The Interactive Fly

Zygotically transcribed genes

Visual signal transduction

  • How does visual signal transduction occur?
  • Photomechanical responses in Drosophila photoreceptors
  • A dPIP5K dependent pool of Phosphatidylinositol 4,5 Bisphosphate (PIP2) is required for G-Protein coupled signal transduction in Drosophila photoreceptors
  • Speed and sensitivity of phototransduction in Drosophila depend on degree of saturation of membrane phospholipids.
  • Functional cooperation between the IP3 receptor and Phospholipase C secures the high sensitivity to light of Drosophila photoreceptors in vivo
  • A new genetic model for calcium induced autophagy and ER-stress in Drosophila photoreceptor cells
  • The GTP- and phospholipid-binding protein TTD14 regulates trafficking of the TRPL ion channel in Drosophila photoreceptor cells
  • Drosophila vision depends on carcinine uptake by an organic cation transporter
  • Evidence for dynamic network regulation of Drosophila photoreceptor function from mutants lacking the neurotransmitter histamine>
  • Phospholipase D activity couples plasma membrane endocytosis with retromer dependent recycling
  • Calcium signalling in Drosophila photoreceptors measured with GCaMP6f
  • CULD is required for rhodopsin and TRPL channel endocytic trafficking and survival of photoreceptor cells
  • The beta-alanine transporter BalaT is required for visual neurotransmission in Drosophila
  • A single residue mutation in the Galphaq subunit of the G protein complex causes blindness in Drosophila
    Proteins involved in visual signal transduction

    Arrestin A (Arr1) - FlyBase ID: FBgn0000120
    A Drosophila homologue of mammalian arrestin, a protein that interacts stoichiometrically with activated rhodopsin,
    inhibiting its ability to interact with the G protein, transducin, thus terminating the visual response.

    Arrestin 2 (Arr2)
    Arr1 and Arr2 follow different time courses of phosphorylation in vivo and have different functional roles in the photoreceptor.
    Arrestin is phosphorylated by CaM kinase II

    voltage sensitive calcium channel that stimulates neurotransmetter release at the presynaptic terminus at the neuromuscular junction

    Calcium/calmodulin dependent protein kinase II
    Targets Arrestin and thus plays a regulatory role in Drosophila photoreceptor light adaptation

    A Ca2+ sensor for a broad diveristy of retinal proteins. Target proteins include TRP, TRPL, NINAC and INAD all known
    to be involved in visual signal transduciton. Other target proteins are Drosophila UNC13 and CRAG, a protein related to Rab3 GTPase exchange proteins.

    Calnexin 99A
    mutations impair the ability of photoreceptor cells to control cytosolic Ca2+ levels following activation of the light-sensitive TRP channels

    facilitates membrane turnover and endocytosis of rhodopsin in photoreceptors

    CUB and LDLa domain
    rhodopsin endocytic trafficking - photoreceptor desensitization and adaptation

    Dopamine receptor
    modulates circadian regulated locomotion

    an β-alanyl-dopamine synthase regulating β-alanyl conjugation of dopamine and histamine, thus 'trapping' these biogenic amines
    preventing their further function - regulates pigmentation, photoreceptor activity and behavioral rhythmicity

    G protein alpha49B - FlyBase ID: FBgn0004435
    Functions as a G-protein-alphaq-subunit - mediates the stimulation by light-activated rhodopsin of the
    norpA-encoded phospholipase C in the visual transduction cascade

    Inactivation no afterpotential C (InaC)
    An eye specific Protein kinase C - functions in adaptation and termination of the photoresponse - targets TRP cation influx channel, the PDZ-containing protein
    inactivation, no afterpotential D (INAD), and the unconventional myosin NINAC

    Inactivation no afterpotential D (InaD)
    An adaptor protein containing PDZ domains that homomultimultimerizes and interacts with NORPA, Rhodopsin, PKC, Calmodulin, TRP and TRPL

    inactivation no afterpotential E
    diacylglycerol lipase involved in visual signal transduction and lipid metabolism

    lipid phosphate phosphohydrolase that functions during phototransduction - along with rdgA regulates amplification
    and response termination during phototransduction

    microtubule star
    catalyzes dephosphorylation of INAD modulating fast deactivation of the visual response

    neither inactivation nor afterpotential C (common alternative name: NinaC)
    motor domain protein involved in adaptation during visual signal transduction - regulates of translocation
    of Arrestin2 - required for stability of INAD and PKC

    Neither inactivation nor afterpotential E A light senstive G protein coupled receptor - The opsin moiety of the major rhodopsin, RH1, which occupies the rhabdomeres
    of the outer six photoreceptor cells R1-R6 in each ommatidium of the adult fly.

    No receptor potential A (NorpA)
    Phospholipase C-beta - a phosphatidylinositol-specific PLC - catalyzes the breakdown of phospholipids to generate inositol trisphosphate and diacylglycerol

    Rhodopsin 3 (Rh3) - FlyBase ID: FBgn0003249
    A light senstive G protein coupled receptor - the opsin moiety of a rhodopsin specific to the rhabdomere of the
    seventh photoreceptor cell. Also expressed in The most anterior staining marks progenitors of the larval eye known as Bolwig's organ.

    Rhodopsin 4 (Rh4) - FlyBase ID: FBgn0003250
    A light senstive G protein coupled receptor - The opsin moiety of a rhodopsin specific to the rhabdomere of the seventh photoreceptor cell;
    Rh3 and Rh4 are expressed in nonoverlapping subsets of ommatidia; the distribution of the two types of R7 cells within the eye is irregular

    Rhodopsin 5 (Rh5) - FlyBase ID: FBgn0014019
    A light senstive G protein coupled receptor - The opsin moiety of a rhodopsin expressed in a subset of R8 photoreceptor cells

    Rhodopsin 6 (Rh6) - FlyBase ID: FBgn0019940
    A light senstive G protein coupled receptor - The opsin moiety of a rhodopsin expressed in a subset of R8 photoreceptor cells

    Shaker cognate b
    slow delayed rectifier K+ channel - upregulated by light stimulation - functions in light adaptation

    A synaptic vesicle protein that may be one of the Ca2+ sensors that functions in release of neurotransmitter

    Transient receptor potential (Trp)
    A Calmodulin binding cation channel that is activated in the visual transduction process

    Transient receptor potential cation channel γ
    TRPγ-TRPL heteromultimers contribute to the photoresponse

    Trp-like (Trpl) - FlyBase ID: FBgn0005614
    A Calmodulin binding cation channel that is activated in the visual transduction process

    How does visual signal transduction take place?

    Drosophila visual signal transduction, the process by which incoming light is converted to neural signals that can be passed to the brain, provides an ideal system for the molecular dissection the process by which extracellular signals are transduced across the plasma membrane leading to neuron activation. In this signal transduction pathway, light stimulates rhodopsin, which activates an eye-specific G protein (Galphaq). Six genes have been identified that code for different Rhodopsins expressed in different sets of photoreceptor cells in the visual system (FlyBase, 1998). The phototransduction cascade is one of the fastest known G protein receptor coupling systems. In Drosophila, the high temporal resolution is evidenced by the less than 20 milli-second latency between photon excitation and photoreceptor cell depolarization (Ranganathan, 1991). Activated Galphaq triggers NorpA (no receptor potential A), a phospholipase C-beta to catalyze the breakdown of phospholipids to generate inositol trisphosphate (IP3) and diacylglycerol. Both Trp (transient receptor potential) and Trp-like are cation channels that are activated in the visual transduction process. These two proteins share homology with alpha-subunits of voltage-gated calcium and sodium channels in vertebrates. The rise in IP3 is thought to result in the release of Ca2+ from the internal Ca2+ stores. However, the release of Ca2+ has been shown not to involved the Inositol 1,4,5,-tris-phosphate receptor, leaving unanswered questions as to the source and regulation of the initial Ca2+ current (Acharya, 1997). It has now been shown, however, that polyunsaturated fatty acids activate the Drosophila light-sensitive channels TRP and TRPL. As arachidonic acid may not be found in Drosophila, it is suggested that another polyunsaturated fatty acid, such as linolenic acid, may be a messenger of excitation in Drosophila photoreceptors (Chyb, 1999). The opening of Ca2+ channels leads to depolarization of photoreceptors. This depolarization leads to the activation of the the Trp store-operated Ca2+ channel. The store-operated calcium channel in the plasma membrane is responsible for replenishing the internal calcium stores depleted during phosphoinositide-mediated process (Shieh, 1997 and references).

    Among the proteins of critical importance in visual phototransduction is Inactivation-no-afterpotential D, a photoreceptor-specific protein containing five repeated protein interaction motifs known as PDZ repeats. InaD is an adaptor protein that homomultimultimerizes and has the ability to interacts with multiple components of the signal transduction pathway, including Rhodopsin, NorpA, PKC, Calmodulin, Trp and Trp-like (Shieh, 1997 and references and Xu, 1998a and references). The view that signaling through G protein-coupled cascades occurs via random stochastic collisions between membrane receptors and effector molecules has been widely held for many years. However, the alternative proposal suggesting that signaling cascades are comprised of components that are physically coupled seems to better reflect reality. Work of several research groups have shown that Drosophila vision is mediated by a massive supramolecular complex and that assembly of such a complex is facilitated by homomultimerization of the scaffold protein InaD. Thus, most of the proteins critical in phototransduction appear to couple directly to InaD. The InaD supramolecular complex may not be a particle, consisting of a single InaD monomer to which a maximum of five target proteins bind. Instead, the visual cascade appears to be mediated through a more complicated higher order signaling web or complex (signalplex) consisting of an extended network of InaD homomultimers to which more than five targets bind. Most of these targets appear to bind to more than one PDZ module and several targets appear to associate with INAD via the same PDZ domains. Thus, the nature of the InaD signalplex appears to be more complicated than a single particle held together by a scaffolding protein (Xu, 1998a and references).

