InteractiveFly: GeneBrief

apolipophorin: Biological Overview | References

BIOLOGICAL OVERVIEW

Wnt and Hedgehog family proteins are secreted signalling molecules (morphogens) that act at both long and short range to control growth and patterning during development. Both proteins are covalently modified by lipid, and the mechanism by which such hydrophobic molecules might spread over long distances is unknown. The Drosophila lipoprotein particle, Lipophorin, bears lipid-linked morphogens on its surface and is required for long-range signaling activity of Wingless and Hedgehog. Wingless, Hedgehog and glycophosphatidylinositol-linked proteins copurify with lipoprotein particles marked by lipophorin, and co-localize with these particles in the developing wing epithelium of Drosophila. In larvae with reduced lipoprotein levels, Hedgehog accumulates near its site of production, and fails to signal over its normal range. Similarly, the range of Wingless signalling is narrowed. A novel function is proposed for lipoprotein particles, in which they act as vehicles for the movement of lipid-linked morphogens and glycophosphatidylinositol-linked proteins (Panakova, 2005).

In the developing wing of Drosophila, Hedgehog activates short-range target gene expression up to five cells away from its source of production, and longer-range targets over more than twelve cell diameters. Wingless can signal through a range of over 30 cell diameters. These morphogens are anchored to the membrane via covalent lipid modification. The mechanisms that allow long-range movement of molecules with such strong membrane affinity are unclear (Panakova, 2005).

Like Wingless and Hedgehog, glycophosphatidylinositol (gpi)-linked proteins transfer between cells with their lipid anchor intact. Gpi-linked green fluorescent protein (GFP) expressed in Wingless-producing cells spreads into receiving tissue at the same rate as Wingless, where it co-localizes with Wingless in endosomes. Thus, it is proposed that these proteins travel together on a membranous particle, which has been called an argosome (Greco, 2001). How might argosomes form? One possibility is that argosomes are membranous exovesicles. Such particles could be generated by plasma membrane vesiculation, or by an exosome-related mechanism. Alternatively, argosomes might resemble lipoprotein particles like low-density lipoprotein (LDL). Vertebrate lipoprotein particles are scaffolded by apolipoproteins and comprise a phospholipid monolayer surrounding a core of esterified cholesterol and triglyceride. Insects construct similar particles called lipophorins (Arrese, 2001; van der Horst, 2002). Lipid-modified proteins of the exoplasmic face of the membrane (such as GFPgpi, Wingless or Hedgehog) might insert into the outer phospholipid monolayer of such a particle via their attached lipid moieties. This study use biochemical fractionation to determine the sort of particle with which lipid-linked proteins associate, and genetic means to address its function (Panakova, 2005).

Lipid-linked proteins copurify with lipophorin: Sedimentation of Wingless, Hedgehog and gpi-linked proteins were compared to that of transmembrane proteins, exosomes and lipophorin particles. To mark exosomes, flies were used expressing a vertebrate CD63:GFP fusion construct. CD63 is a tetraspanin that localizes to internal vesicles of multivesicular endosomes, and is released on exosomes (Escola, 1998; Wubbolts, 2003). In Drosophila imaginal discs, CD63:GFP localizes to late endosomes in producing cells, consistent with vertebrate studies. It is released and endocytosed by neighbouring cells between one and three cell diameters away, indicating that it is present on exosomes (Panakova, 2005).

To mark lipoprotein particles, antibodies were made to Drosophila apolipophorins I and II (ApoLI and ApoLII); these proteins are generated by cleavage of the precursor pro-Apolipophorin (Sundermeyer, 1996; Kutty, 1996). Lipophorin is produced in the fat body (Kutty, 1996); consistent with this, apolipophorin transcripts cannot be detected in imaginal discs. Nevertheless, the ApoLI and ApoLII proteins are as abundant in discs as in the fat body (Panakova, 2005).

Plasma membrane and exosomal markers are completely pelleted after centrifugation for 3 h at 120,000g, whereas most ApoLII remains in the supernatant. Most Wingless:GFP and Hedgehog is present in the pellet, as are the gpi-linked proteins Fasciclin, Connectin, Klingon and Acetylcholineasterase; this is not unexpected, because these proteins localize to the plasma membrane and internal membrane compartments. Surprisingly, however, some Wingless:GFP (6%), Hedgehog (2%) and gpi-linked proteins (14%-22%) remain in the supernatant (Panakova, 2005).

The 120,000g supernatant (S120) contains both free soluble proteins and lipoprotein particles. To separate them, isopycnic density centrifugation was performed. In these gradients, lipophorin moves to the top low-density fraction whereas soluble proteins are present in higher-density fractions. Gpi-linked proteins are found almost entirely in the top fraction with lipophorin. Treating the S120 with Phosphatidylinositol-specific phospholipase C (PI-PLC) before density centrifugation shifts their migration to higher-density fractions. This suggests that gpi-linked proteins associate with low-density particles via their gpi anchor (Panakova, 2005).

Similarly, when S120s from larvae that express Wingless:GFP or Hedgehog:HA in imaginal discs are subjected to isopycnic density centrifugation, these proteins are found in the lowest-density fraction with ApoLII, as is endogenous Hedgehog. Antibodies to endogenous Wingless detect a doublet in the top fraction and a band of somewhat higher mobility in high-density fractions. These data indicate that non-membrane-bound Wingless and Hedgehog associate with low-density particles in imaginal discs in vivo; other larval tissues may secrete Wingless in a non-lipophorin-associated form (Panakova, 2005).

To ask whether lipid-linked proteins associate with lipophorin, or with some other low-density particle, ApoLII was immunoprecipitated from larval S120s and precipitates were probed for Wingless, Hedgehog or GFPgpi. These proteins are immunoprecipitated by anti-ApoLII. Hedgehog and Fas-1 also immunoprecipitate with ApoLII from the more purified top fraction of KBr gradients. Thus, lipid-linked morphogens and gpi-linked proteins associate directly with lipophorin particles (Panakova, 2005).

Lipophorin-RNAi perturbs lipid transport: To assess the role of lipophorin in larval growth and development, the levels of ApoLI and II were reduced by RNA interference directed against two different regions of the apolipophorin messenger RNA. Similar phenotypes were produced by each construct. To express double-stranded (ds)RNA, a modified GAL4:UAS system was used in which expression of inverted repeats can be temporally controlled by heat-shock-dependent excision of an intervening HcRed cassette by the flippase (FLP) recombinase. Extracts were tested from wild-type larvae or larvae harbouring hs-flp, GAL4 driver and UAS dsRNA constructs at various times after heat shock to see how fast lipophorin levels were reduced. Larvae of the latter genotype made only 50% of the wild-type level of ApoLII, even in the absence of heat shock; basal activity of the heat-shock promoter in the fat body causes HcRed excision in approximately 50% of fat-body cells, although excision strictly depends on heat shock in other larval tissues. Although they survive less frequently, these flies have no obvious phenotype (Panakova, 2005).

After heat shock, all fat-body cells excise the HcRed cassette and ApoLII levels decrease further. After four days, ApoLII is reduced to 5% of wild-type levels. ApoLI levels are reduced with similar kinetics. These animals prolong the third larval instar and rarely pupariate. All the experiments described below were performed on third-instar larvae 4-6 days after heat shock (Panakova, 2005).

