rdgB knockdown in neurons reduced nocturnal sleep in Drosophila melanogaster.

Recent studies revealed behaviorally defined sleep is conserved across broad species from insect to human. For evolutional analysis, it is critical to determine how homologous genes regulate the homologous function among species. Drosophila melanogaster shares numerous sleep related genes with mammals including Sik3, salt-inducible kinase 3, whose mutation caused long sleep both in mouse and fruit fly. The Drosophila rdgB (retinal degeneration B) encodes a membrane-associated phosphatidylinositol transfer protein and its mutation caused light-induced degeneration of photoreceptor cells. rdgB mutation also impaired phototransduction and olfactory behavior, indicating rdgB is involved in the normal neural transmission. Mammalian rdgB homologue, Pitpnm2 (phosphatidylinositol transfer protein membrane-associated 2) was discovered as one of SNIPPs (sleep-need index phosphoproteins), suggesting its role in sleep. Here, we show that rdgB is involved in sleep regulation in Drosophila. Pan-neuronal and mushroom body (MB) specific rdgB knockdown decreased nocturnal sleep. MB neurons play a dominant role, since the rescue of rdgB expression only in MB neurons in pan-neuronal knockdown reversed the sleep reducing effect of rdgB knockdown. These results revealed the sleep-related function of rdgB in Drosophila which may be conserved across species.

RDGBα, a PtdIns-PtdOH transfer protein, regulates G-protein-coupled PtdIns(4,5)P2 signalling during Drosophila phototransduction.

Many membrane receptors activate phospholipase C (PLC) during signalling, triggering changes in the levels of several plasma membrane lipids including phosphatidylinositol (PtdIns), phosphatidic acid (PtdOH) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. It is widely believed that exchange of lipids between the plasma membrane and endoplasmic reticulum (ER) is required to restore lipid homeostasis during PLC signalling, yet the mechanism remains unresolved. RDGBα (hereafter RDGB) is a multi-domain protein with a PtdIns transfer protein (PITP) domain (RDGB-PITPd). We find that, in vitro, the RDGB-PITPd binds and transfers both PtdOH and PtdIns. In Drosophila photoreceptors, which experience high rates of PLC activity, RDGB function is essential for phototransduction. We show that binding of PtdIns to RDGB-PITPd is essential for normal phototransduction; however, this property is insufficient to explain the in vivo function because another Drosophila PITP (encoded by vib) that also binds PtdIns cannot rescue the phenotypes of RDGB deletion. In RDGB mutants, PtdIns(4,5)P2 resynthesis at the plasma membrane following PLC activation is delayed and PtdOH levels elevate. Thus RDGB couples the turnover of both PtdIns and PtdOH, key lipid intermediates during G-protein-coupled PtdIns(4,5)P2 turnover.

The Drosophila photoreceptor as a model system for studying signalling at membrane contact sites.

Several recent studies have demonstrated the existence of membrane contact sites (MCS) between intracellular organelles in eukaryotic cells. Recent exciting studies have also demonstrated the existence of biomolecular interactions at these contact sites in mediating changes in the membrane composition of the cellular compartments. However, the role of such contact sites in regulating organelle function and physiological processes remains less clear. In this review we discuss the existence of a contact site between the plasma membrane (PM) and the endoplasmic reticulum (ER) inDrosophilaphotoreceptors. Further, we discuss the role of specific proteins present at this location in regulating phospholipid turnover and its impact in regulating a physiological process, namely phototransduction.

RdgBα reciprocally transfers PA and PI at ER-PM contact sites to maintain PI(4,5)P2 homoeostasis during phospholipase C signalling in Drosophila photoreceptors.