    An important Ca2+ sensor in Drosophila vision appears to be Calmodulin, since a reduction in levels of retinal Calmodulin causes defects in adaptation and termination of the photoresponse. These functions of Calmodulin appear to be mediated, at least in part, by four calmodulin-binding proteins: the Trp and Trp-like ion channels, NinaC and InaD. Eight additional Calmodulin-binding proteins have been found to be expressed in the Drosophila retina. These included six targets that were related to proteins implicated in synaptic transmission. Among these six are a homolog of the diacylglycerol-binding protein, UNC13, and a protein, CRAG, related to Rab3 GTPase exchange proteins. Both UNC13 and CRAG are implicated in the regulation of synaptic vesicle exocytosis. Two other calmodulin-binding proteins included Pollux, a protein with similarity to a portion of a yeast Rab GTPase activating protein, and Calossin, an enormous protein of unknown function conserved throughout animal phylogeny. The full-length sequences corresponding to three of the calmodulin-binding proteins have been described previously. These corresponded to two Calmodulin-dependent protein kinases, MLCK and CaM kinase II, as well as to one of the two previously described Calcineurin proteins. A third calmodulin-dependent protein kinase is expressed in the Drosophila retina, CaM kinase I. No CaM kinase I had previously been reported from any invertebrate raising the possibility that this protein kinase was specific to vertebrates. Nevertheless, the Drosophila CaM kinase I was highly related, ~60% identical over 349 amino acids, to vertebrate CaM kinase I. Thus, it appears that calmodulin functions as a Ca2+ sensor for a broad diversity of retinal proteins, some of which are implicated in synaptic transmission, and others directly in phototransduction (Xu, 1998b).

    Photomechanical responses in Drosophila photoreceptors

    Phototransduction in Drosophila microvillar photoreceptor cells is mediated by a G protein-activated phospholipase C (PLC). PLC hydrolyzes the minor membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2), leading by an unknown mechanism to activation of the prototypical transient receptor potential (TRP) and TRP-like (TRPL) channels. This study found that light exposure evokes rapid PLC-mediated contractions of the photoreceptor cells and modulates the activity of mechanosensitive channels introduced into photoreceptor cells. Furthermore, photoreceptor light responses are facilitated by membrane stretch and are inhibited by amphipaths, which alter lipid bilayer properties. These results indicate that, by cleaving PIP2, PLC generates rapid physical changes in the lipid bilayer that lead to contractions of the microvilli, and suggest that the resultant mechanical forces contribute to gating the light-sensitive channels (Hardie, 2012).

    In most invertebrate photoreceptor cells, the visual pigment (rhodopsin) and other components of the phototransduction cascade are localized within tightly packed microvilli (tubular membranous protrusions), together forming a light-guiding rod-like stack (rhabdomere). After photoisomerization, rhodopsin activates a heterotrimeric guanine nucleotide-binding protein (Gq protein), releasing its guanosine triphosphate-bound α subunit, which in turn activates phospholipase C (PLC). How PLC activity leads to gating of the light-sensitive transient receptor potential channels (TRP and TRPL) in the microvilli is unresolved. PLC hydrolyzes the minor membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), yielding soluble inositol 1,4,5-trisphosphate (InsP3), diacylglycerol (DAG, which remains in the inner leaflet of the microvillar lipid bilayer), and a proton. The light-sensitive channels in Drosophila photoreceptors can be activated by a combination of PIP2 depletion and protons, but it remains unclear how PIP2 depletion might contribute to channel gating. It has been speculated that changes in membrane properties play a role, and members of the TRP ion channel family have been repeatedly, although controversially, implicated as mechanosensitive channels. This led the current study to ask whether cleavage of the bulky, charged inositol head group of PIP2 from the inner leaflet might alter the physical properties of the lipid bilayer in the microvilli, resulting in mechanical forces that contribute to channel gating (Hardie, 2012).

    Remarkably, Drosophila photoreceptors responded to light flashes with small (<1 μm) but rapid contractions that are directly visible in dissociated cells via bright-field microscopy. To obtain improved temporal and spatial resolution, these photomechanical responses were recorded with an atomic force microscope (AFM), positioning the AFM cantilever on the distal tips of photoreceptors in a whole excised retina glued to a coverslip. Contact force (~100 pN) was maintained constant, so that changes in sample height resulted in immediate, matching changes in the cantilever's z-position. Contractions were elicited indefinitely by repeated brief flashes of modest intensity, with kinetics similar to those of electrical responses recorded from dissociated photoreceptors. The latencies of contractions induced by the brightest stimuli (4.9 ± 0.9 ms) were significantly shorter than the latencies of voltage responses to the same stimuli (6.6 ± 0.6 ms). Only a few hundred effectively absorbed photons per photoreceptor are required to elicit detectable contractions, which saturate with flashes containing ~106 photons, corresponding to ~30 effectively absorbed photons per microvillus. This intensity dependence overlapped with that of the electrical response. Like the electrical responses, the contractions were eliminated in mutants lacking PLC, which shows that they too required PLC activity (Hardie, 2012).

    Because PLC activity is normally terminated by Ca2+ influx through the light-sensitive channels, net PIP2 hydrolysis is enhanced when the light-sensitive current is blocked. Therefore contractions were measured in trpl mutants expressing only TRP light-sensitive channels before and after blocking TRP channel activity with La3+ and ruthenium red (RR). Indeed, after blocking the light-sensitive channels the contractions are enhanced, more sensitive to light, and saturate at lower intensities, corresponding to only ~1 to 5 effectively absorbed photons per microvillus. In the absence of Ca2+ influx, such intensities deplete virtually all microvillar PIP2, resulting in temporary loss of sensitivity to light. After such saturating flashes, the photomechanical response is also temporarily refractory, recovering sensitivity with a time course (t1/2 ~ 40 s) similar to that of PIP2 resynthesis. By contrast, without channel blockers, sensitivity recovers within ~10 s (Hardie, 2012).

    Blockade of all light-sensitive current in these experiments also shows that the contractions cannot result from any downstream effects of Ca2+ influx or osmotic changes caused by ion fluxes associated with the light response. Therefore, these results indicate that the contractions result from hydrolysis of PIP2. Although other downstream effects of PLC cannot be excluded, the speed of the contractions supports a simple and direct mechanism. Cleavage of the bulky head groups from PIP2 molecules, which represent 1% to 2% of lipids in the plasma membrane, leaves DAG in the membrane, which occupies a substantially smaller area than PIP2. This should increase membrane tension, leading to shrinkage of the microvillar diameter, as reported for the action of PLC on the diameter of artificial liposomes. Integrated over the stack of ~30,000 microvilli, such a mechanism seems capable of accounting for the observed macroscopic contractions, which represent at most ~0.5% of the rhabdomere length. Within each microvillus, it is suggested that the alteration to the mechanical properties of the lipid bilayer may contribute to channel gating (Hardie, 2012).

    To test whether phototransduction generates sufficient mechanical forces to gate mechanosensitive channels (MSCs), whole-cell patch-clamp recordings were made from dissociated photoreceptors lacking all light-sensitive channels (trpl;trp double mutants, or trpl mutants exposed to La3+ and RR). The photoreceptors were then perfused with gramicidin, a monovalent (Ca2+-impermeable) cation channel and one of the best-characterized MSCs, which is known to be regulated by changes in bilayer physical properties. Incorporation of gramicidin channels into the membrane generated a constitutive inward current that stabilized after a few minutes. Despite having replaced the native light-sensitive channels with MSCs, the photoreceptors still responded to light, with a rapid increase in the gramicidin-mediated current. Like the photomechanical responses recorded after blocking Ca2+ influx through the light-sensitive channels, these gramicidin-mediated responses inactivate slowly, are temporarily refractory to further stimulation and have a similar intensity dependence (Hardie, 2012).

    To test whether the light-sensitive channels were mechanically sensitive, membrane tension was manipulated osmotically. Channels were not directly activated by perfusing cells with hyper- or hypo-osmotic solutions; however, it was reasoned that it would be impossible to mimic the exact physical effects of PIP2 hydrolysis, which would include a specific combination of changes in membrane tension, curvature, thickness, lateral pressure profile, charge, and pH. Therefore whether osmotic manipulation could enhance or suppress light-sensitive channel activity was tested. In wild-type photoreceptors, light-induced currents are rapidly and reversibly facilitated by ~50% after perfusion with hypo-osmotic solutions (200 mOsm), which, like PIP2 depletion, would be expected to alleviate crowding between phospholipids, increase tension, and reduce membrane thickness. Conversely, responses in hyperosmotic (400 mOsm) solutions were about half those in control solutions. Analysis of single-photon responses (quantum bumps) indicated that modulation resulted from changes in both quantum efficiency (fraction of rhodopsin photoisomerizations generating a quantum bump) and bump amplitude. Recordings from trp and trpl mutants showed that both TRP and TRPL channels were modulated, although facilitation of currents mediated by TRPL channels (in trp mutants) was more pronounced. Modulation of the light response by osmotic manipulation was at least as pronounced in Ca2+-free bath, indicating that facilitation by membrane stretch did not result from leakage of Ca2+ into the cell from the extracellular space (Hardie, 2012).

    To test whether modulation might have been mediated by effects on upstream components of the cascade such as PLC, the activity of spontaneously active TRP channels was measured in recordings made with pipettes lacking adenosine triphosphate. Under these conditions, PIP2 becomes depleted (thereby removing PLC’s substrate), sensitivity to light is lost, and the TRP channels enter a constitutively active ('rundown') state uncoupled from the phototransduction cascade. Nonetheless, the channels were still similarly modulated by osmotic manipulation, whereas single-channel conductance, estimated by noise analysis, was unaffected. These results indicate that osmotic pressure directly modulates the open probability of both TRP and TRPL channels (Hardie, 2012).

