and Df(3R)lpr1/2 germ-line clones (M) and is normal in Df(3R)lpr1 egg chambers (L). The Df(3R)lpr1/2 egg chamber shown in (K) was dissected from young females in which a few egg chambers in each ovary escaped degeneration at mid oogenesis. (N, O) Egg chambers of wild-type (N) and Df(3R)lpr1/2 (O) ovaries stained with DAPI to reveal the nuclei. Several stage 10 egg chambers display nuclear fragmentation in Df(3R)lpr1/2 females (arrows in O). Scale bars: 100 µm. (F–M) shown at the same magnification.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3037410&req=5

pgen-1001297-g002: Lpr2 is required for the uptake of neutral lipids during Drosophila vitellogenesis.(A–B) lpr1 (A) and lpr2 (B) expression in wild-type ovarioles detected by in situ hybridization. Transcripts of both genes were first visible in the nurse cells (n) of stage 8 egg chambers (asterisk) and their levels increased thereafter. lrp1 transcripts were also detected in the follicle cells of mature egg chambers (A, arrow in inset). (C–E) Immunostaining showing Lpr2 protein localization during oogenesis. Lpr2 is first detected at low levels in stage 8 egg chambers, coinciding with the start of vitellogenesis (asterisks in C). (D) Magnification of a stage 10 egg chamber showing expression at the nurse cells (n) and oocyte (o) membranes. Maximal expression was detected at stage 11 egg chambers (E and E', two focal planes). (F–M) Nile red staining of egg chambers (F–H and J–M) and one blastoderm stage embryos (I) to reveal lipid droplets (yellow, nile red dye fluorescence was captured in the green and red channels). (F–I) Wild-type (wt) genotype. Neutral lipids start to accumulate at vitellogenic stages and reach a maximum in blastoderm embryos. Note that near the end of vitellogenesis, nurse cells degenerate and dump their content into the oocyte. (F) stage 9 (asterisk), (G) stage 10 and (H) stage 11 egg chambers. (J–M) stage 10 egg chambers of the indicated genotypes. Accumulation of neutral lipids is reduced in Df(3R)lpr2 (J), Df(3R)lpr1/2 (K, egg chamber outlined) and Df(3R)lpr1/2 germ-line clones (M) and is normal in Df(3R)lpr1 egg chambers (L). The Df(3R)lpr1/2 egg chamber shown in (K) was dissected from young females in which a few egg chambers in each ovary escaped degeneration at mid oogenesis. (N, O) Egg chambers of wild-type (N) and Df(3R)lpr1/2 (O) ovaries stained with DAPI to reveal the nuclei. Several stage 10 egg chambers display nuclear fragmentation in Df(3R)lpr1/2 females (arrows in O). Scale bars: 100 µm. (F–M) shown at the same magnification.

Mentions: To examine the role of the lipophorin receptors in lipid metabolism, we first generated three small deletions in the lpr1-lpr2 genomic region (Figure 1A). Df(3R)lpr1 completely deletes lpr1; Df(3R)lpr2 deletes exons 1 to 8 of lpr2, including the promoters and translation initiation codons, while Df(3R)lpr1/2 affects both genes. The breakpoints for each deficiency were confirmed by PCR analysis. Even though several lpr2 exons are still present in Df(3R)lpr2 and Df(3R)lpr1/2 chromosomes, we did not detect Lpr2 protein expression in either deficiency using an antibody which recognizes Lpr2 intracellular domain, which is common to all Lpr2 isoforms (Figure S2C–S2F and not shown). This data strongly suggests that Df(3R)lpr2 is a allele for lpr2 and Df(3R)lpr1/2 is a allele for both lpr1 and lpr2. Flies with mutations in individual lipophorin receptor genes as well as the double mutant Df(3R)lpr1/2 were homozygous viable and displayed fertility phenotypes: Df(3R)lpr1 females laid eggs which hatched at rates similar to wild-type females (85.5% hatching rate for Df(3R)lpr1 compared to 87.5% for the wild-type stock Oregon R, n = 200). Df(3R)lpr2 females laid eggs but most of them failed to hatch (0.5% hatching rate; n = 200). Df(3R)lpr1/2 females were completely sterile, where the few eggs laid by young flies failed to hatch. These results indicate that lpr2, and to a lesser extent lpr1, are required for normal oogenesis. The Drosophila ovarian follicle is composed of a 16-cell germ-line cyst with one oocyte and 15 nurse cells, which is surrounded by somatic follicle cells. During vitellogenesis, the oocyte and nurse cells increase in volume and accumulate large amounts of yolk proteins and neutral lipids (Figure 2F–2I) that are captured from the surrounding hemolymph [21]. The Yolkless (Yl) receptor mediates the endocytic uptake of yolk proteins [22], [23]. However, no receptor involved in lipid uptake has been reported. To analyze whether lpr1 or lpr2 mediate lipid uptake during vitellogenesis, we first examined the lipid content of ovaries from wild-type and Df(3R)lpr2 females with the lipophilic nile red dye. Accumulation of neutral lipids was first visible in stage 9 wild-type egg chambers and reached a maximum by the end of vitellogenesis at stage 11 (Figure 2F–2I). A marked decrease in lipid droplets was observed in Df(3R)lpr2 egg chambers (Figure 2J, compare to Figure 2G. Figure S3A) or when this deficiency was combined with the double mutant Df(3R)lpr1/2 (not shown). Most of the embryos originating from Df(3R)lpr2 mutant oocytes could not complete embryogenesis and died at various stages of development, showing generalized apoptosis and pleiotropic phenotypes such as muscle detachment and nervous system malformations (not shown). A small number of Df(3R)lpr2 egg chambers exhibited higher lipid levels (Figure S3A). It seems likely that the few embryos that successfully hatched from Df(3R)lpr2 females (0.5%) were derived from these egg chambers.