To investigate the requirement for lipophorin in lipid transport, the accumulation of neutral lipids in larval tissues was assessed by staining them with Nile Red. Cells of the posterior midgut normally contain many small lipid droplets. Lipophorin reduction causes a dramatic expansion of these droplets, suggesting that lipophorin is required for the efficient extraction of lipid from the midgut (Panakova, 2005).

The wild-type fat body contains both small and large lipid droplets. Fat bodies of lipophorin-RNAi larvae are reduced in size and have fewer small lipid droplets, although larger droplets appeared normal. These data suggest that lipophorin delivers lipid to the fat body (Panakova, 2005).

Lipid droplets in discs from lipophorin-RNAi larvae are fewer and smaller than in the wild type. Their discs are also reduced in size, particularly in the wing pouch. Thus, discs require lipophorin for accumulation of lipid droplets and for growth. Neither Caspase3 activation nor membrane phosphatidylinositol 3,4,5-phosphate (PIP₃) accumulation is altered in lipophorin- RNAi discs , suggesting that their small size is not due to cell death or reduced insulin signalling (Panakova, 2005).

Hedgehog function requires lipophorin: To test whether lipophorin association is required for Hedgehog function, Hedgeghog distribution and signalling was examined in lipophorin-RNAi larval discs. In wild-type discs, Hedgehog expressed in the posterior compartment moves across the anterior-posterior (AP) compartment boundary and activates transcription of short and long-range target genes. Cells closest to the source respond by activating the transcription of collier and patched. Further away, Hedgehog activates transcription of decapentaplegic. Levels of Collier and a decapentaplegic reporter construct (dpplacZ) were monitored in wild-type and lipophorin-RNAi discs stained in parallel and imaged under identical conditions. Discs from lipophorin-RNAi larvae activate collier at least as efficiently as those of the wild type. In contrast, the range of activation of dppLacZ is significantly narrowed in lipophorin RNAi discs. dppLacZ is expressed up to 11 cells away from the AP boundary in wild-type discs, but only up to six cells away in lipophorin-RNAi larvae. These data suggest that lipophorin knockdown decreases the range of Hedgehog signalling (Panakova, 2005).

To discover whether Hedgehog trafficking was altered, discs were stained for Hedgehog and Patched. In wild-type discs, Hedgehog moves into the anterior compartment, where it is found in endosomes, often with Patched. Patched-mediated endocytosis is thought to sequester Hedgehog and limit its spread. Hedgehog is most abundant up to five cell rows away from the AP boundary; although Hedgehog signals over a wider range, specific staining there cannot be distinguished from background. In lipophorin-RNAi discs, Hedgehog accumulates to abnormally high levels in the first five rows of anterior cells. Hedgehog spots were found in the most apical 10 µm of the wild-type disc. The lipophorin-RNAi disc contained 1,208 Hedgehog spots in the same region. Most accumulated Hedgehog colocalizes with Patched in endosomes. Furthermore, Patched co-accumulates more extensively with Hedgehog in endosomes than it does in wild-type. These data indicate that lipophorin RNAi either increases the susceptibility of Hedgehog to Patched-mediated endocytosis, or prevents subsequent degradation of the protein (Panakova, 2005).

Drosophila cannot synthesize sterols and relies on dietary sources. To assess whether reduced uptake of sterols or other lipids might cause the changes seen, the effects of lipid deprivation on larval development were explored. Larvae were allowed to hatch and feed on sucrose/agarose plates supplemented with yeast for 2-3 days, then transferred to plates containing chloroform-extracted yeast autolysate, rather than yeast. These larvae are developmentally delayed; after 7 days of lipid deprivation, their discs are much smaller than those of younger late-third-instar larvae. In contrast, yeast-fed siblings pupariate and begin to eclose by this time. Those flies that infrequently eclose after larval lipid depletion are small (35%-60% of normal body weight) but normally patterned. Thus, lipid depletion stalls imaginal growth (Panakova, 2005).

To discover whether lipid starvation affected Hedgehog trafficking or signalling, larvae were deprived of lipid 2 days after hatching and their discs were stained 6 days later. No changes in Hedgehog or Patched distribution are apparent in these discs compared with younger yeast-fed discs of similar size. Furthermore, the range of dpp and collier expression does not differ in lipid-starved and yeast-fed discs. Thus, lipid starvation does not mimic the effects of lipophorin knockdown. It is speculated that lipid-starvation-induced growth arrest prevents membrane sterol from dropping to levels that would interfere with the Hedgehog pathway. Thus, lipophorin does not indirectly affect the Hedgehog pathway via lipid deprivation (Panakova, 2005).

Wingless function requires lipophorin: To discover whether lipophorin RNAi perturbed Wingless trafficking, Wingless distribution was examined. In lipophorin-RNAi discs, extracellular Wingless is less abundant on both the apical and basolateral epithelial surfaces and spreads over shorter distances. However, no consistent alterations were detected in intracellular Wingless. Thus, lipophorin promotes accumulation of extracellular Wingless (Panakova, 2005).

To investigate whether Wingless signalling requires lipophorin, the activation of two target genes was examined. Senseless is produced only in cells near the Wingless source and its expression is unaffected by lipophorin RNAi. Distalless is normally produced in a gradient throughout most of the wing pouch. In lipophorin-RNAi discs, the Distalless gradient is abnormally narrow. This suggests that lipophorin knockdown specifically perturbs long-range Wingless signalling (Panakova, 2005).

Conclusions: This study establishes the principle that lipid-linked proteins of the exoplasmic face of the membrane associate with lipoproteins. These include many gpi-linked proteins with diverse functions, as well as the lipid-linked morphogens Wingless and Hedgehog. The mechanism allowing long-range dispersal of lipid-linked proteins is not yet understood. The finding that these proteins exist in both membrane-associated and lipoprotein-associated forms suggests reversible binding to lipoprotein particles as a plausible mechanism for intercellular transfer, and the consequences of lowering lipoprotein levels in Drosophila larvae supports this idea (Panakova, 2005).

Lipophorin knockdown narrows the range of both Wingless and Hedgehog signalling. Hedgehog accumulates to an abnormally high level in cells near the source of production and long-range signalling is inhibited; short-range target genes, however, are expressed normally. These data suggest that Hedgehog does not move as far when lipophorin levels are low. The range over which Hedgehog moves is normally restricted by Patched-mediated endocytosis. In discs from lipophorin RNAi larvae, accumulated Hedgehog co-localizes with Patched in endosomes, suggesting that it is more efficiently sequestered by Patched. How might lipophorin antagonize Patched-mediated sequestration and promote long-range movement? (Panakova, 2005).

The data are consistent with the idea that lipophorin is continuously needed for movement, rather than required only for the release of morphogens. If lipophorin were important only for Hedgehog secretion, lipophorin RNAi would be expected to decrease the amount of Hedgehog found in receiving tissue; this seems not to be the case. Furthermore, altered Hedgehog trafficking in receiving tissue is consistent with a model in which lipophorin is required at each step of intercellular transfer. The idea is favored that reversible association of Hedgehog with lipophorin particles facilitates its transfer from the plasma membrane of one cell to that of the next. This model predicts that lowering lipophorin levels should increase the length of time that Hedgehog spends in the plasma membrane before becoming associated with lipophorin. This would slow its rate of transfer and increase the probability of Patched endocytosing Hedgehog before it moved to the next cell. Hedgehog would then signal efficiently in the short range, but be so efficiently sequestered by Patched that very little protein would travel far enough to activate long-range target genes. These predictions are completely consistent with the current observations (Panakova, 2005).