    MSCs such as gramicidin are sensitive to amphiphilic compounds, which insert into the lipid bilayer. Because they are attracted to anionic phospholipids, cationic amphipaths insert preferentially into the inner leaflet, where they increase crowding, promote negative (concave) curvature, and decrease membrane stiffness. This study found that four structurally unrelated cationic amphipaths were all effective, reversible inhibitors of the light-induced current. Neither light-induced PLC activity (measured using a genetically targeted PIP2-sensitive biosensor to monitor PIP2 hydrolysis) nor single-channel conductance were substantially affected. The 50% inhibitory concentrations (IC50 values) were much higher than those of their traditional drug targets and ranged over approximately three orders of magnitude. However, after correcting for pKa and partitioning, the effective concentration of the compounds in the membrane was similar (~5 mM) in each case. Thus, their mode of action is likely related to their physicochemical properties rather than conventional drug-receptor interactions. Because cationic amphipaths are also lipophilic weak bases, and because it is proposed that protons are also critical for activating the light-sensitive channels, an alternative but not mutually exclusive possible mechanism of action is as lipophilic pH buffers of the membrane environment. It was also noted that polyunsaturated fatty acids (PUFAs) such as arachidonic and linolenic acid (effective activators of both TRP and TRPL) are not only anionic amphipaths (predicted to have opposite effects to cationic amphipaths) but also, as weak acids, natural protonophores; such a dual action could account for their agonist effect (Hardie, 2012).

    The mechanism of activation of the light-sensitive channels in invertebrate microvillar photoreceptors has long remained an enigma. Neither InsP3 nor DAG -- the two obvious products of PIP2 hydrolysis -- are reliable agonists for the light-sensitive channels. Although PUFAs are effective agonists and might be generated from DAG, a DAG lipase with the appropriate specificity has not been found in the photoreceptors. By contrast, two neglected consequences of PLC activity -- the depletion of its substrate (PIP2) together with protons released by PIP2 hydrolysis -- were recently shown to potently activate the light-sensitive channels in a combinatorial manner (Huang, 2010). The current results support the hypothesis that the effect of PIP2 depletion is mediated mechanically by changes to the physical properties of the lipid bilayer, thereby introducing the concept of mechanical force as an intermediate or 'second messenger' in metabotropic signal transduction (Hardie, 2012).

    A dPIP5K dependent pool of Phosphatidylinositol 4,5 Bisphosphate (PIP2) is required for G-Protein coupled signal transduction in Drosophila photoreceptors

    Multiple PIP2 dependent molecular processes including receptor activated phospholipase C activity occur at the neuronal plasma membranes, yet levels of this lipid at the plasma membrane are remarkably stable. Although the existence of unique pools of PIP2 supporting these events has been proposed, the mechanism by which they are generated is unclear. In Drosophila photoreceptors, the hydrolysis of PIP2 by G-protein coupled phospholipase C activity is essential for sensory transduction of photons. This study identified dPIP5K as an enzyme essential for PIP2 re-synthesis in photoreceptors. Loss of dPIP5K causes profound defects in the electrical response to light and light-induced PIP2 dynamics at the photoreceptor membrane. Overexpression of dPIP5K was able to accelerate the rate of PIP2 synthesis following light induced PIP2 depletion. Other PIP2 dependent processes such as endocytosis and cytoskeletal function were unaffected in photoreceptors lacking dPIP5K function, and are probably carried out by Skittles. These results provide evidence for the existence of a unique dPIP5K dependent pool of PIP2 required for normal Drosophila phototransduction. These results define the existence of multiple pools of PIP2 in photoreceptors generated by distinct lipid kinases and supporting specific molecular processes at neuronal membranes (Chakrabarti, 2015).

    The detection and conversion of external stimuli into physiological outputs is a fundamental property of neurons and depends on intracellular signal transduction pathways. Phosphoinositides, the seven phosphorylated derivatives of phosphatidylinositol are key signalling molecules and of these the most abundant PIP2 has multiple roles in neurons. Several neuronal receptors (such as the metabotropic glutamate, growth factor and sensory receptors) transduce stimuli into cellular information using the hydrolysis of PIP2 by phospholipase C enzymes. Additionally, within the context of neuronal cell biology PIP2 has several roles including cytoskeletal function and several ion channels and transporters require PIP2 for their activity. At the pre-synaptic terminal, a regulated cycle of PIP2 turnover is essential to regulate synaptic vesicle cycling. Thus PIP2 plays multiple roles at the plasma membrane of neurons; hence not surprisingly, changes in phosphoinositide metabolism have been linked to several inherited diseases of the human nervous system. Finally, one of the molecular targets of lithium, used in the treatment of bipolar disorders, is inositol monophosphatase a key regulator of PIP2 turnover in neurons (Chakrabarti, 2015).

    Given the multiple functions of PIP2 at the plasma membrane, it is unclear if a common pool of PIP2 supports all these functions. Alternatively, if there are distinct pools, it is unclear how these are generated and sequestered on the nanoscale structure of the membrane. In principle, PIP2 can be generated by the activity of two classes of phosphatidylinositol phosphate kinase (PIPK) enzymes, designated PIP5K and PIP4K; PIP5K phosphorylates PI4-P at position 5 of the inositol ring, whereas PIP4K phosphorylates PI5-P at position 4. Although PIP4K and PIP5K synthesize the same end product, they are not functionally redundant and studies of the mammalian enzymes has defined the molecular basis of substrate specificity. Genes encoding PIP5K are present in all sequenced eukaryotes; however PIP4K appears to be a feature of metazoans; mammalian genomes contain three distinct genes for each of these two activities. However, the functional importance of these two classes of enzymes in generating plasma membrane PIP2 has remained unclear (Chakrabarti, 2015).

    Drosophila photoreceptors are a well-established model for analyzing phosophoinositide signaling in-vivo. In these cells, the absorption of photons is transduced into neuronal activity by G-protein coupled, phospholipase Cβ (PLCβ) mediated PIP2 hydrolysis (NorpA). Thus, during phototransduction, PIP2 needs to be resynthesized to match consumption by ongoing PLCβ activity. PIP2 turnover is tightly regulated in photoreceptors; mutants in molecules that regulate PIP2 turnover show defects in phototransduction. However the role of PIPK enzymes in regulating PIP2 synthesis during phototransduction is unknown. This study analyzed each of the three PIPK encoded in the Drosophila genome that could generate PIP2 in the context of phototransduction. This analysis defines three pools of PIP2 supporting distinct molecular processes in photoreceptors (Chakrabarti, 2015).

    The hydrolysis of PIP2 by PLC in response to receptor activation is a widespread mechanism of signalling at the plasma membrane. In some cells such as neurons, activation of cell surface receptors by neurotransmitter ligands (e.g glutamate, Ach) or sensory stimuli triggers high rates of PLC activation and rapid consumption of PIP2. Under these conditions, it is essential that levels of PIP2, the substrate for PLC are maintained as failure to do so would likely result in desensitization. In mammalian cells, multiple classes of PIPK, the enzymes that resynthesize PIP2 have been described; yet the contribution of these enzymes to PIP2 resynthesis following PLC activation during cell signalling in vivo remains unclear. Broadly two classes of PIPK can synthesize PIP2 have been described; PIP5K that phosphorylates PI4P at position 5 or PIP4K that can phosphorylate PI5P at position 4. This study has analyzed the consequence of loss of each of these two classes of PIPK to resynthesis following PLC mediated PIP2 depletion during Drosophila phototransduction (Chakrabarti, 2015).

    Loss of dPIP5K function results in profound defects in the light activated electrical response as well as slower recovery of plasma membrane PIP2 levels. Conversely overexpression of dPIP5K was able to substantially accelerate the recovery of PIP2 levels following stimulation with a bright flash of light. dPIP5K is localized to the microvillar plasma membrane, the site at which PIP2 needs to be produced to support ongoing light induced PLC activity. Finally, this study found that loss of dPIP5K enhances the ERG defect in a hypomorphic allele of rdgB, a gene with a well-established defect in the response to light. Collectively these observations strongly suggest that dPIP5K activity underlies the conversion of PI4P to PIP2 at the microvillar membrane where it is then available as a substrate for light induced PLCβ activity. By contrast loss of the only PIP4K enzyme in the Drosophila genome has minimal effects on phototransduction and this enzyme is not targeted to the microvillar plasma membrane. These findings also imply that dPIP4K activity (and hence the conversion of PI5P into PIP2) is dispensable for maintaining PIP2 levels during Drosophila phototransduction. This is consistent with a previous study which found no reduction in the levels of PIP2 in flies lacking dPIP4K function. These observations validate the conclusion from biochemical studies in mammalian cells that the levels of PI5P are substantially lower than those of PIP2 and hence it is unlikely to be the source of the majority of PIP2 in cells. The identity of the PI4K isoform that generates the substrate, PI4P used by dPIP5K remains unknown although a recent study in mammalian systems suggests that PI4KIIIα is likely to be the relevant isoform (Chakrabarti, 2015).

    Although the ERG response is severely affected in the dPIP5K18 variant, it is not abolished as seen in null mutants of PLCβ (norpA) that are not able to hydrolyse PIP2. Additionally, the resting levels of PIP2 as detected by the PIP2 biosensor are comparable to wild type and following a bright flash of light that depletes PIP2, its levels do recover albeit at a slower rate than in wild type photoreceptors. Given that dPIP5K18 is a protein null allele, these observations imply that there must be a second pool of PIP2 in dPIP5K18 cells that is able to support phototransduction and microvillar PIP2 re-synthesis albeit with lower efficiency. This second pool of PIP2 is likely available with low efficiency for PLC activity in the absence of the dPIP5K dependent pool thus accounting for the residual light response and observed PIP2 dynamics in dPIP5K18 photoreceptors (Chakrabarti, 2015).

