Padinjat Raghu

Visual transduction in Drosophila.

The brains capacity to analyse and interpret information is limited ultimately by the input it receives. This sets a premium on information capacity of sensory receptors, which can be maximized by optimizing sensitivity, speed and reliability of response. Nowhere is selection pressure for information capacity stronger than in the visual system, where speed and sensitivity can mean the difference between life and death. Phototransduction in flies represents the fastest G-protein-signalling cascade known. Analysis in Drosophila has revealed many of the underlying molecular strategies, leading to the discovery and characterization of signalling molecules of widespread importance.

Polyunsaturated fatty acids activate the Drosophila light-sensitive channels TRP and TRPL.

Phototransduction in invertebrate microvillar photoreceptors is thought to be mediated by the activation of phospholipase C (PLC), but how this leads to gating of the light-sensitive channels is unknown,. Most attention has focused on inositol-1,4,5-trisphosphate, a second messenger produced by PLC from phosphatidylinositol-4,5-bisphosphate; however, PLC also generates diacylglycerol, a potential precursor for several polyunsaturated fatty acids, such as arachidonic acid and linolenic acid. Here we show that both of these fatty acids reversibly activate native light-sensitive channels (transient receptor potential (TRP) and TRP-like (TRPL)) in Drosophila photoreceptors as well as recombinant TRPL channels expressed in Drosophila S2 cells. Recombinant channels are activated rapidly in both whole-cell recordings and inside-out patches, with a half-maximal effector concentration for linolenic acid of ∼10 µM. Four different lipoxygenase inhibitors, which might be expected to lead to build-up of endogenous fatty acids, also activate native TRP and TRPL channels in intact photoreceptors. As arachidonic acid may not be found in Drosophila, we suggest that another polyunsaturated fatty acid, such as linolenic acid, may be a messenger of excitation in Drosophila photoreceptors.

Calcium Influx via TRP Channels Is Required to Maintain PIP2 Levels in Drosophila Photoreceptors

The trp (transient receptor potential) gene encodes a Ca2+ channel responsible for the major component of the phospholipase C (PLC) mediated light response in Drosophila. In trp mutants, maintained light leads to response decay and temporary total loss of sensitivity (inactivation). Using genetically targeted PIP2-sensitive inward rectifier channels (Kir2.1) as biosensors, we provide evidence that trp decay reflects depletion of PIP2. Two independent mutations in the PIP2 recycling pathway (rdgB and cds) prevented recovery from inactivation. Abolishing Ca2+ influx in wild-type photoreceptors mimicked inactivation, while raising Ca2+ by blocking Na+/Ca2+ exchange prevented inactivation in trp. The results suggest that Ca2+ influx prevents PIP2 depletion by inhibiting PLC activity and facilitating PIP2 recycling. Without this feedback one photon appears sufficient to deplete the phosphoinositide pool of approximately 4 microvilli.

Constitutive activity of the light-sensitive channels TRP and TRPL in the Drosophila diacylglycerol kinase mutant, rdgA

Mutations in the Drosophila retinal degeneration A (rdgA) gene, which encodes diacylglycerol kinase (DGK), result in early onset retinal degeneration and blindness. Whole-cell recordings revealed that light-sensitive Ca2+ channels encoded by the trp gene were constitutively active in rdgA photoreceptors. Early degeneration was rescued in rdgA;trp double mutants, lacking TRP channels; however, the less Ca2+-permeable light-sensitive channels (TRPL) were constitutively active instead. No constitutive activity was seen in rdgA;trpI;trp mutants lacking both classes of channel, although, like rdgA;trp, these still showed a residual slow degeneration. Responses to light were restored in rdgA;trp but deactivated abnormally slowly, indicating that DGK is required for response termination. The findings suggest that early degeneration in rdgA is caused by uncontrolled Ca2+ influx and support the proposal that diacylglycerol or its metabolites are messengers of excitation in Drosophila photoreceptors.

Normal Phototransduction in Drosophila Photoreceptors Lacking an InsP3 Receptor Gene

The Drosophila light-sensitive channels TRP and TRPL are prototypical members of an ion channel family responsible for a variety of receptor-mediated Ca(2+) influx phenomena, including store-operated calcium influx. While phospholipase Cbeta is essential, downstream events leading to TRP and TRPL activation remain unclear. We investigated the role of the InsP(3) receptor (InsP(3)R) by generating mosaic eyes homozygous for a deficiency of the only known InsP(3)R gene in Drosophila. Absence of gene product was confirmed by RT-PCR, Western analysis, and immunocytochemistry. Mutant photoreceptors underwent late onset retinal degeneration; however, whole-cell recordings from young flies demonstrated that phototransduction was unaffected, quantum bumps, macroscopic responses in the presence and absence of external Ca(2+), light adaptation, and Ca(2+) release from internal stores all being normal. Using the specific TRP channel blocker La(3+) we demonstrated that both TRP and TRPL channel functions were unaffected. These results indicate that InsP(3)R-mediated store depletion does not underlie TRP and TRPL activation in Drosophila photoreceptors.

Molecular Basis of Amplification in Drosophila Phototransduction: Roles for G Protein, Phospholipase C, and Diacylglycerol Kinase

In Drosophila photoreceptors, the amplification responsible for generating quantum bumps in response to photoisomerization of single rhodopsin molecules has been thought to be mediated downstream of phospholipase C (PLC), since bump amplitudes were reportedly unaffected in mutants with greatly reduced levels of either G protein or PLC. We now find that quantum bumps in such mutants are reduced approximately 3- to 5-fold but are restored to near wild-type values by mutations in the rdgA gene encoding diacylglycerol kinase (DGK) and also by depleting intracellular ATP. The results demonstrate that amplification requires activation of multiple G protein and PLC molecules, identify DGK as a key enzyme regulating amplification, and implicate diacylglycerol as a messenger of excitation in Drosophila phototransduction.