This model differs significantly from the original concept of argosome function. It was initially speculated that argosomes were exosome-like particles with an intact membrane bilayer, and that lipid-linked morphogens needed to be assembled on these particles to be secreted by producing cells. Instead, it was found that argosomes are exogenously derived lipoproteins that facilitate the movement of morphogens through the epithelium. Many questions remain as to how morphogens become associated with argosomes, and how the spread and cell-interactions of these particles are regulated. Clearly, heparan sulphate proteoglycans are essential for the movement of Hedgehog and Wingless into receiving tissue. Because heparan sulphate binds to vertebrate lipoprotein particles, one might speculate that heparan sulphate proteoglygans (HSPGs) facilitate morphogen movement through lipoprotein binding. Conversely, many gpi-linked proteins, including the HSPG's Dally and Dally-like, are found on lipoprotein particles themselves. These associated proteins have the potential to modulate the cellular affinities or trafficking properties of lipoproteins and the morphogens they carry (Panakova, 2005).

The data suggest that lipophorin particles not only mediate intercellular transfer of Hedgehog, but may also be endocytosed together with the morphogen. Interestingly, LDL-receptor-related proteins Arrow and Megalin have demonstrated roles in Wingless signalling and Hedgehog endocytosis, respectively. It is intriguing to speculate that these receptors might be important for interaction with the lipoprotein-associated form of the morphogen (Panakova, 2005).

Cholesterol has the potential to modulate the activity of the Hedgehog pathway at many different points. Whether changes in the level of cellular cholesterol normally play a role in regulating the activity of the pathway is unclear. This study shows that Hedgehog interacts with the particle that delivers sterol to cells. This observation raises the possibility that internalization of Hedgehog is linked to sterol uptake, and suggests new mechanisms to link nutrition, growth and signalling during development (Panakova, 2005).

Lipoprotein-heparan sulfate interactions in the Hh pathway; Lipophorin interacts with the heparan sulfate moieties of the glypicans Dally and Dally/-like

The Drosophila lipoprotein particle, Lipophorin, bears lipid-linked morphogens on its surface and is required for long-range signaling activity of Wingless and Hedgehog. Heparan sulfate proteoglycans are also critical for trafficking and signaling of these morphogens. Lipophorin interacts with the heparan sulfate moieties of the glypicans Dally and Dally-like. Membrane-associated glypicans can recruit Lipophorin to disc tissue, and remain associated with these particles after they are released from the membrane by cleavage of their gpi anchors. The released form of Dally colocalizes with Patched, Hedgehog, and Lipophorin in endosomes and increases Hedgehog signaling efficiency without affecting its distribution. These data suggest that heparan sulfate proteoglycans may influence lipid-linked morphogen signaling, at least in part, by binding to Lipophorin. They further suggest that the complement of proteins present on lipoprotein particles can regulate the activity of morphogens (Eugster, 2007).

Controlling the spread and signaling of secreted morphogens is of critical importance for pattern formation during development. Morphogens of the Wnt and Hedgehog families undergo covalent lipid modification; these lipid moieties are essential for normal trafficking and signaling. It has been shown that the cell biological mechanisms they influence are not completely understood. The lipid-linked morphogens Wingless (Wg) and Hedgehog (Hh) can be released from the plasma membrane on the Drosophila lipoprotein Lipophorin, and Lipophorin is important for their long-range but not short-range signaling activity (Panáková, 2005). How might Lipophorin association influence morphogen signaling activity? One function of Lipophorin may be to mobilize otherwise membrane-bound molecules for long-range movement. Furthermore, a variety of gpi-linked proteins are also found on these particles, raising the possibility that morphogen signaling could be subject to additional regulation by other particle-associated proteins (Eugster, 2007).

Heparan sulfate proteoglycans (HSPGs) are also essential for normal trafficking and signaling of morphogens. Sterol-modified Hh cannot accumulate in or move through tissue that does not synthesize heparan sulfate (HS). In contrast, truncated Hh that cannot be sterol modified spreads freely through tissue missing HS. The effects of HS on the Hh pathway are mediated at least in part by the glypicans Dally and Dally-like (Dlp). Whereas Dlp is required for Hh signaling in S2 cells, it appears to act redundantly with Dally in imaginal discs. Only clones of cells mutant for both dally and dlp show autonomous reduction in Hh signaling (Eugster, 2007).

While autonomous functions of Dally and Dlp are presumably exerted by membrane-linked forms of the proteins, Dlp can also be shed from the plasma membrane. Notum, an enzyme in the α/β hydrolase family, removes the gpi anchor from Dlp expressed in S2 cells, releasing the protein into the supernatant. Whether Dally can be detached from its gpi anchor by Notum or other enzymes is unresolved. Some gpi-anchored proteins are released from cell membranes by associating with lipoprotein via their lipid anchors (Panáková, 2005), suggesting yet another mechanism for glypican shedding. It is not yet clear how and to what extent shedding occurs in vivo. Furthermore, specific roles for shed glypicans would be difficult to detect genetically because mutant clones have access to released glypicans produced by wild-type tissue (Eugster, 2007).

This study examined the relationship between glypicans and Lipophorin in the Hh pathway. Lipophorin was found to bind to the HS moieties of glypicans and can be recruited to disc tissue by these proteins. Lipophorin remains associated with glypicans when they are released from the plasma membrane by gpi removal. The released form of Dally is found in endosomes containing Lipophorin, Hh, and the Hh receptor Patched (Ptc) and increases Hh signaling efficiency (Eugster, 2007).

One consequence of Lipophorin particles binding to the heparan sulfate moieties of glypicans is that membrane-associated Dally and Dlp can recruit Lipophorin particles to imaginal disc tissue. Indeed, HSPGs are autonomously required for Lipophorin's interaction with the basal side of the disc epithelium. This raises the possibility that HSPGs may recruit lipid-linked morphogens to receiving cells, at least in part, by binding to morphogen-bearing lipoprotein particles. Although Hh is present in both apical and basal pools in the wing disc, it accumulates much more strongly on the basal surface when endocytosis is blocked. This suggests that the majority of Hh protein may spread along the basal surface. ttv, botv mutant cells that cannot recruit Lipophorin basally may be compromised in their ability to interact with Hh-bearing Lipophorin particles. This idea is also consistent with the observation that Hh mutants that cannot be sterol modified (and are presumably not associated with lipoproteins) no longer depend on HSPGs to bind to receiving cells (Eugster, 2007).

These data suggest that Lipophorin must be transcytosed from the basal side of the disc to the apical lumen. Although membrane-associated Dally and Dlp are found on the basolateral membrane, removal of the gpi anchor results in apical secretion. It will be interesting to investigate whether glypican endocytosis and subsequent release might play a role in Lipophorin transcytosis. The presence of transcytosed particles in the disc lumen may explain why removal of HS from clones of cells only disrupts basal Lipophorin accumulation. Particles transcytosed by other cells would be available for apical internalization by HS-independent mechanisms (Eugster, 2007).