    The ultrastructure of dPIP5K18 photoreceptors was essentially normal. This was particularly surprising given that in addition to phototransduction, PIP2 at the microvillar membrane is also expected to regulate multiple processes required to maintain normal microvillar structure including dynamin dependent endocytosis as well as cytoskeletal function. However, using multiple readout, molecular readouts of endocytosis and cytoskeletal function were found to be unaffected in dPIP5K18 photoreceptors. These observations imply that the PIP2 required for these processes is not dependent on dPIP5K activity; rather PIP2 generated by a separate PIPK supports these processes. Thus far, dPIP4K has not been detected on the microvillar plasma membrane, dPIP4K29 photoreceptors show normal ultrastructure on eclosion and do not undergo light dependent microvillar degeneration; thus dPIP4K is unlikely to be the critical enzyme that generates the PIP2 required to support dynamin dependent endocytosis, p-Moesin localization, or phototransduction. The Drosophila genome encodes an additional PIP5K activity, sktl that is expressed at low levels in the adult retina but is localized to both the microvillar and basolateral membrane and hence could synthesize PIP2 at both these locations. Complete loss of sktl function is cell lethal and overexpression of sktl in developing photoreceptors results in a severe block in rhabdomere biogenesis whereas overexpression of sktl results in light dependent retinal degeneration in post-development photoreceptors. These findings presumably reflect an essential and non-redundant role for SKTL in supporting fundamental PIP2 dependent cellular processes such as endocytosis and cytoskeletal function that are not dependent on PIP2 hydrolysis by PLC. This model is consistent with the cell-lethal phenotype of photoreceptors that are null for sktl and previous studies showing a role for sktl in supporting cytoskeletal function and endocytosis in other Drosophila tissues and processes such as spermiogenesis and oogenesis (Chakrabarti, 2015).

    Collectively, these observations imply that there are at least two pools of PIP2 in photoreceptors; one generated by dPIP5K that is required to support a normal electrical response to light but is dispensable for non-PLC dependent functions of PIP2 in photoreceptors and another that is generated by enzymes other than dPIP5K (most likely SKTL) that is also capable of supporting PIP2 synthesis during the light response albeit with reduced efficiency. In summary the PIP2 pool synthesized by dPIP5K is unique in that it is required for a normal light response and apparently dispensable for other PIP2 dependent functions/processes. It also reflects the existence of distinct/segregated pools of PIP2 on the same microvillar plasma membrane that are maintained by distinct kinases (Chakrabarti, 2015).

    A number of previous studies have shown that in multiple eukaryotic cell types, plasma membrane PIP2 levels are remarkably stable, undergoing transient fluctuations despite ongoing PLC mediated PIP2 hydrolysis. However the reasons for this remarkable finding have remained unclear although pharmacological studies have suggested the importance of PIP2 resynthesis in this process. One potential explanation for this idea is the existence at the plasma membrane of two pools of PIP2, a larger but less dynamic pool of that is not normally accessed by PLC and supporting non-PLC dependent functions of this lipid and a second, quantitatively smaller but more dynamic pool that is the substrate for PLC activity. What underpins such pools of PIP2? The existence of separate enzymes that generate unique pools of PIP2 has been previously suggested but there have been limited experimental studies to support this model. In murine platelets where thrombin induced PIP2 hydrolysis appears to be dependent on PIP5K1β but not PIP5Kγ; since both these enzymes are expressed in platelets this implies the existence of two pools of PIP2 in these cells of which the PIP5K1β dependent pool is available for thrombin dependent PIP2 turnover (Chakrabarti, 2015).

    This finding together with this study in Drosophila photoreceptors implies that the plasma membrane in general may contain a specific pool of PIP2 dedicated for the use of receptor dependent PLC signalling and synthesized by a specific PIPK. It is possible that given the high rates of PLC activated PIP2 turnover at the plasma membrane (such as the microvillar membrane in photoreceptors) eukaryotic cells have evolved a mechanism to generate distinct PIP2 pool for this purpose so that other PIP2 dependent functions at the plasma membrane remain unaffected by ongoing receptor activated PIP2 hydrolysis. It is likely that dPIP5K and mammalian PIP5K1β represent PIP5K enzymatic activities required to support such a pool of PIP2 at the plasma membrane (Chakrabarti, 2015).

    It is presently unclear what properties might make dPIP5K more suitable for generating PIP2 in the context of receptor triggered PLC activity. One possibility is that the kinetic properties of the enzyme encoded by dPIP5K is distinct from that encoded by sktl allowing it to function in the context of high rates of PIP2 turnover. Alternatively (or additionally) within the nanoscale organization of the microvillar plasma membrane, it is possible that dPIP5K is segregated such that PIP2 generated by this enzyme is available within molecular distances of the phototransduction machinery. Interestingly, Drosophila photoreceptors contain within their microvillar membrane a macromolecular signalling complex organized by the PDZ domain protein INAD. It is presently not known if dPIP5K is part of a similar complex but the existence of such mechanisms has been previously shown for mammalian PIP5K1γ in the context of focal adhesion function. Interestingly, it has been reported that the INAD protein complex that includes PLCβ is recruited to detergent resistant membranes during light stimulation which themselves have been previously implicated in the formation of PIP2 microdomains and receptor activated PIP2 turnover. It is possible that the two PIPKs, SKTL and dPIP5K show differential localization to such domains thus generating and segregating such pools of PIP2 and further studies in this direction are likely to provide insight into this issue. Nevertheless this study has provided evidence for the concept of distinct PIPK enzymes as the basis for functionally distinct pools of PIP2 at the plasma membrane. Further analysis in this system is likely to reveal the molecular basis for the organization of PIP2 pools at cellular membranes (Chakrabarti, 2015).

    Speed and sensitivity of phototransduction in Drosophila depend on degree of saturation of membrane phospholipids.

    Drosophila phototransduction is mediated via a G-protein-coupled PLC cascade. Recent evidence, including the demonstration that light evokes rapid contractions of the photoreceptors, suggested that the light-sensitive channels (TRP and TRPL) may be mechanically gated, together with protons released by PLC-mediated PIP2 hydrolysis. If mechanical gating is involved this study predicted that the response to light should be influenced by altering the physical properties of the membrane. To achieve this, the study used diet to manipulate the degree of saturation of membrane phospholipids. In flies reared on a yeast diet, lacking polyunsaturated fatty acids (PUFAs), mass spectrometry showed that the proportion of polyunsaturated phospholipids was sevenfold reduced (from 38% to ~5%) but rescued by adding a single species of PUFA (linolenic or linoleic acid) to the diet. Photoreceptors from yeast-reared flies showed a 2- to 3-fold increase in latency and time to peak of the light response, without affecting quantum bump waveform. In the absence of Ca(2+) influx or in trp mutants expressing only TRPL channels, sensitivity to light was reduced up to ~10-fold by the yeast diet, and essentially abolished in hypomorphic G-protein mutants (Gαq). PLC activity appeared little affected by the yeast diet; however, light-induced contractions measured by atomic force microscopy or the activation of ectopic mechanosensitive gramicidin channels were also slowed ~2-fold. The results are consistent with mechanosensitive gating and provide a striking example of how dietary fatty acids can profoundly influence sensory performance in a classical G-protein-coupled signaling cascade (Randall, 2015).

    The ability to manipulate fatty acid composition of phospholipids in flies by diet has been reported previously. However, there are no convincing reports of physiological consequences. This study found that the proportionof polyunsaturated phospholipids in flies reared on yeast was reduced ~7-fold, and that this was associated with pronounced effects on visual performance, with photoreceptor responses slowed 2- to 3-fold,without significantly affecting quantum bump waveform, Na+/Ca2+ exchange, or PLC activity. Although absolute sensitivity was little affected by diet in wild-type photoreceptors, this could be attributed to compensation by Ca2+-dependent positive feedback acting on TRP channels. In the absence of Ca2+ influx or in trp mutants expressing only TRPL channels, sensitivity was reduced up to ~10-fold, and essentially abolished in hypomorphic Gαq1 mutants. The results indicate that these effects, all of which were rescued by the addition of a single species of PUFA tothe yeast diet, were mediated predominantly downstream of PLC and were associated with slowing of photomechanical responses (Randall, 2015).

    The macroscopic light response in Drosophila reflects the summation of quantum bumps, each arising from activation of most of the ~20 TRP channels in a single microvillus. Briefly, a single activated rhodopsin is believed to activate ~5 or so Gq-proteins by random diffusional encounters, and each released Gq α-subunit diffuses further before binding and activating PLC. Each PLC molecule rapidly hydrolyzes PIP2, building up sufficient excitatory 'messenger' to overcome a finite threshold required to activate the first TRP channel with a stochastically variable latency of ~15-100 ms. The resulting Ca2+ influx raises Ca2+ throughout the microvillus into the micromolar range within milliseconds, facilitating activation of the remaining channels, and generating an 'all-or-none' quantum bump localized to a single microvillus. This raises Ca2+ within the affected microvillus to ~1 μm, terminating the bump by Ca2+-dependent inactivation of the channels and preceding steps of the cascade. This highly nonlinear positive and negative feedback cycle, which shapes the quantum bump waveform, was apparently not influenced by diet. However, bump latency, i.e., the time taken to activate the first channel, was clearly profoundly delayed in flies reared on the YF diet (Randall, 2015).

    Which products of PLC activity are responsible for this initial gating remains controversial. InsP3 and Ca2+ stores apparently play no role, because mutants of the InsP3 receptor have normal phototransduction. Alternative candidates include DAG or PUFAs, which might be released from DAG by an appropriate lipase. Of these, DAG has generally proved ineffective as an agonist when exogenously applied; however, there is genetic evidence, based on mutants of DAG kinase (rdgA), for DAG as an excitatory messenger. One lab has also reported that DAG can activate TRP channels in excised patches from dissociated rhabdomeres; however, activation was very sluggish with delays of up to 60 s, in a preparation that is in a physiologically severely compromised state (Randall, 2015).

    In contrast, there is universal consensus that PUFAs are very effective agonists when exogenously applied to both native channels in the photoreceptors and heterologously expressed TRPL channels. Apparent support for endogenous PUFAs came from the reduced light response found in a Drosophila DAG lipase mutant, inaE. However, inaE encodes an sn-1 DAG lipase, which rather than PUFAs, releases mono-acyl glycerols (MAGs), which are at best weak and slowly acting channel agonists when applied exogenously (Hardie, unpublished results). For PUFA generation, either an sn-2 DAG lipase or an additional enzyme (MAG lipase) would be required, but there is no evidence for either in photoreceptors. Furthermore, the inaE gene product immunolocalizes to the cell body with, at most, occasional puncta in the rhabdomere, and there is no evidence that PUFAs are generated in response to illumination (Randall, 2015).