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)

Lpr2 is required for the uptake of neutral lipids during Drosophila vitellogenesis.(A–B) lpr1 (A) and lpr2 (B) expression in wild-type ovarioles detected by in situ hybridization. Transcripts of both genes were first visible in the nurse cells (n) of stage 8 egg chambers (asterisk) and their levels increased thereafter. lrp1 transcripts were also detected in the follicle cells of mature egg chambers (A, arrow in inset). (C–E) Immunostaining showing Lpr2 protein localization during oogenesis. Lpr2 is first detected at low levels in stage 8 egg chambers, coinciding with the start of vitellogenesis (asterisks in C). (D) Magnification of a stage 10 egg chamber showing expression at the nurse cells (n) and oocyte (o) membranes. Maximal expression was detected at stage 11 egg chambers (E and E', two focal planes). (F–M) Nile red staining of egg chambers (F–H and J–M) and one blastoderm stage embryos (I) to reveal lipid droplets (yellow, nile red dye fluorescence was captured in the green and red channels). (F–I) Wild-type (wt) genotype. Neutral lipids start to accumulate at vitellogenic stages and reach a maximum in blastoderm embryos. Note that near the end of vitellogenesis, nurse cells degenerate and dump their content into the oocyte. (F) stage 9 (asterisk), (G) stage 10 and (H) stage 11 egg chambers. (J–M) stage 10 egg chambers of the indicated genotypes. Accumulation of neutral lipids is reduced in Df(3R)lpr2 (J), Df(3R)lpr1/2 (K, egg chamber outlined) and Df(3R)lpr1/2 germ-line clones (M) and is normal in Df(3R)lpr1 egg chambers (L). The Df(3R)lpr1/2 egg chamber shown in (K) was dissected from young females in which a few egg chambers in each ovary escaped degeneration at mid oogenesis. (N, O) Egg chambers of wild-type (N) and Df(3R)lpr1/2 (O) ovaries stained with DAPI to reveal the nuclei. Several stage 10 egg chambers display nuclear fragmentation in Df(3R)lpr1/2 females (arrows in O). Scale bars: 100 µm. (F–M) shown at the same magnification.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1001297-g002: Lpr2 is required for the uptake of neutral lipids during Drosophila vitellogenesis.(A–B) lpr1 (A) and lpr2 (B) expression in wild-type ovarioles detected by in situ hybridization. Transcripts of both genes were first visible in the nurse cells (n) of stage 8 egg chambers (asterisk) and their levels increased thereafter. lrp1 transcripts were also detected in the follicle cells of mature egg chambers (A, arrow in inset). (C–E) Immunostaining showing Lpr2 protein localization during oogenesis. Lpr2 is first detected at low levels in stage 8 egg chambers, coinciding with the start of vitellogenesis (asterisks in C). (D) Magnification of a stage 10 egg chamber showing expression at the nurse cells (n) and oocyte (o) membranes. Maximal expression was detected at stage 11 egg chambers (E and E', two focal planes). (F–M) Nile red staining of egg chambers (F–H and J–M) and one blastoderm stage embryos (I) to reveal lipid droplets (yellow, nile red dye fluorescence was captured in the green and red channels). (F–I) Wild-type (wt) genotype. Neutral lipids start to accumulate at vitellogenic stages and reach a maximum in blastoderm embryos. Note that near the end of vitellogenesis, nurse cells degenerate and dump their content into the oocyte. (F) stage 9 (asterisk), (G) stage 10 and (H) stage 11 egg chambers. (J–M) stage 10 egg chambers of the indicated genotypes. Accumulation of neutral lipids is reduced in Df(3R)lpr2 (J), Df(3R)lpr1/2 (K, egg chamber outlined) and Df(3R)lpr1/2 germ-line clones (M) and is normal in Df(3R)lpr1 egg chambers (L). The Df(3R)lpr1/2 egg chamber shown in (K) was dissected from young females in which a few egg chambers in each ovary escaped degeneration at mid oogenesis. (N, O) Egg chambers of wild-type (N) and Df(3R)lpr1/2 (O) ovaries stained with DAPI to reveal the nuclei. Several stage 10 egg chambers display nuclear fragmentation in Df(3R)lpr1/2 females (arrows in O). Scale bars: 100 µm. (F–M) shown at the same magnification.