Dally and Dlp continue to associate with Lipophorin via HS moieties after cleavage of the gpi anchor. The fact that both glypicans remain bound to Lipophorin after shedding raises the possibility that released glypicans influence signaling from lipoprotein particles. This analysis of the role of released Dally supports this idea (Eugster, 2007).

The data indicate that released Dally is required for full-strength Hh signaling, but does not affect the range over which Hh spreads. Hh signaling promotes Ci-dependent target gene activation by three separable mechanisms that are differentially sensitive to Hh levels. Ci stabilization requires the lowest levels of Hh signaling, and thus occurs over the broadest range. Ci stabilization is insufficient for target gene activation, however. To activate transcription, full-length Ci must be released with Suppressor of Fused (Su[Fu]) from cytoplasmic complexes containing Fused and Costal-2 and translocate to the nucleus. This occurs over shorter distances than Ci stabilization. Still higher levels of Hh signaling induce other processes needed for full activity of the Ci-Su(Fu) heterodimer within the nucleus. These are less well understood, but may involve phosphorylation of Su(Fu). In dally mutant discs, Ci stabilization and nuclear translocation, which require lower levels of Hh signaling, are normal. Only Ci activation is impaired by loss of dally. Thus, released Dally appears to increase the quantitative output of the Hh signaling pathway without increasing the amount of Hh (Eugster, 2007).

Although the possibility cannot be ruled out that a small, non-particle-associated fraction of released Dally gives rise to the phenotypes that were see, the extensive cofractionation and colocalization of released Dally with Lipophorin suggests that Lipophorin may act by influencing the behavior of these particles. For example, interaction of Lipophorin with the HS moieties of released Dally might reduce the affinity of Lipophorin particles for cell-surface HS, promoting transfer of Hh-bearing particles to Ptc. Alternatively, Dally HS moieties on Hh-bearing lipoprotein particles might promote the formation of ligand-receptor complexes. IHog acts as a coreceptor with Ptc and, like released Dally, is necessary for full-strength Hh signaling (Yao, 2006). Adding soluble heparin induces IHog dimerization and increases IHog-Hh binding in vitro (McLellan, 2006). Presentation of Dally HS on the same particle with Hh may greatly increase the efficiency with which these complexes form by bringing HS, Hh, and IHog into close proximity in vivo. Furthermore, lipoprotein particles that carried multiple copies of Dally and Hh would have the potential to induce receptor crosslinking. In this way, lipoprotein particles may act as scaffolding platforms, bringing together specific ligands and increasing the diversity of combinatorial signals available for patterning during development (Eugster, 2007).

Neurexin regulates visual function via mediating retinoid transport to promote rhodopsin maturation

Neurexins are cell adhesion molecules involved in synapse formation and synaptic regulation. Mutations in the neurexin genes are linked to a number of neurodevelopmental disorders such as autism. This study shows that the Drosophila homolog of alpha-Neurexin is critical for fly visual function. Lack of Neurexin leads to significantly impaired visual function due to reduced rhodopsin levels. The decreased chromophore levels cause deficits in rhodopsin maturation and that Neurexin is required for retinoid transport. Using yeast two-hybrid screening, Neurexin was shown to interact with apolipoprotein I (ApoL I), a product generated by cleavage of retinoid- and fatty acid-binding glycoprotein (RFABG) that functions in retinoid transport. Finally, it was demonstrated that Neurexin is essential for the apolipoproteins level. These results reveal a role for Neurexin in mediating retinoid transport and subsequent rhodopsin maturation and suggest that Neurexin regulates lipoprotein function (Tian, 2013).

This study has shown that Neurexin mediates Rh1 maturation through regulating retinoid transport, which is essential for rhodoposin maturation (see Model for Neurexin Action in the Maturation of Rhodopsin). It was further demonstrated that the intracellular region of Neurexin interacts with ApoL I and is required for the stability of ApoL I and II, key proteins that function in transporting retinoids in the retina. The results reveal a role for Neurexin in mediating retinoid transport and subsequent rhodopsin maturation and suggest that Neurexin regulates lipoprotein function (Tian, 2013).

Membrane receptors are responsible for translating extracellular stimuli into intracellular responses. The successful intracellular transport of rhodopsin to light sensory organelles is essential for photoreceptor function and survival, as defects in rhodopsin transport lead to severe retinal degeneration. Several proteins play a role in Rh1 maturation, and defects in a number of steps in the biosynthetic pathway may affect Rh1 production. The present study shows that Drosophila Neurexin is required for Rh1 maturation. Loss of Neurexin leads to reduced Rh1 levels and impaired visual function. Eye-specific expression of Neurexin rescues the impaired Rh1 level and visual function in the mutant. This study provides the compelling evidence that the cell adhesion molecule is required for rhodopsin maturation and function (Tian, 2013).

Previous studies have shown that Neurexin-1α is expressed in both embryonic chick retina and embryonic mice retina. This work show that Drosophila Neurexin is localized in the rhabdomeres in photorecepoters and photoreceptor-specific expression of Neurexin is able to rescue the impaired Rh1 level in the mutant. These results reveal that photoreceptor-derived Neurexin is essential for Rh1 maturation. The canonical binding partners of Neurexins, Neuroligins, are thought to be important for establishing the asymmetry of the synapse. However, unlike with Neurexin, loss of Neuroligin did not alter Rh1 levels. Taken together, these results suggest that Neurexin is probably activating via a Neuroligin and synapse-independent manner to regulated Rh1 maturation in the fly eye (Tian, 2013).

Drosophila is a good model system for genetic and molecular studies of vitamin A metabolism, because vitamin A is not required for fly viability but is critical for the generation of chromophores and for the synthesis of visual pigments. Several mutants affecting vitamin A production have been identified by PDA screening. Using HPLC analysis, this study showed that the chromophore levels are dramatically decreased in nrx^δ83 mutants. This finding represents the evidence that Neurexin is linked to retinoid transport and subsequent rhodopsin maturation (Tian, 2013).

In carotenoid-deprived mutants of Drosophila, defective chromophore production observed outside the retina can be rescued by supplying vitamin A in food. However, restoring Rh1 levels in nrx^δ83 mutants by supplying all-trans retinal in food was unsuccessful. In contrast, Rh1 levels could be restored by expressing Neurexin or RFABG in the photoreceptors. These results further support the conclusion that Neurexin functions inside the retina to facilitate chromophore generation or transport (Tian, 2013).

Neurexins are single-pass transmembrane proteins and the intracellular domain of Neurexin interacts with a number of exocytotic proteins, such as Velis, Munc18, and CASK. This study reveals that expression of the intracellular region of Neurexin is sufficient to restore the Rh1 level. In a yeast two-hybrid screen, two overlapping cDNAs were isolated of ApoL I binding with the intracellular domains of Neurexin. It was further shown that the ApoL protein levels are reduced in nrx^δ83 mutant retina and overexpression of Neurexin is able to restore ApoL protein levels in the mutant eye. It has been reported that IRBP undergoes rapid turnover (half-life, 10.7 hr) in the Xenopus interphotoreceptor matrix. The current results provide evidence that the intracellular region of Neurexin plays an important role in stabilizing ApoL proteins (Tian, 2013).