    An alternative hypothesis comes from evidence showing that the channels can be activated by the strict combination of two further consequences of PLC action, namely PIP2 depletion and proton release. Furthermore it was suggested that PIP2's role is mediated not by ligand binding/unbinding but by the physical effects of PIP2 depletion on the lipid bilayer. Thus, removal of PIP2's inositol headgroup from the inner leaflet effectively reduces membrane area, and it was proposed that the resulting mechanical effects (on, e.g., membrane tension, curvature, or lateral pressure) lead to mechanical gating of the channels, in combination with protons. Evidence supporting this included: (1) the ability of light to activate ectopic mechanosensitive channels (gramicidin); (2) facilitation of light responses by hypotonic solutions; and (3) rapid contractions of the photoreceptors in response to light, interpreted as the concerted contraction of microvilli due to PIP2 hydrolysis in the inner leaflet (Randall, 2015).

    The present study used diet to increase the degree of saturation of the phospholipids, which should make membranes stiffer and less flexible, and it ws predicted that this might suppress mechanosensitive channel activity. Lipidomic analysis confirmed a drastic sevenfold reduction in PUFA content across all phospholipid species in YF-reared flies. This was associated with a 2- to 3-fold longer latency and a marked reduction in open probability once Ca2+-dependent positive feedback was eliminated. In principle PLC activity might be suppressed by the YF diet, because membrane fluidity, and hence diffusion (e.g., of G-proteins, or PIP2), might be slower in a membrane dominated by saturated phospholipids. However, molecular dynamic simulations predict that lateral diffusion coefficients in fact depend only weakly on the degree of phospholipid saturation. Importantly, the rate of PIP2 hydrolysis monitored by proton release appeared unaffected by diet suggesting that diet was acting primarily downstream of PLC. That dietary manipulation had indeed resulted in alteration to the mechanical properties of the membrane was supported by AFM measurements of the light-induced contractions and responses mediated by ectopic mechanosensitive gramicidin channels. These indicated that, despite the lack of effect on PLC activity, YF-reared flies generated slower photomechanical responses (Randall, 2015).

    Although consistent with the mechanical gating hypothesis, by themselves these data do not exclude the alternative suggestions, that the excitatory messenger is DAG or a PUFA. According to a recent study (Delgado, 2014), DAG generated by light-induced PIP2 hydrolysis in Drosophila photoreceptor membrane includes both monounsaturated and polyunsaturated species (32:1, 32:2, 34:1, 34:2, 36:3, and 36:4) suggesting at least some derive from polyunsaturated PIP2 species. In turn, should any fatty acids be released from DAG, they would most likely include PUFAs. However, the near absence of polyunsaturated PIP2 in flies reared on YF diets implies that DAG generated in response to light would now be predominantly saturated or monounsaturated. If different species of DAG and/or PUFA had different potencies for activating the channels, then in principle this might also explain the results. In this respect, the otherwise equivocal evidence for DAG and PUFAs as messengers notwithstanding, it should be noted that heterologously expressed TRPL channels are activated more potently by linolenic than by oleic acid and that polyunsaturated DAG species have been reported to be more effective activators of PKC in mammalian cells (Randall, 2015).

    In conclusion, these results confirm that fatty acid composition of phospholipids is strongly influenced by diet in Drosophila. Increasing the degree of saturation leads to pronounced slowing of the electrical light response, which is associated with slower photomechanical responses. These findings are consistent with the suggestion that TRP and TRPL channel activation in Drosophila photoreceptors are mediated, together with protons, by mechanical forces in the membrane induced by PIP2 hydrolysis by PLC. To what extent visual performance in flies might be affected by dietary fatty acids under natural conditions is unknown. Although Drosophila thrive on a yeast diet in the laboratory, their typical food is rotting plant material, which will contain PUFAs to varying degrees. Interestingly, in food-preference studies, Drosophila larvae show preference for unsaturated over saturated fatty acids, but this preference is reversed in adult flies. The fatty acid composition of mammalian phospholipids is also influenced by diet, and the effects of fatty acid intak on health and disease are extensively documented. Certain PUFAs such as ω-3 (e.g., α-linolenic acid) and ω-6 (e.g., linoleic acid) can only be supplied by dietary intake in mammals. Of these, α-linolenic acid is also precursor for docosahexaenoic acid (22:6), which is the most abundant ω-3 fatty acid in mammalian brain and retina (though absent in flies). ω-3 and ω-6 deficiency is associated with cardiovascular disease and diabetes, can lead to cognitive defects in rodents, and within the mammalian retina leads to a reduced a-wave in the ERG. However, the underlying mechanisms are poorly understood, and in this respect, the current results provide a striking and novel example of how dietary fatty acids can profoundly and specifically influence in vivo performance and behavior via a defined step within the context of a classical G-protein-coupled signaling cascade. Interestingly, the importance of the mechanical properties of membranes containing polyunsaturated phospholipids has also been highlighted in recent studies of mechanosensation and rapid endocytosis (Randall, 2015).

    Functional cooperation between the IP3 receptor and Phospholipase C secures the high sensitivity to light of Drosophila photoreceptors in vivo

    Drosophila phototransduction is a model system for the ubiquitous phosphoinositide signaling. In complete darkness, spontaneous unitary current events (dark bumps) are produced by spontaneous single Gqα activation, while single-photon responses (quantum bumps) arise from synchronous activation of several Gqα molecules. Recent studies have shown that most of the spontaneous single Gqα activations do not produce dark bumps, because of a critical phospholipase Cβ (PLCβ) activity level required for bump generation. Surpassing the threshold of channel activation depends on both PLC activity and cellular [Ca(2+)], which participates in light excitation via a still unclear mechanism. This study shows that in IP3 receptor (IP3R)-deficient photoreceptors, both light-activated Ca(2+) release from internal stores and light sensitivity were strongly attenuated. This was further verified by Ca(2+) store depletion, linking Ca(2+) release to light excitation. In IP3R-deficient photoreceptors, dark bumps were virtually absent and the quantum-bump rate was reduced, indicating that Ca(2+) release from internal stores is necessary to reach the critical level of PLCβ catalytic activity and the cellular [Ca(2+)] required for excitation. Combination of IP3R knockdown with reduced PLCbeta catalytic activity resulted in highly suppressed light responses that were partially rescued by cellular Ca(2+) elevation, showing a functional cooperation between IP3R and PLCβ via released Ca(2+). These findings suggest that in contrast to the current dogma that Ca(2+) release via IP3R does not participate in light excitation, this study shows that released Ca(2+) plays a critical role in light excitation. The positive feedback between PLCβ and IP3R found here may represent a common feature of the inositol-lipid signaling (Kohn, 2015).

    In this study, in vivo light-response suppression was accompanied by reduced Ca2+ release from IP3-sensitive stores. In addition, the rate of spontaneously produced dark bumps, which is highly sensitive to Gqα-dependent PLCβ catalytic activity and cellular Ca2+ level, was virtually abolished in IP3R-deficient photoreceptors. This dark-bump elimination indicates that the suppressed Ca2+ release from IP3-sensitive stores underlies the suppressed catalytic activity of PLCβ, leading to suppressed light response in IP3R-deficient photoreceptors. Further evidence that the suppressed light response arises from inhibition of Ca2+ release from IP3-sensitive stores came from blocking the Ca2+ pump by Tg, which mimicked the phenotype of the IP3R-deficient photoreceptors in WT flies. The above findings indicate that IP3R-mediated Ca2+ release has a critical role in light excitation of Drosophila photoreceptors. The combination of the PLCβ mutant norpAH43 with IP3R-deficient photoreceptors, which synergistically suppressed the light response, strongly suggests that there is functional cooperation between the IP3R and PLCβ in generation of the light response (Kohn, 2015).

    It has been previously shown that an increase in cytosolic Ca2+ participates in light excitation as evidenced by enhancement of the light response following photo release of caged Ca2+ at the rising phase of the light response. The target of Ca2+ action has not been entirely resolved. PLCβ is an important target for Ca2+ action and the regulation of its catalytic activity by Ca2+ has been thoroughly investigated. These studies showed that the positive charge of Ca2+ is used to counterbalance local negative charges formed in the active site during the course of the catalytic reaction. Accordingly, Ca2+ performs electrostatic stabilization of both the substrate and the transition state, thus providing a twofold contribution to lower the activation energy of the enzyme reaction (Kohn, 2015).

    The following model explains how functional cooperation between the IP3R and PLCβ via the released Ca2+ operates and secures quantum-bump production: absorption of a single photon, which induces activation of several PLCβ molecules, is initially insufficient at resting Ca2+ levels to reach the critical level of PLCβ activity required for TRP/TRPL channel activation. Nevertheless, the IP3 molecules produced by the given PLCβ activity are able to activate the nearby IP3Rs, mobilize Ca2+ from the stores, and elevate PLCβ activity above the threshold required for TRP/TRPL channel activation. In addition, the released Ca2+ may also reduce the threshold of TRP/TRPL channel activation and allow bump generation. According to this model, the following enzymatic reactions may explain the current findings. Each Gqα-activated PLCβ has low catalytic activity due to the relatively low (<160 nM) resting Ca2+ concentration in the cytosol. In addition, each activated PLCβ remains active for only a short (approximately several tens of milliseconds) time due to the GTPase-activating protein activity of PLCβ that causes a rapid hydrolysis of Gqα-GTP followed by inactivation of PLCβ. The initial low catalytic activity of PLCβ is apparently below the threshold required for activation of the TRP and TRPL channels, but this low activity still results in hydrolysis of PIP2 producing IP3. Since there are no IP3 buffers in the microvilli and the IP3 degradation time is relatively slow (~1 s), the produced IP3 molecules diffuse fast along the microvillus at an estimated time of ~1 ms along 1 microm long microvillus and bind to IP3R located at the nearby submicrovillar cisternae (SMC; the photoreceptors' extensions of smooth ER). IP3R channels residing at the SMC, which are large channels with high sensitivity for IP3 and thus can be activated at low PLCβ activity, open and release Ca2+ juxtaposed to the base of the microvillus. The released Ca2+ steeply raises the local Ca2+ concentration, probably to the microM range, because of the very small aqueous volume of the microvillus and the relatively large local Ca2+ elevation via the release mechanism (Kohn, 2015).