Phosphatidylinositol (PI) is the precursor lipid for the synthesis of PI 4,5-bisphosphate [PI(4,5)P2] at the plasma membrane (PM) and is sequentially phosphorylated by the lipid kinases, PI 4-kinase and phosphatidylinositol 4-phosphate (PI4P)-5-kinase. Receptor-mediated hydrolysis of PI(4,5)P2 takes place at the PM but PI resynthesis occurs at the endoplasmic reticulum (ER). Thus PI(4,5)P2 resynthesis requires the reciprocal transport of two key intermediates, phosphatidic acid (PA) and PI between the ER and the PM. PI transfer proteins (PITPs), defined by the presence of the PITP domain, can facilitate lipid transfer between membranes; the PITP domain comprises a hydrophobic cavity with dual specificity but accommodates a single phospholipid molecule. The class II PITP, retinal degeneration type B (RdgB)α is a multi-domain protein and its PITP domain can bind and transfer PI and PA. In Drosophila photoreceptors, a well-defined G-protein-coupled phospholipase Cß (PLCß) signalling pathway, phototransduction defects resulting from loss of RdgBα can be rescued by expression of the PITP domain provided it is competent for both PI and PA transfer. We propose that RdgBα proteins maintain PI(4,5)P2 homoeostasis after PLC activation by facilitating the reciprocal transport of PA and PI at ER-PM membrane contact sites.

The expanding roles of PI4P and PI(4,5)P2 at the plasma membrane: Role of phosphatidylinositol transfer proteins.

Phosphoinositides are phosphorylated derivatives of phosphatidylinositol, a phospholipid that is synthesised at the endoplasmic reticulum. The plasma membrane contains the enzymes to phosphorylate phosphatidylinositol and is therefore rich in the phosphorylated derivatives, PI4P and PI(4,5)P2. PI(4,5)P2 is a substrate for phospholipase C and during cell signaling, PI(4,5)P2 levels are reduced. Here I discuss a family of proteins, phosphatidylinositol transfer proteins (PITPs) that can restore PI(4,5)P2 levels.

ITPA (inosine triphosphate pyrophosphatase): from surveillance of nucleotide pools to human disease and pharmacogenetics.

Cellular nucleotide pools are often contaminated by base analog nucleotides which interfere with a plethora of biological reactions, from DNA and RNA synthesis to cellular signaling. An evolutionarily conserved inosine triphosphate pyrophosphatase (ITPA) removes the non-canonical purine (d)NTPs inosine triphosphate and xanthosine triphosphate by hydrolyzing them into their monophosphate form and pyrophosphate. Mutations in the ITPA orthologs in model organisms lead to genetic instability and, in mice, to severe developmental anomalies. In humans there is genetic polymorphism in ITPA. One allele leads to a proline to threonine substitution at amino acid 32 and causes varying degrees of ITPA deficiency in tissues and plays a role in patients' response to drugs. Structural analysis of this mutant protein reveals that the protein is destabilized by the formation of a cavity in its hydrophobic core. The Pro32Thr allele is thought to cause the observed dominant negative effect because the resulting active enzyme monomer targets both homo- and heterodimers to degradation.

Opsin Expression in the Central Nervous System of the Mantis Shrimp Neogonodactylus oerstedii.

Visual pigments, each composed of an opsin protein covalently bound to a chromophore molecule, confer light sensitivity for vision. The eyes of some species of stomatopod crustaceans, or mantis shrimp, can express dozens of different opsin genes. The opsin diversity, along with spectral filters and unique tripartite eye structure, bestow upon stomatopods unusually complex visual systems. Although opsins are found in tissues outside typical image-forming eyes in other animals, extraocular opsin expression in stomatopods, animals well known for their diversity of opsins, was unknown. Caudal photoreception in the central nervous system of decapod crustaceans, a group closely related to stomatopod crustaceans, is thought to be opsin based. However, electrophysiological data suggest that stomatopods do not have caudal photoreceptors. In this study, we identified mRNAs that could encode four different opsins and several components of a potential Gq-mediated phototransduction pathway in the central nervous system of the Caribbean mantis shrimp Neogonodactylus oerstedii. The four opsins are abundantly expressed in the cerebral ganglion, or brain, with little or no expression in the remainder of the ventral nerve cord. Our data suggest that there are previously undiscovered cerebral photoreceptors in stomatopods.