Mentions: To examine the role of the lipophorin receptors in lipid metabolism, we first generated three small deletions in the lpr1-lpr2 genomic region (Figure 1A). Df(3R)lpr1 completely deletes lpr1; Df(3R)lpr2 deletes exons 1 to 8 of lpr2, including the promoters and translation initiation codons, while Df(3R)lpr1/2 affects both genes. The breakpoints for each deficiency were confirmed by PCR analysis. Even though several lpr2 exons are still present in Df(3R)lpr2 and Df(3R)lpr1/2 chromosomes, we did not detect Lpr2 protein expression in either deficiency using an antibody which recognizes Lpr2 intracellular domain, which is common to all Lpr2 isoforms (Figure S2C–S2F and not shown). This data strongly suggests that Df(3R)lpr2 is a allele for lpr2 and Df(3R)lpr1/2 is a allele for both lpr1 and lpr2. Flies with mutations in individual lipophorin receptor genes as well as the double mutant Df(3R)lpr1/2 were homozygous viable and displayed fertility phenotypes: Df(3R)lpr1 females laid eggs which hatched at rates similar to wild-type females (85.5% hatching rate for Df(3R)lpr1 compared to 87.5% for the wild-type stock Oregon R, n = 200). Df(3R)lpr2 females laid eggs but most of them failed to hatch (0.5% hatching rate; n = 200). Df(3R)lpr1/2 females were completely sterile, where the few eggs laid by young flies failed to hatch. These results indicate that lpr2, and to a lesser extent lpr1, are required for normal oogenesis. The Drosophila ovarian follicle is composed of a 16-cell germ-line cyst with one oocyte and 15 nurse cells, which is surrounded by somatic follicle cells. During vitellogenesis, the oocyte and nurse cells increase in volume and accumulate large amounts of yolk proteins and neutral lipids (Figure 2F–2I) that are captured from the surrounding hemolymph [21]. The Yolkless (Yl) receptor mediates the endocytic uptake of yolk proteins [22], [23]. However, no receptor involved in lipid uptake has been reported. To analyze whether lpr1 or lpr2 mediate lipid uptake during vitellogenesis, we first examined the lipid content of ovaries from wild-type and Df(3R)lpr2 females with the lipophilic nile red dye. Accumulation of neutral lipids was first visible in stage 9 wild-type egg chambers and reached a maximum by the end of vitellogenesis at stage 11 (Figure 2F–2I). A marked decrease in lipid droplets was observed in Df(3R)lpr2 egg chambers (Figure 2J, compare to Figure 2G. Figure S3A) or when this deficiency was combined with the double mutant Df(3R)lpr1/2 (not shown). Most of the embryos originating from Df(3R)lpr2 mutant oocytes could not complete embryogenesis and died at various stages of development, showing generalized apoptosis and pleiotropic phenotypes such as muscle detachment and nervous system malformations (not shown). A small number of Df(3R)lpr2 egg chambers exhibited higher lipid levels (Figure S3A). It seems likely that the few embryos that successfully hatched from Df(3R)lpr2 females (0.5%) were derived from these egg chambers.

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