    Accordingly, a single IP3R channel can release ∼104 Ca2+ ions in 1 ms channel opening and Ca2+-induced Ca2+ release mechanism is a property of the IP3R channels and of the ryanodine receptors, which reside in the ER. Ca2+ released via IP3R of the WT SMC diffuse back toward the activated PLCβ and the TRP/TRPL channels in the microvillus. Although Ca2+ diffuses ∼20-fold slower than IP3 due to strong buffering, the diffusion constant strongly depends on Ca2+ concentration. Accordingly, at ~250 μM the Ca2+ diffusion coefficient is as large as that of IP3. Once a single TRP channel is activated, the large Ca2+ influx through this channel is sufficient to facilitate the rest of the active PLCβ molecules or reduce the threshold for TRP/TRPL channel activation in this microvillus and produce a bump that reflects activation of the entire microvillus . When there is abnormally low Ca2+ release via the IP3R because of low IP3R expression levels (IP3R-RNAi), there is not enough Ca2+ to increase PLC activity or to reduce TRP activation threshold, and activated PLC in this microvillus does not produce a bump, leading to abnormally low frequency of dark bumps (Kohn, 2015).

    The invasive whole-cell recording technique, which was used in previous studies and avoided Ca2+ buffering of the pipette solution, most likely resulted in abnormally elevated cytosolic Ca2+ concentration, which also allowed the Ca2+ pump to keep the stores full. This artificially elevated cytosolic [Ca2+] together with the constitutive Ca2+ leak from the full stores, bypassed the need to mobilize Ca2+ via functional IP3R to facilitate PLCβ activity and reach its critical catalytic activity level needed to activate the TRP/TRPL channels. In the present study in the intact eye, a significant reduction in light-response amplitude was observed when the IP3R level was reduced. Furthermore, when cellular [Ca2+] was reduced by prolonged extracellular EGTA application, the light response of the IP3R-deficient flies was further suppressed. Moreover, when using invasive patch-clamp whole-cell recordings without Ca2+ buffering of the pipette solution, no significant difference between WT and IP3R-deficient flies was observed, as found in the previous study. However, when pipette Ca2+ was reduced with EGTA, the phenotype of reduced light excitation was observed in both reduced quantum-bump frequency as well as in macroscopic light-response suppression. Unlike quantum bumps, dark bumps were virtually eliminated even without buffering the pipette Ca2+ in IP3R-deficient flies, indicating that in the dark the IP3R-deficiency led to abnormally low cytosolic [Ca2+], possibly due to reduced Ca2+ leak from stores leading to cellular [Ca2+] below the critical level required for PLC activation observed in WT flies. Alternatively, the positive feedback between the released Ca2+ and PLC may function at single PLC molecules. Hence the nominal pipette Ca2+ is not sufficient to allow PLC activity to pass the threshold of channel activation, but the released Ca2+ via IP3R activation together with pipette Ca2+ allows PLC activity to pass this threshold and generate dark-bump (Kohn, 2015).

    There is a striking functional similarity between, the cerebellar Purkinje cell (PC) proteins of the IP signaling and Drosophila photoreceptors, but the link of cerebellar mGluR1 receptor to TRPC3 activation is not clear. Interestingly, in PC neurons, stromal interaction molecule 1 (STIM1) was proved an essential regulator of Ca2+ level in neuronal endoplasmic reticulum Ca2+ stores. Accordingly, STIM1-specific deletion caused impairments in slow synaptic current and cerebellar motor behavior. Strikingly, refilling empty Ca2+ stores through increased Ca2+ level in the cytosol partially rescued the phenotype of the stim1 knock-out mice, reminiscent of the rescue of the phenotype of the IP3R-deficient fly by artificially elevated cytosolic Ca2+. Thus, the facilitatory role of released Ca2+ on PLC in light excitation of Drosophila photoreceptors represents an essential mechanism that operates in other PI systems (Kohn, 2015).

    A new genetic model for calcium induced autophagy and ER-stress in Drosophila photoreceptor cells

    Cytoplasmic Ca2+ overload is known to trigger autophagy and ER-stress. Furthermore, ER-stress and autophagy are commonly associated with degenerative pathologies, but their role in disease progression is still a matter of debate, in part, owing to limitations of existing animal model systems. The Drosophila eye is a widely used model system for studying neurodegenerative pathologies. Recently, the Drosophila protein, Calphotin, has been characterized as a cytosolic immobile Ca2+ buffer, which participates in Ca2+ homeostasis in Drosophila photoreceptor cells. Exposure of calphotin hypomorph flies to continuous illumination, which induces Ca2+ influx into photoreceptor cells, results in severe Ca2+-dependent degeneration. This study shows that this degeneration is autophagy and ER-stress related and thus provides a new model in which genetic manipulations trigger changes in cellular Ca2+ distribution. This model constitutes a framework for further investigations into the link between cytosolic Ca2+, ER-stress and autophagy in human disorders and diseases (Weiss, 2015).

    Autophagy and ER-stress are highly conserved during evolution as many other essential cell maintenance pathways. Autophagy has been implicated in several pathologies, including protein aggregate disorders, neurodegeneration and cancer. Both autophagy and ER-stress are commonly associated with degenerative pathologies, but their role in disease progression is still a matter of debate. Many human genes, implicated in degenerative diseases, have orthologs in Drosophila. Studies in fly models have identified novel factors that regulate these diseases; therefore, Drosophila is a powerful model system for studying these genes (Weiss, 2015).

    An illuminating example is the Drosophila model for class III autosomal dominant retinitis pigmentosa (ADRP), in which ER-stress has been shown to play a protective role against retinal degeneration. Moreover, it has been shown that when Drosophila cells (both photoreceptors and S2 cells) are pre-exposed to a mild ER-stress, they are protected from treatments with various cell death agents. It has also been shown recently that activation of mild ER stress is neuroprotective, both in Drosophila and mouse models of Parkinson disease, and this protection is a consequence of autophagy activation. Accordingly, it is well established that activation of the ER-stress response by accumulation of misfolded proteins (unfolded protein response, UPR) can trigger autophagy (Weiss, 2015).

    It is well established that elevation of intracellular Ca2+ can trigger autophagy. The stimulation of autophagy by elevated cytosolic [Ca2+] has been studied mainly in tissue culture cells, in which mobilization of Ca2+ is triggered by application of various Ca2+ mobilizing agents (e.g. thapsigargin, ionomycin, vitamin D). However, since increased cytosolic [Ca2+] also activates ER-stress which induces autophagy, a question arises as to whether the activation of autophagy by Ca2+ is induced directly or indirectly via activation of ER-stress. The fact that thapsigargin-induced autophagy occurs in UPR-deficient cells, suggests a direct involvement of Ca2+ in the induction of autophagy. Although Ca2+ regulation of autophagy has been found to be mediated through the activation of Bcl-2 and calmodulin-dependent kinase kinase-β, the target by which Ca2+ activates autophagy directly is yet to be elucidated. These unsolved issues can be conveniently addressed in the new model of Ca2+ induced autophagy in Drosophila photoreceptor cells (Weiss, 2015).

    Neurodegenerative diseases, such as Parkinson, Alzheimer and Huntington’s diseases are accompanied by the accumulation of large aggregates of mutant proteins and are autophagy associated diseases. These autophagy associated diseases, which are characterized by abnormal protein aggregations, have highly developed model systems of Drosophila. The increased autophagosome formation observed in these diseases, may play a pro- tective role by degrading misfolded proteins. Many of the above diseases, including Parkinson and Alzheimer diseases, are also associated with reduced cellular Ca2+ buffering and impairments in cellular Ca2+ homeostasis reminiscent of Drosophila photoreceptors with reduced calphotin. Therefore, combining the Drosophila models of autophagy associated diseases with calphotin hypomorph can be very useful for studying the possible link between reduced cellular Ca2+ buffering and neurodegeneration (Weiss, 2015).

    In the genetic fly model of calphotin hypomorph, reduced calphotin levels, which trigger changes in cellular Ca2+ homeostasis allows induction of Ca2+ induced autophagy, simply by changing the illumination conditions of the flies’ environment. Accordingly, both the severity and progression of the observed degeneration can be highly controlled and manipulated. The degeneration process of this experimental model is sufficiently slow, allowing monitoring the progression of the degeneration process and elucidation of the involved proteins. Thus, the Drosophila Cpn hypomorph photoreceptor cells constitutes a powerful model, in which genetic manipulation combined with illumination determine the level of sustained cellular Ca2+ that trigger the induction of Ca2+ dependent autophagy, ER- stress and cell death. This model can provide a framework for further investigations into the link between cytosolic Ca2C, ER-stress and autophagy in human disorders and diseases (Weiss, 2015).

    The GTP- and phospholipid-binding protein TTD14 regulates trafficking of the TRPL ion channel in Drosophila photoreceptor cells

    Recycling of signaling proteins is a common phenomenon in diverse signaling pathways. In photoreceptors of Drosophila, light absorption by rhodopsin triggers a phospholipase Cβ-mediated opening of the ion channels Transient receptor potential (TRP) and TRP-like (TRPL) and generates the visual response. The signaling proteins are located in a plasma membrane compartment called rhabdomere. The major rhodopsin (Rh1) and TRP are predominantly localized in the rhabdomere in light and darkness. In contrast, TRPL translocates between the rhabdomeral plasma membrane in the dark and a storage compartment in the cell body in the light, from where it can be recycled to the plasma membrane upon subsequent dark adaptation. This study identified the gene mutated in trpl translocation defective 14 (ttd14) (CG30118), which is required for both TRPL internalization from the rhabdomere in the light and recycling of TRPL back to the rhabdomere in the dark. TTD14 is highly conserved in invertebrates and binds GTP in vitro. TTD14 is a cytosolic protein and binds to PtdIns(3)P, a lipid enriched in early endosome membranes, and to phosphatidic acid. In conclusion, TTD14 is a novel regulator of TRPL trafficking, involved in internalization and subsequent sorting of TRPL into the recycling pathway that enables this ion channel to return to the plasma membrane (Cerny, 2015).