Drosophila RFABG is thought to be the functional homolog of vertebrate IRBP, and lack of IRBP causes delayed transfer of newly synthesized chromophores from the RPE to photoreceptors in mice. This phenotype resembles that of nrx^δ83 mutant flies with gradual increase in the Rh1 level after eclosion. Drosophila lipophorins have been shown to play an important role in the transport of lipid-linked morphogens and glycophosphatidylinositol-linked proteins. This study has shown that ApoL protein levels are reduced in nrx^δ83 mutant retina and Rh1 levels are restored upon overexpression of RFABG in the mutant eye. Sustained overexpression of RFABG might compensate the reduced stability of ApoL proteins in the mutant eye. These observations are consistent with the Neurexin rescue experiments, which show expression of Neurexin in photoreceptors is sufficient for restoring Rh1 level in the mutant. This study reveals the linker between and Neurexin and retinoid transport (Tian, 2013).

Nutritional and environmental factors play important roles in Autism Spectrum Disorders. (ASD), and fatty acid metabolism and abnormal membrane fatty acid composition may contribute to this disorder. It has been reported that apolipoproteins, especially Apo B-100, are reduced in children with ASD. Drosophila RFABG show high similarity in its domain structure with vertebrate Apo B-100. This study has shown that the region aa 1,390-1,480 of RFABG is sufficient for the interaction with Neurexin. The sequence aa 1,390-1,480 is lysine enriched (13 out of 90 residues). These highly charged residues could be important in mediating the Neurexin/ApoL interaction. In addition, this region is conserved between Drosophila RFABG and vertebrate IRBP and Apo B-100 (20% identity), implying that the Neurexin/ApoL I interaction may be conserved among various species. The revealed interaction and function correlation between Neurexin and lipoproteins have put a step forward for understanding of pathological relations of Neurexin mutations and perturbed fatty acid metabolism in ASD patients (Tian, 2013).

Lipoproteins in Drosophila melanogaster--assembly, function, and influence on tissue lipid composition

Interorgan lipid transport occurs via lipoproteins, and altered lipoprotein levels correlate with metabolic disease. However, precisely how lipoproteins affect tissue lipid composition has not been comprehensively analyzed. This study identified the major lipoproteins of Drosophila melanogaster; genetics and mass spectrometry were used to study their assembly, interorgan trafficking, and influence on tissue lipids. The apoB-family lipoprotein Lipophorin (Lpp) is the major hemolymph lipid carrier. It is produced as a phospholipid-rich particle by the fat body, and its secretion requires Microsomal Triglyceride Transfer Protein (MTP). Lpp acquires sterols and most diacylglycerol (DAG) at the gut via Lipid Transfer Particle (LTP), another fat body-derived apoB-family lipoprotein. The gut, like the fat body, is a lipogenic organ, incorporating both de novo-synthesized and dietary fatty acids into DAG for export. This study identified distinct requirements for LTP and Lpp-dependent lipid mobilization in contributing to the neutral and polar lipid composition of the brain and wing imaginal disc. These studies define major routes of interorgan lipid transport in Drosophila and uncover surprising tissue-specific differences in lipoprotein lipid utilization (Palm, 2012).

The major inter-organ lipid transport routes in Drosophila are executed by a single lipoprotein, Lpp, which is scaffolded by the apoB homologue apoLpp. Its major polar lipid constituents are long-chain PE and sterols, and its major neutral lipid is medium-chain DAG. Lpp lipidation takes place in two consecutive steps, which require distinct lipid transfer proteins, MTP and LTP, and take place in different organs: fat body and gut. ApoLpp is translated and lipidated in the fat body by an MTP-dependent mechanism, resulting in the formation of dense Lpp particles rich in PE. These are recruited to the gut, where they are further loaded with DAG and sterols through the activity of LTP. Thus, although Lpp originates in the fat body, it is loaded both with fat body and gut lipids (Palm, 2012).

Lipidation of mammalian apoB, like that of Drosophila apoLpp, proceeds in two distinct steps, formation of primordial phospholipid-rich lipoprotein particles, and subsequently acquisition of bulk neutral lipid. However, this process occurs entirely in the secretory pathway of producing cells. MTP has been proposed to be required both for initial transfer of phospholipids, and for the recruitment of TAG to the ER lumen for incorporation into lipoproteins. Interestingly, Drosophila MTP has been shown to promote the secretion of apoB-containing lipoproteins from COS cells, and to transfer phospholipids, but not TAG, between liposomes. This suggested that MTP acquired the ability to transfer TAG in the vertebrate lineage. Experiments described in this study show that Drosophila MTP is required for the production of the two Drosophila apoB-family lipoproteins Lpp and LTP in vivo; they further show that MTP is insufficient to load Lpp with normal quantities of DAG, the major neutral lipid of Lpp. These data support the idea that MTP originally evolved to promote the assembly of phospholipid-rich apoB-family lipoproteins (Palm, 2012).

The novel Drosophila apoB-family lipoprotein LTP shares many properties with the Lipid Transfer Particle purified from the hemolymph of several insects, including Manduca and Locusta. The scaffolding proteins of Drosophila LTP, apoLTPI and apoLTPII, are generated from a single precursor, apoLTP. Orthologous apoB-family proteins of other insects are therefore plausible candidates for the scaffolding proteins of their LTP particles. Insect LTPs were shown to contain a third, small protein subunit, apoLTPIII. Biochemical experiments do not address whether Drosophila LTP might contain an apoLTPIII subunit, because LTP is of such low abundance that silver staining barely detects the much larger apoLTPI. Sequence analysis of apoLTP does not suggest the existence of a protease cleavage site that could give rise to a protein of the size of apoLTPIII, and neither apoLTPI nor apoLTPII antibodies detect an additional protein of this size. Thus, if apoLTPIII exists in Drosophila, it is not likely to be derived from the apoLTP precursor (Palm, 2012).

The function of LTP as a lipid transfer protein rather than a carrier of bulk hemolymph lipid uncovers surprising evolutionary plasticity of the apoB lipoprotein family. Insect LTPs have been studied in vitro in a wide range of systems. In different contexts, they have been shown to facilitate the exchange of DAG and phospholipids between Lpp and fat body or gut, and even between insect and human lipoproteins of different densities. Studies of feeding Drosophila larvae indicate that only a subset of the lipid transfer activities of LTP may be relevant under specific metabolic conditions in vivo. LTP moves DAG and sterols from the larval gut onto Lpp. However, it does not facilitate significant net transfer of fat body lipids to Lpp. Consistent with this, radiolabeling experiments showed that the rate of DAG transfer from larval Manduca fat body to Lpp exceeds the rate of the reverse process. This may reflect a dominance of nutritional lipid uptake and fat storage in feeding larvae (Palm, 2012).

Although no Drosophila HDL-like lipoprotein was identified, it is noted that LTP and Lpp share some functional features with mammalian HDL, despite being scaffolded by unrelated apolipoproteins. Together, Lpp and LTP mediate efflux of sterols from the gut to circulation. Conceivably, other tissues that recruited both lipoproteins might efflux sterol for reverse transport (Palm, 2012).