Activation of heterologously expressed Drosophila TRPL channels: Ca2+ is not required and InsP3 is not sufficient

Light-sensitive channels encoded by the Drosophila transient receptor potential-like gene (trpl) are activated in situ by an unknown mechanism requiring activation of Gq and phospholipase C (PLC). Recent studies have variously concluded that heterologously expressed TRPL channels are activated by direct Gq-protein interaction, InsP3 or Ca2+. In an attempt to resolve this confusion we have explored the mechanism of activation of TRPL channels co-expressed with a PLC-specific muscarinic receptor in a Drosophila cell line (S2 cells). Simultaneous whole-cell recordings and ratiometric Indo-1 Ca2+ measurements indicated that agonist (CCh)-induced activation of TRPL channels was not always associated with a rise in Ca2+. Internal perfusion with BAPTA (10 mM) reduced, but did not block, the response to agonist. In most cases, releasing caged Ca2+ facilitated the level of spontaneous channel activity, but similar concentrations (200-500 nM) could also inhibit TRPL activity. Releasing caged InsP3 invariably released Ca2+ from internal stores but had only a minor influence on TRPL activity and none at all when Ca2+ release was buffered with BAPTA. Caged InsP3 also failed to activate any light-sensitive channels in situ in Drosophila photoreceptors. Two phospholipase C inhibitors (U-73122 4 microM and bromo-phenacyl bromide 50 microM) reduced both spontaneous and agonist-induced TRPL activity in S2 cells. The results suggest that, as in situ, TRPL activation involves G-protein and PLC; that Ca2+ can both facilitate and in some cases inhibit TRPL channels, but that neither Ca2+ nor InsP3 is the primary activator of the channel.

lazaro Encodes a Lipid Phosphate Phosphohydrolase that Regulates Phosphatidylinositol Turnover during Drosophila Phototransduction

An essential step in Drosophila phototransduction is the hydrolysis of phosphatidylinositol 4,5 bisphosphate PI(4,5)P2 by phospholipase Cbeta (PLCbeta) to generate a second messenger that opens the light-activated channels TRP and TRPL. Although the identity of this messenger remains unknown, recent evidence has implicated diacylglycerol kinase (DGK), encoded by rdgA, as a key enzyme that regulates its levels, mediating both amplification and response termination. In this study, we demonstrate that lazaro (laza) encodes a lipid phosphate phosphohydrolase (LPP) that functions during phototransduction. We demonstrate that the synergistic activity of laza and rdgA regulates response termination during phototransduction. Analysis of retinal phospholipids revealed a reduction in phosphatidic acid (PA) levels and an associated reduction in phosphatidylinositol (PI) levels. Together our results demonstrate the contribution of PI depletion to the rdgA phenotype and provide evidence that depletion of PI and its metabolites might be a key signal for TRP channel activation in vivo.

Rhabdomere biogenesis in Drosophila photoreceptors is acutely sensitive to phosphatidic acid levels

Phosphatidic acid (PA) is postulated to have both structural and signaling functions during membrane dynamics in animal cells. In this study, we show that before a critical time period during rhabdomere biogenesis in Drosophila melanogaster photoreceptors, elevated levels of PA disrupt membrane transport to the apical domain. Lipidomic analysis shows that this effect is associated with an increase in the abundance of a single, relatively minor molecular species of PA. These transport defects are dependent on the activation state of Arf1. Transport defects via PA generated by phospholipase D require the activity of type I phosphatidylinositol (PI) 4 phosphate 5 kinase, are phenocopied by knockdown of PI 4 kinase, and are associated with normal endoplasmic reticulum to Golgi transport. We propose that PA levels are critical for apical membrane transport events required for rhabdomere biogenesis.

Phosphatidylinositol Transfer Protein, Cytoplasmic 1 (PITPNC1) Binds and Transfers Phosphatidic Acid

Background: Phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1) (alternative name, RdgBβ) promotes metastatic colonization and angiogenesis in humans. Results: We demonstrate that RdgBβ is a phosphatidic acid (PA)- and phosphatidylinositol-binding protein and binds PA derived from the phospholipase D pathway. Conclusion: RdgBβ is the first lipid-binding protein identified that can bind and transfer PA. Significance: PA bound to RdgBβ is a likely effector downstream of phospholipase D. Phosphatidylinositol transfer proteins (PITPs) are versatile proteins required for signal transduction and membrane traffic. The best characterized mammalian PITPs are the Class I PITPs, PITPα (PITPNA) and PITPβ (PITPNB), which are single domain proteins with a hydrophobic cavity that binds a phosphatidylinositol (PI) or phosphatidylcholine molecule. In this study, we report the lipid binding properties of an uncharacterized soluble PITP, phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1) (alternative name, RdgBβ), of the Class II family. We show that the lipid binding properties of this protein are distinct to Class I PITPs because, besides PI, RdgBβ binds and transfers phosphatidic acid (PA) but hardly binds phosphatidylcholine. RdgBβ when purified from Escherichia coli is preloaded with PA and phosphatidylglycerol. When RdgBβ was incubated with permeabilized HL60 cells, phosphatidylglycerol was released, and PA and PI were now incorporated into RdgBβ. After an increase in PA levels following activation of endogenous phospholipase D or after addition of bacterial phospholipase D, binding of PA to RdgBβ was greater at the expense of PI binding. We propose that RdgBβ, when containing PA, regulates an effector protein or can facilitate lipid transfer between membrane compartments.