    Drosophila vision depends on carcinine uptake by an organic cation transporter

    Recycling of neurotransmitters is essential for sustained neuronal signaling, yet recycling pathways for various transmitters, including histamine, remain poorly understood. In the first visual ganglion (lamina) of Drosophila, photoreceptor-released histamine is taken up into perisynaptic glia, converted to carcinine, and delivered back to the photoreceptor for histamine regeneration. This study identified an organic cation transporter, CarT (carcinine transporter), that transports carcinine into photoreceptors during histamine recycling. CarT mediated in vitro uptake of carcinine. Deletion of the CarT gene caused an accumulation of carcinine in laminar glia accompanied by a reduction in histamine, resulting in abolished photoreceptor signal transmission and blindness in behavioral assays. These defects were rescued by expression of CarT cDNA in photoreceptors, and they were reproduced by photoreceptor-specific CarT knockdown. These findings suggest a common role for the conserved family of CarT-like transporters in maintaining histamine homeostasis in both mammalian and fly brains (Chaturvedi, 2016).

    Evidence for dynamic network regulation of Drosophila photoreceptor function from mutants lacking the neurotransmitter histamine>

    Synaptic feedback from interneurons to photoreceptors can help to optimize visual information flow by balancing its allocation on retinal pathways under changing light conditions. But little is known about how this critical network operation is regulated dynamically. This study investigated this question by comparing signaling properties and performance of wild-type Drosophila R1-R6 photoreceptors to those of the HdcJK910 mutant, which lacks the neurotransmitter histamine and therefore cannot transmit information to interneurons. Recordings show that HdcJK910 photoreceptors sample similar amounts of information from naturalistic stimulation to wild-type photoreceptors, but this information is packaged in smaller responses, especially under bright illumination. Analyses reveal how these altered dynamics primarily resulted from network overload that affected HdcJK910 photoreceptors in two ways. First, the missing inhibitory histamine input to interneurons almost certainly depolarized them irrevocably, which in turn increased their excitatory feedback to HdcJK910 R1-R6s. This tonic excitation depolarized the photoreceptors to artificially high potentials, reducing their operational range. Second, rescuing histamine input to interneurons in HdcJK910 mutant also restored their normal phasic feedback modulation to R1-R6s, causing photoreceptor output to accentuate dynamic intensity differences at bright illumination, similar to the wild-type. These results provide mechanistic explanations of how synaptic feedback connections optimize information packaging in photoreceptor output and novel insight into the operation and design of dynamic network regulation of sensory neurons (Dau, 2016).

    Phospholipase D activity couples plasma membrane endocytosis with retromer dependent recycling

    During illumination, the light sensitive plasma membrane (rhabdomere) of Drosophila photoreceptors undergoes turnover with consequent changes in size and composition. However the mechanism by which illumination is coupled to rhabdomere turnover remains unclear. This study found that photoreceptors contain a light-dependent phospholipase D (PLD) activity. During illumination, loss of PLD resulted in an enhanced reduction in rhabdomere size, accumulation of Rab7 positive, rhodopsin1-containing vesicles (RLVs) in the cell body and reduced rhodopsin protein. These phenotypes were associated with reduced levels of phosphatidic acid, the product of PLD activity and were rescued by reconstitution with catalytically active PLD. In wild type photoreceptors, during illumination, enhanced PLD activity was sufficient to clear RLVs from the cell body by a process dependent on Arf1-GTP levels and retromer complex function. Thus, during illumination, PLD activity couples endocytosis of RLVs with their recycling to the plasma membrane thus maintaining plasma membrane size and composition (Thakur, 2016).

    Calcium signalling in Drosophila photoreceptors measured with GCaMP6f

    Drosophila phototransduction is mediated by phospholipase C leading to activation of cation channels (TRP and TRPL) in the 30000 microvilli forming the light-absorbing rhabdomere. The channels mediate massive Ca2+ influx in response to light, but whether Ca2+ is released from internal stores remains controversial. Flies were generated expressing GCaMP6f in their photoreceptors and measured Ca2+ signals from dissociated cells, as well as in vivo by imaging rhabdomeres in intact flies. In response to brief flashes, GCaMP6f signals had latencies of 10-25ms, reached 50% Fmax with approximately 1200 effectively absorbed photons and saturated (DeltaF/F0 approximately 10-20) with 10000-30000 photons. In Ca2+ free bath, smaller (DeltaF/F0 approximately 4), long latency ( approximately 200ms) light-induced Ca2+ rises were still detectable. These were unaffected in InsP3 receptor mutants, but virtually eliminated when Na+ was also omitted from the bath, or in trpl;trp mutants lacking light-sensitive channels. Ca2+ free rises were also eliminated in Na+/Ca2+ exchanger mutants, but greatly accelerated in flies over-expressing the exchanger. These results show that Ca2+ free rises are strictly dependent on Na+ influx and activity of the exchanger, suggesting they reflect re-equilibration of Na+/Ca2+ exchange across plasma or intracellular membranes following massive Na+ influx. Therefore, there were no significant light-induced release of Ca2+ from internal stores (Asteriti, 2017).

    CULD is required for rhodopsin and TRPL channel endocytic trafficking and survival of photoreceptor cells

    Endocytosis of G-protein-coupled receptors (GPCRs) and associated channels contributes to desensitization and adaptation of a variety of signaling cascades. In Drosophila, the major light sensing rhodopsin, Rh1, and the downstream ion channel, Transient Receptor Potential Like (TRPL), are endocytosed in response to light, but the mechanism is unclear. Using an RNA-Sequencing approach, this study discovered CULD (CG17352), a photoreceptor-cell enriched CUB- and LDLa-domain transmembrane protein that is required for endocytic trafficking of Rh1 and TRPL. CULD localized to endocytic Rh1- or TRPL-positive vesicles. Mutations in culd resulted in the accumulation of Rh1 and TRPL within endocytic vesicles, and disrupted the regular turnover of endocytic Rh1 and TRPL. In addition, loss of CULD induced light- and age-dependent retinal degeneration, and reduced levels of Rh1 but not TRPL suppressed retinal degeneration in culd null mutant flies. These data demonstrate that CULD plays an important role in the endocytic turnover of Rh1 and TRPL, and suggest that CULD-dependent rhodopsin endocytic trafficking is required for maintaining photoreceptor integrity (Xu, 2016)

    G-protein-coupled receptors (GPCRs) are the largest family of membrane receptors and, therefore, transduce signals from a wide variety of hormones, cytokines, neurotransmitters, as well as sensory stimuli. Each of these interactions triggers distinct intracellular responses through heterotrimeric G proteins. Upon continuous stimulation, GPCRs are deactivated by arrestins, and internalized through dynamin-dependent endocytosis. Many internalized GPCRs undergo lysosomal degradation and/or recycling, leading to downregulation of receptor levels, which is important for reducing the strength and duration of cellular responsiveness following various stimuli (Xu, 2016).

    The Drosophila phototransduction cascade is a model pathway for the dissection of GPCR signaling and associated regulatory processes. Proteins of the visual signal transduction cascade are found within rhabdomeres, which are specialized compartments within photoreceptor cells that contain tightly packed microvilli. Light-induced activation of rhodopsin triggers the phototransduction cascade by stimulating the vision protein phospholipase C, which is encoded by the no receptor potential A (norpA) gene, through the α subunit of the heterotrimeric G protein DGq. This opens the transient receptor potential (TRP) channel and the TRP-like (TRPL) Ca2+/cation channel, and depolarizes the photoreceptor neurons. Meanwhile, activated rhodopsin, which is referred to as metarhodopsin, is immediately bound by arrestin and deactivated. After inactivation, metarhodopsin is either photoconverted back into rhodopsin or internalized for degradation. Although the majority of internalized metarhodopsin is degraded, with newly synthesized rhodopsin replenishing the pool, it has recently been reported that internalized rhodopsin (Rh1; encoded by ninaE in Drosophila melanogaster) can be recycled upon stimulation with light. The principle arrestin, Arr2, plays a pivotal role in deactivating rhodopsin, whereas Arr1 binds and internalizes rhodopsin (Xu, 2016).

    Long-term adaptation to light stimuli also involves the dynamic activity-dependent translocation of signaling proteins that are not GPCRs. As seen with mammalian Rod photoreceptors, light induces the movement of Arr2 and Arr1 into the rhabdomeres. In Drosophila, TRP and TRPL function as the primary light-activated channels. TRP stably localizes to the rhabdomeres by forming a multiprotein signaling complex, the signalplex with inactivation-no-after-potential D protein (INAD), a protein that contains five PDZ domains. In contrast, illumination results in TRPL translocating from the rhabdomeres to an intracellular storage compartment within the cell body. However, the mechanisms that underlie light-induced translocation and trafficking of rhodopsin and TRPL are not yet fully understood. Furthermore, it is unclear whether this endocytic trafficking of TRPL plays a physiological role in maintaining the integrity of photoreceptor cells (Xu, 2016).

    By using an RNA-Sequencing (RNA-Seq) approach, this study identified a so-far-unknown gene that is enriched in photoreceptors, and encodes a transmembrane protein with both a CUB and an LDLa domain. This protein was named CULD (CUB- and LDLa-domain protein). CULD mainly localized to the endocytic TRPL- or Rh1-positive vesicles. Mutations in culd led to endosomal accumulation of Rh1 and TRPL, which disrupted the light sensitivity of photoreceptors; blocking of Arr1-mediated endocytosis eliminated the intracellular accumulation of Rh1. Moreover, culd mutants underwent light-dependent retinal degeneration, and resulted in a phenotype that could be rescued by reducing the levels of Rh1. These data indicate that CULD is essential for the function and survival of photoreceptor cells by promoting the endocytic turnover of Rh1 and TRPL (Xu, 2016).