While it is clear that dietary lipids do contribute to Lpp DAG, the gut does not directly incorporate dietary fatty acids into DAG destined for export. The long-chain fatty acids that predominate in the diet strikingly differ from the medium-chain fatty acids in Lpp DAG. A possible explanation is that the gut remodels dietary fatty acids, conceivably via limited β-oxidation. Interestingly, the gut is also a lipogenic organ and a significant fraction of the medium-chain fatty acids found in Lpp DAG derives from de novo fatty acid synthesis in this organ. In more primitive animals, such as Caenorhabditis elegans, lipid uptake, storage and lipogenesis all occur in the gut. More complex animals, including Drosophila, have developed separate organ systems for lipid storage and lipogenesis. However, the data show that this separation of functions is not absolute in the fly. Rather, other nutrients such as amino acids or sugars might be partially converted to lipid by the gut, instead of being transported intact into circulation. It would be interesting to ask what circumstances favor this conversion. Intriguingly, de novo lipogenesis has been observed in the mammalian gut, especially under conditions of insulin resistance, and has been proposed to contribute to the postprandial dyslipidemia observed in this state. Drosophila may be a useful model to explore this problem (Palm, 2012).

Gut and fat body differ in how they respond to blockage of lipid export to Lpp. Enterocytes vastly and rapidly expand their normally moderate stores of medium-chain DAG and TAG. This occurs even in the absence of dietary lipids, when exported lipids are derived from endogenous fatty acid synthesis. Thus, the gut has a flexible capacity for lipid storage. In contrast, the larval fat body maintains its neutral lipid stores within tight limits. When lipoprotein transport is blocked, endogenous lipid synthesis from other dietary components may suffice to build the large TAG stores of this organ. Furthermore, even though the fat body normally supplies the entire animal with large amounts of lipoproteins, TAG stores hardly increase when Lpp is not produced. Homeostatic mechanisms must maintain fat body TAG levels. In this way, the fat body differs from the gut, which accumulates fat when lipoprotein export is blocked, similar to mammalian gut and liver (Palm, 2012).

Peripheral tissues cannot maintain normal TAG levels in the absence of Lpp. The wing disc depends on Lpp for a large fraction of its fat stores. Interestingly, this work indicates that lipid delivery from the fat body and gut differently contributes to wing disc neutral lipids. TAG species containing medium-chain fatty acids depends on LTP and Lpp-mediated DAG mobilization from the gut. TAG species containing long-chain fatty acids also depend on Lpp-mediated lipid delivery, but are less affected by a blockage of DAG export from the gut. As Lpp is produced in the fat body, this suggests that long-chain TAG in wing discs may be derived from lipids supplied by the fat body. The most abundant source of long-chain fatty acids in Lpp is PE, which raises the possibility that wing discs use Lpp phospholipids to build cellular fat stores. Consistent with this, cultured murine hepatocytes convert a significant fraction of LDL or HDL-derived PC to TAG, although the in vivo relevance of this pathway remains to be explored. However, Lpp still contains reduced amounts of medium-chain DAG when LTP-mediated lipid loading is impaired. Thus, long-chain fatty acids in wing disc TAG might also derive from elongation of medium-chain fatty acids. Interestingly, although medium-chain DAG is the most abundant lipid transported through circulation, tissues store only minor amounts of neutral lipid containing medium-chain fatty acids. This would be consistent with the idea that tissues either elongate these fatty acids or subject them to β-oxidation (Palm, 2012).

The brain also requires Lpp-mediated lipid delivery to build its TAG stores. Interestingly, the brain stores normal levels of TAG when gut lipid mobilization is inhibited. While this does not exclude the possibility that the brain may directly acquire lipids from the gut under normal conditions, it indicates that TAG levels in this organ are more resistant to fluctuations in nutritional conditions than those in the wing disc (Palm, 2012).

In addition to providing fatty acids for neutral lipid storage, lipoproteins also influence the phospholipid composition of wing disc and gut: Lpp knock-down specifically reduces those PE species that are most abundant in Lpp. This suggests that Lpp might directly deliver PE to the cellular membranes of wing disc and gut. It further raises the possibility that phospholipid synthesis in other tissues could have organism-wide effects on membrane lipid composition. Since PE-rich Lpp particles are assembled in the fat body, this tissue is a likely source of these lipids. However, the brain does not depend on Lpp to maintain its normal membrane phospholipid composition. Furthermore, previous work suggested that the brain is more resistant to sterol depletion than other tissues. In general, these data indicate that the lipid composition of the brain is more tightly and autonomously controlled than that of other tissues (Palm, 2012).

In mammals, cellular lipid synthesis and lipid supply from circulation are coordinated through the SREBP pathway. Since Drosophila SREBP is regulated by PE instead of sterols, it will be interesting to explore whether altered PE levels in Lpp-deprived wing discs might activate SREBP signaling and increase lipid synthesis or lipoprotein uptake. If true, coordination of cellular lipid synthesis with lipid supply through lipoproteins is an evolutionarily conserved function of the SREBP pathway (Palm, 2012).

Lipoproteins transport large amounts of lipids through circulation - including many of the polar and neutral lipid species present in cells. These data indicate that in Drosophila, individual organs utilize lipoprotein-derived lipids not only for fat storage but also for membrane homeostasis. ApoB-deficient human patients, and patients with dyslipidemia suffer from various abnormalities in peripheral tissues. The data suggest that it may be worthwhile to examine how these perturbations alter the membrane lipid composition of affected tissues (Palm, 2012).

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

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. This study 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, the 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 (Parra-Peralbo, 2014).

Most metazoans accumulate triacylglycerol (TAG), a strongly hydrophobic molecule with a high energy content, as the main substrate for energy storage. Large amounts of TAG are stored in fat body cells, the Drosophila equivalent of adipocytes, but most other cell types also accumulate limited amounts of it as intracellular lipid droplets. Because of their hydrophobicity, the extracellular transport of lipids requires dedicated mechanisms to increase their solubility in extracellular fluids. In mammals, lipids are packed into several types of lipoprotein particles which contain a hydrophobic core of neutral lipids (mostly TAG and esterified cholesterol) surrounded by a monolayer of phospholipids. In addition, apolipoproteins stabilize and regulate these particles. Similar lipoproteins, named lipophorins, are also found in insects. They share the same basic structure and play similar functions as mammalian lipoproteins. In Drosophila, apolipophorins are exclusively synthesized in the fat body, where they are partially lipidated and released into the hemolymph. It has been suggested that lipophorins act as a reusable shuttle in lipid transport. Lipids, primarily diacylglycerol (DAG), derived from the digestion of food in the gut or from the mobilization of lipids in the fat body, are loaded onto pre-formed, circulating lipophorins, then transported through the body via the hemolymph and unloaded upon reaching peripheral tissues for use as a source of energy and phospholipids. During this cycling process, negligible degradation of apolipophorin occurs (Parra-Peralbo, 2014).

In mammals, the Low Density Lipoprotein Receptor (LDLR) and other related proteins mediate endocytosis and the clearance of lipoproteins from plasma. Similar proteins belonging to the LDLR family, known as lipophorin receptors, were subsequently identified in insects. They can bind to lipophorins and mediate their endocytosis both in cell culture systems and in vivo. Because of these properties, it has been suggested that lipophorin receptors may play an important role in insect lipid metabolism (Parra-Peralbo, 2014 and references therein).