    A microarray analysis has previously been used to compare the genes expressed in wild-type heads with heads from a mutant fly that lacked eyes in order to identify eye-enriched genes, which led to the further identification of some genes functioning in phototransduction. However, owing to multiple cell types in the compound eye, many genes identified in this analysis might not function in photoreceptor cells. This study describes an RNA-Seq screen to identify genes expressed predominantly in photoreceptors. Among the 58 genes identified, 36 genes were known to function in photoreceptor cells, representing most of the genes that play major roles in phototransduction or retinal degeneration. However, 22 genes had not been described as being enriched in photoreceptor cells previously. Among them, cg9935 (Eye-enriched kainate receptor: Ekar) has been recently reported to regulate the retrograde glutamate signal in photoreceptor cells and contribute to light-evoked depolarization during phototransduction (Hu, 2015). This study further characterized the new photoreceptor cell-enriched gene culd as being required for turnover of Rh1 and TRPL. Although culd had also been identified as an eye-enriched gene in the earlier microarray analysis that compared RNA expression in wild-type and eyeless heads, 93 other eye-enriched candidates prevented focusing on CULD. In this RNA-Seq screen, only photoreceptor-cell-enriched genes can be identified, and a reasonable number of candidates might represent new factors functioning in phototransduction. However, some eye-enriched genes important for phototransduction might be missed in this screen. For example, recently identified polyglutamine-binding protein 1 (PQBP1) was not found as a photoreceptor-cell-specific gene in the RNA-Seq screen. This might be because PQBP1 is also expressed in other non-photoreceptor retinal cells. Overall, this screen for photoreceptor-enriched genes sheds a light on further understanding of phototransduction and mechanisms of retinal degeneration (Xu, 2016).

    Appropriate signals cause arrestins to translocate to the plasma membrane where they bind to activated GPCRs, thereby inhibiting G-protein-dependent signaling and regulating GPCR endocytosis and trafficking. In Drosophila there are two arrestins within photoreceptors, Arr1 and Arr2. Although Arr2 binds to Rh1, it is Arr1 that primarily colocalizes with Rh1 in internalized vesicles. Therefore, Arr1 might mediate light-dependent endocytosis of Rh1, whereas Arr2 functions to quench activated Rh1. In culd mutant flies, Rh1 was immobilized within endocytic vesicles and Arr1 colocalized with the endocytic Rh1; blocking the Arr1-medicated endocytosis in culd mutant cells eliminated the abnormal intracellular accumulation of Rh1. These data strongly suggest that CULD functions downstream of Arr1-mediated endocytosis of Rh1 (Xu, 2016).

    Early endosomes containing Rab5 serve as a focal point of the endocytic pathway. Sorting events initiated in early endosomes determine the subsequent fate of internalized proteins, that is, whether they will be recycled to the plasma membrane or degraded within lysosomes. Rh1 and TRPL share the same internalization pathway, and during light stimulation Rab5 initially mediates this vesicular transport pathway. In wild-type photoreceptors, however, Rh1 and TRPL have different fates from common Rab5-positive early endosomes. The majority of Rh1 is eventually delivered to lysosomes for degradation, whereas most internalized TRPL tends to be stored. In wild-type cells, the photoreceptor-enriched protein CULD colocalized with the endocytic TRPL or Rh1 vesicles, and the majority of CULD-positive vesicles were also Rab5-positive. This spatial pattern indicates that CULD is required for the endocytic trafficking of TRPL and Rh1 after they are internalized (Xu, 2016).

    CULD functions during the early steps of endocytosis that immediately follow internalization, which is a pathway involved in rhodopsin and TRPL endocytic turnover. Eliminating CULD had profound effects on the photoreceptor physiology. In both vertebrates and invertebrates, the light sensitivity of photoreceptor cells is primarily determined by functional rhodopsin. The culd mutant flies exhibited a gradual reduction in light sensitivity, which suggests that the amount of functional rhodopsin is reduced in culd mutant flies. As the amount of the monomer form of Rh1 was not affected and a large fraction of Rh1 accumulated within intracellular vesicles in culd mutant photoreceptor cells, the rhabdomeral Rh1 levels might be reduced. It is also likely that the endocytic degradation of Rh1 scavenges damaged Rh1 molecules, and blocking this process might lead to the accumulation of dysfunctional Rh1 in rhabdomeres (Xu, 2016).

    TRPL has been reported to translocate from rhabdomeres to intracellular compartments for storage during prolonged light stimulation. However, a recent study suggests that some endocytic TRPL proteins are also delivered to lysosomes for degradation. Mutations in culd impaired TRPL endocytic trafficking upon light stimulation, leading to the retention of TRPL in Rab7-positive vesicles. TRPL protein levels were increased in culd mutants and this is probably due to decreased TRPL degradation (Xu, 2016).

    As a major light sensor within photoreceptor cells, a small amount of activated Rh1 is internalized and degraded upon light stimulation. This is followed by replenishment of the rhabdomeric Rh1 pool. Therefore, a balance between Rh1 endocytosis and replenishment is required for Rh1 homeostasis under light conditions. Prolonged exposure to blue light triggers massive endocytosis of Rh1 and leads to a gradual loss of Rh1. Mutations in culd blocked Rh1 degradation during prolonged light treatment, indicating that the loss of CULD inhibited the Rh1-degradation pathway. Unlike TRPL, Rh1 levels were not increased, which suggests that Rh1 replenishment is strictly controlled. Given that, in Drosophila, rhodopsin levels are regulated by both the synthesis of the opsin and the chromophore subunits, it might be reasonable that in culd mutant cells, the chromophore is not released from the accumulated rhodopsin, and the reduction of free retinal pool might limit the synthesis of new rhodopsin (Xu, 2016).

    Both vertebrates and invertebrates have a family of transmembrane proteins that contain both CUB- and LDLa- domains. However, only a few CUB/LDLa proteins have been functionally characterized. Among these proteins, NETO1 and NETO2 (see Drosophila Neto) have been intensively studied. NETO1 functions as an auxiliary subunit of ionotropic glutamate receptors, N-methyl-D-aspartate receptors and kainate receptors, modulating the channel properties of these glutamate receptors. NETO2 maintains normal levels of the neuron-specific K+-Cl- co-transporter KCC2 (also known as SLC12A5), and loss of NETO2-KCC2 interactions reduces KCC2-mediated Cl- extrusion, and decreases synaptic inhibition in hippocampal neurons. Moreover, in both Drosophila and C. elegans, the CUB/LDLa proteins NETO and SOL-2 are required for the clustering and functioning of glutamine receptors, thereby contributing to neuronal signaling pathways. This study cloned a new gene culd, which encodes a member of the CUB/LDLa family proteins specifically expressed in the Drosophila photoreceptor cell; this protein containing a CUB domain, an LDLa domain and one predicted transmembrane motif. CULD was not directly required for the activity of receptors or channels, but instead mediated the endocytic trafficking of Rh1 and TRPL. Loss of CULD led to the accumulation of Rh1 and TRPL in endocytic vesicles, and subsequent retinal degeneration. Therefore, this study revealed a new function of the CUB/LDLa family proteins, namely the endocytic turnover of receptors and channels (Xu, 2016).

    It has been proposed that the accumulation of Rh1-Arr2 complexes in late endosomes triggers cell death of photoreceptor cells. Internalized Rh1-Arr2 complexes are not degraded but instead accumulate in late endosomes of norpA mutant photoreceptor cells. Similar rhodopsin accumulations are seen in mutations that affect the trafficking of late endosomes to lysosomes, which causes light-dependent retinal degeneration. Toxic Rh1-Arr2 complexes also induce retinal degeneration in rdgC, rdgB and fatp mutant flies. The culd mutations caused the accumulation of Rh1-Arr1 complexes and TRPL in endocytic vesicles and light-dependent retinal degeneration, suggesting that endosomal accumulation of either channels or receptors induced cell death. In addition, the evidence that ninaEP332 but not trpl302 rescued photoreceptor degeneration of the culd1 mutants suggests that abnormal Rh1-Arr1 accumulation induces cell degeneration, whereas intracellular accumulation of TRPL does not contribute to the neuronal degeneration (Xu, 2016).

    The beta-alanine transporter BalaT is required for visual neurotransmission in Drosophila

    The recycling of neurotransmitters is essential for sustained synaptic transmission. In Drosophila, histamine recycling is required for visual synaptic transmission. Synaptic histamine is rapidly taken up by laminar glia, and is converted to carcinine. After delivered back to photoreceptors, carcinine is hydrolyzed to release histamine and β-alanine. This histamine is repackaged into synaptic vesicles, but it is unclear how the β-alanine is returned to the laminar glial cells. This study identified a new beta-alanine transporter, which has been named BalaT (Beta-alanine Transporter). Null balat mutants exhibited lower levels of β-alanine, as well as less β-alanine accumulation in the retina. Moreover, BalaT is expressed and required in retinal pigment cells for maintaining visual synaptic transmission and phototaxis behavior. These results provide the first genetic evidence that retinal pigment cells play a critical role in visual neurotransmission, and suggest that a BalaT-dependent beta-alanine trafficking pathway is required for histamine homeostasis and visual neurotransmission (Han, 2017).

    A single residue mutation in the Galphaq subunit of the G protein complex causes blindness in Drosophila

    Heterotrimeric G proteins play central roles in many signaling pathways, including the phototransduction cascade in animals. However, the degree of involvement of the G protein subunit Galphaq is not clear since animals with strong loss of function mutations previously reported remain responsive to light stimuli. This study recovered a new allele of Galphaq in Drosophila that abolishes light response in a conventional ERG assay, and reduces sensitivity in whole-cell recordings of dissociated cells by at least 5 orders of magnitude. In addition, mutant eyes demonstrate a rapid rate of degeneration in the presence of light. The new allele is likely the strongest hypomorph described to date. Interestingly, the mutant protein is produced in the eyes but carries a single amino acid change of a conserved hydrophobic residue that has been assigned to the interface of interaction between Galphaq and its downstream effector PLC. This study thus uncovered possibly the first point mutation that specifically affects this interaction in vivo (Cao, 2017).


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    list of proteins involved in visual signal transduction

    date revised: 20 April 2017

    Zygotically transcribed genes

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