This study examined the function of Drosophila lipophorin receptors in the uptake of neutral lipids. This organism has two lipophorin receptor genes, the lipophorin receptor 1 (lpr1) and lpr2, which are translated into multiple, functionally diverse isoforms. lpr1 and lpr2 are required for neutral lipid uptake in imaginal disc cells and oocytes. These results suggest a model where lpr1 and lpr2 promote the extracellular hydrolysis of neutral lipids contained in lipoprotein particles (Parra-Peralbo, 2014).

The Drosophila genome contains two closely related genes (lpr1 and lpr2, homologous to other described lipophorin receptors in insects like locust, mosquitoes, cockroaches, silkworm, wax moth or bees. Insect lipophorin receptors were first isolated because of their homology to the mammalian LDLR and subsequently shown to be involved in insect lipid metabolism. In particular, the locust Lipophorin Receptor, by far the best characterized member of the family, was able to induce the endocytic uptake of labeled lipophorins when expressed in mammalian cells. Moreover, it was required for the endocytosis of lipophorins by locust fat body cells. This study generated several novel mutations in Drosophila which disrupted lpr1, lpr2 and, in view of a possible functional redundancy between the two receptors, a deficiency was generated that affects both genes simultaneously (Df(3R)lpr1/2), representing a null mutation for lipophorin receptor function. Despite the critical role lipophorins play in lipid transport in insects and the embryonic lethal phenotype of a null mutation in the single Drosophila apolipophorin gene (Rfabg), complete disruption of both Drosophila lipophorin receptors does not affect the viability of flies. Moreover, no significant change were detected in total body TAG content when animals with mutations in lpr1, lpr2 or the double mutant were compared with their isogenic controls. Moreover, the fat body cells of mutant and control animals were indistinguishable, containing similar number of lipid droplets and of equivalent sizes. The rate of lipid mobilization under starvation conditions was also unaffected in lpr1 and lpr2 mutants. Taken together, these results clearly demonstrated that Drosophila lipophorin receptors are not essential for the storage of TAG in the fat body or for its mobilization. Despite this lack of a requirement, lpr1 and lpr2 expression was detected in the adult fat body, and lipophorin receptors have been identified in the fat body of other insects. Thus, the lipophorin receptors probably have functions in the fat body that are unrelated to the uptake of neutral lipids. Significantly, it has recently been reported that lpr2 is involved in immune response in Drosophila as a regulator of the serpin Necrotic metabolism. In addition, it was shown in microarray experiments that lpr2 transcription changes upon immune challenge (De Gregorio, 2001). Since the fat body is a key immunological organ in the fly, it is possible that lpr2 expression in this tissue is related to immunity (Parra-Peralbo, 2014).

This study has shown that lpr1 and lpr2 have a key role in neutral lipid uptake in two Drosophila organs: the imaginal disc and the ovaries. In both cases, they are required to attain high levels of intracellular TAG. lpr1 and lpr2 are expressed in the wing pouch region of wing imaginal discs and mediate the uptake of neutral lipids by these cells. Another protein involved in lipid storage as a component of lipid droplets, the perilipin-like protein Lipid storage droplet-2 (Lsd-2), is also preferentially expressed and required in the wing pouch region for the accumulation of intracellular lipid droplets. Thus, lipid accumulation in this region of the disc appears to be regulated at multiple levels. Unfortunately, the functional relevance of this lipid accumulation is still unknown (Parra-Peralbo, 2014).

The results indicate that lpr1 and lpr2 genes are transcribed as multiple isoforms each with dramatically different properties. Only those isoforms transcribed from the distal promoters and containing the LA-1 module mediate lipid uptake. Similarly, the lipophorin receptor gene from the mosquito Aedes aegypti has been shown to be translated into fat body and oocyte specific isoforms from two alternative promoters. Several members of the mammalian LDLR family are similarly processed by alternative splicing. Variations in the O-glycosylation region and the LA domains in mammalian VLDLR and ApoER2 have been related to differential sensitivity to proteolytic processing by gamma-secretases, respectively. Thus, the multiple lpr1 and lpr2 isoforms might have different ligand binding and/or stability properties, allowing these receptors to be involved in processes as diverse as neutral lipid uptake, regulation of the immune system and regulation of neurite outgrowth (Parra-Peralbo, 2014).

During vitellogenesis, the nurse cells and the oocyte grow rapidly accumulating large amounts of yolk proteins and lipids from the hemolymph over approximately 18 hours. Work from the Mahowald lab has shown that Yolkless, an LDLR family protein, mediates the endocytic uptake of yolk proteins in Drosophila. This study demonstrates that a different receptor type, the lipophorin receptor, is essential for the uptake of neutral lipids during vitellogenesis. This is clearly shown in Df(3R)lpr2 females and in double mutant lpr1^-, lpr2^-germ-line clones. In both cases, the mutant egg chambers accumulate low levels of neutral lipids. In addition to impaired lipid uptake during vitellogenesis, a second phenotype in was observed Df(3R)lpr1/2 double mutant females, where most of the egg chambers degenerated at mid-oogenesis. A simple explanation for this phenotype would be that degeneration was triggered by the low lipid content of Df(3R)lpr1/2 egg chambers. In fact, it is known that multiple challenges like starvation, extreme temperatures or chemical treatments, trigger a mid-oogenesis checkpoint and induce apoptosis at this stage. Significantly, flies with a mutation in the gene midway, which encodes an acyl coenzyme A: diacylglycerol acyltransferase required for the synthesis of TAG, were described to have severely reduced levels of neutral lipids in the germ-line and displayed apoptosis at mid oogenesis, thus paralleling the Df(3R)lpr1/2 phenotype. However, it was difficult to fully attribute degeneration to low lipid levels, as some experimental conditions were observed that resulted in egg chambers with very low levels of neutral lipids but that did not undergo degeneration. In particular, in Df(3R)lpr1/2 germ-line clones degeneration was absent even though the neutral lipid content of the egg chambers was low. Similarly, expression of UAS-lpr1J exclusively in the follicle cells of Df(3R)lpr1/2 females abolished egg chamber degeneration even though neutral lipid accumulation in the nurse cells and oocytes was low. These experiments suggest that the lipophorin receptors might have an additional function in the follicle cells which is necessary to avoid egg chamber degeneration. Accordingly, lpr1 expression was detected in the follicular epithelium. In this direction, it has recently been described that blocking the nutrient sensing TOR pathway in follicle cells induced apoptosis at mid oogenesis. Thus, Lpr1 could be required to maintain elevated levels of TOR activity in follicle cells. In interpreting these results, the non-autonomous effects of lipophorin receptors should also be considered. This study has shown that expression of UAS-lpr2E exclusively in the oocyte and nurse cells increases lipid uptake in the follicle cells, which could potentially impact on their nutritional status and restore their putative anti-apoptotic activity. Conversely, expression of the transgene in the follicle cells might slightly increase lipid uptake by the oocyte and nurse cells, even though it has not been possible to detect this effect, and thus provide enough lipids to bypass the mid-oogenesis checkpoint. More studies will be required to assess the role of the lipophorin receptors in the follicular epithelium (Parra-Peralbo, 2014).

Drosophila Lpr1 and Lpr2 are bona fide members of the LDLR family, sharing a similar organization of proteins domains with the human LDLR, ApoER2 and VLDLR. The human LDLR is the archetypical endocytic receptor. It is expressed in the liver where it mediates the endocytosis of cholesterol-rich LDL, regulating LDL concentration in serum. Endocytosis of LDL results in the catabolic processing of both, the lipidic and proteic moieties of LDL in lysosomes. Other members of the LDLR family are also well known endocytic receptors with a broad variety of ligands. Drosophila lipophorin receptors can also mediate endocytosis of their ligands. It has recently been reported that Lpr1 is expressed in garland cells and pericardial athrocytes where it is critical for the endocytic clearance of serpin/protease complexes from the hemolymph, thus regulating the innate immune response. Overexpression of Lpr1 and Lpr2 in imaginal discs also induced the endocytosis of lipophorins, which colocalized with endocytic markers. Similarly, the locust lipophorin receptor mediated lipophorin endocytosis in the fat body and in cell culture. Despite this well documented endocytic activity of LDLRs, the current data demonstrates that neutral lipid uptake mediated by Drosophila lipophorin receptors does not require the endocytosis of lipophorin particles. Three lines of evidence support this conclusion: (1) Blocking endocytosis did not affect lipid uptake in the egg chambers; (2) overexpression of Lpr2E in groups of imaginal disc cells induced lipid uptake both in cells expressing the receptor and in a 1-2 cell diameter region of adjacent cells and (3) expression of Lpr2E in the oocyte and nurse cells promoted lipid uptake in the adjacent, somatic follicular epithelium. These results also indicate that Lpr2E is able to locally increase the concentration of lipophorins in the extracellular space. Taking into account this data, the following model is proposed for lipophorin receptor-mediated neutral lipid uptake: lipophorin receptors interact with lipophorins at the cell surface and promote the extracellular hydrolysis of their DAG core by facilitating the activity of an as-yet-unidentified lipase, associated with the extracellular matrix. The free fatty acids generated during DAG hydrolysis could diffuse a few cell diameters away before being captured by cells, explaining why lipophorin receptors can promote lipid uptake non-autonomously. Significantly, physiological data obtained from studies of flight muscles and oocytes in insects indicated that lipid uptake mostly occurs without the concomitant degradation of the apolipophorin, which is consistent with the current hypothesis. Moreover, a lipophorin-specific lipase activity associated with muscle and oocyte cell membranes has been detected. The model offers a possible explanation to understand why only a subset of lpr1 and lpr2 isoforms mediates lipid uptake, whereby only the lipid-uptake promoting isoforms can stabilize lipophorins in the extracellular matrix. Alternatively, if lipophorin receptors must interact with both, a lipophorin particle and a lipase to generate a ternary complex and facilitate lipolysis, then the lipid uptake-defective isoforms might lack the ability to interact with the lipase. Identification of such putative lipase(s) will be necessary to test this hypothesis (Parra-Peralbo, 2014).

The proposed model displays a number of resemblances to the lipolytic processing of triglyceride-rich lipoproteins in the microvascular endothelium of adipose tissue, heart and striated muscles in mammals. Circulating triglyceride-rich lipoproteins, chylomicrons from the intestine and VLDL synthesized by the liver, reach the capillary endothelium where they interact with lipoprotein lipase at the luminal surface. Lipoprotein lipase is essential for the lipolytic processing of chylomicrons and VLDL, generating non-esterified fatty acids from the TAG fraction of lipoproteins. The free fatty acids are then transported to the underlying adipocytes and myocytes by specific transporters such as CD-36. Once inside these cells they are re-esterified into newly synthesized TAG stores or enter the β-oxidation cycle. Recent data indicated that the extracellular lipolysis of TAG-rich lipoproteins is strongly potentiated by the endothelial protein GPIHBP1. This protein is essential for the transcytosis of lipoprotein lipase from the basolateral to the apical capillary endothelial surface. In addition, it has been suggested that it may facilitate lipolysis by simultaneously interacting with lipoprotein lipase and chylomicrons in the luminal surface of capillaries, providing a molecular platform for lipolysis to occur. In agreement with this essential functions, Gpihbp1-deficient mice manifested severe hyperchylomicronemia. The VLDLR, which is also expressed at the capillary endothelium, seems to participate in the lipolytic processing of TAG-rich lipoproteins in similar ways. The VLDLR can mediate the transcytosis of lipoprotein lipase across cultured endothelial cells and interacts with both, lipoprotein lipase and ApoE containing TAG-rich lipoproteins, potentially tethering them to the endothelium surface and thus promoting the action of lipoprotein lipase. These potential functions were supported by the phenotype of vldlr^- mice, which showed delayed clearance of TAG-rich lipoproteins after a meal and increased plasma TAG levels under a high fat diet but normal lipoprotein profiles under regular feeding conditions. Unfortunately, these weak phenotypes have hampered the elucidation of the precise roles that VLDLR plays during the processing of TAG-rich lipoproteins in vivo. It is proposed that in Drosophila, lipophorin receptors have an activity similar to the bridging role proposed for GPIHBP1 and VLDLR in mammals, bringing lipophorins and a putative lipophorin-specific lipase into close contact on the cell surface and promoting in this way the lipolysis of lipophorins. It is speculated that during evolution, a protein related to VLDLR had a critical role in promoting the extracellular hydrolysis of lipoproteins. In insects, this function is carried out by the lipophorin receptors whereas in mammals, GPIHBP1 appears to have taken most of this function, with VLDLR retaining a minor role. The data supports an ancient function for the LDLR family in promoting the extracellular lipolytic processing of lipoproteins (Parra-Peralbo, 2014).

Molecular characterization and developmental expression of a retinoid- and fatty acid-binding glycoprotein from Drosophila

A detailed understanding of the mechanism of lipid transport in insects has been hampered by the inability to identify the proapolipophorin gene that encodes apolipophorins I and II, the principal protein components of lipophorin, the lipid transport vehicle. The Drosophila gene encodes a retinoid- and fatty acid-binding glycoprotein (RFABG) that it is a member of the proapolipophorin gene family. The gene, localized to the chromosome 4 (102 F region), encodes a 3351-amino acid protein that could serve as the precursor for the approximately 70-kDa and >200-kDa polypeptides associated with RFABG. The N-terminal sequence of the approximately 70-kDa polypeptide and that predicted for the >200-kDa polypeptide showed high sequence similarity to blowfly apolipophorin II and apolipophorin I, respectively. The RFABG precursor contains a signal peptide and exhibits a multidomain mosaic protein structure, which is typical of extracellular proteins. It has structural domains similar to lipid-binding proteins, namely vitellogenins and apolipoprotein B. The protein also contains a domain similar to the D domain of von Willebrand factor and mucin. The gene is expressed in the Drosophila embryo during development in cells that make up the amnioserosa and fat bodies. Immunolocalizations using specific antibodies against RFABG reveal that the protein is initially dispersed through the embryonic amnioserosa sac and latter concentrated at skeletal muscle-epidermis apodemeal contact junctions during larval development. This novel gene may play an important role in the transport of lipids, including retinoids and fatty acids, in insects (Kutty, 1996; full text of article).

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date revised: 20 June 2023

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