Studies of theDrosophila norpA phototransduction mutant

Summary ThenorpA ^H44 phototransduction mutant ofDrosophila melanogaster, an allele that, on eclosion, does not exhibit a receptor potential was found, at later ages, to undergo light and temperature dependent degeneration of its photoreceptors as well as decreases in rhodopsin concentration. Pseudopupil measurements and light and electron microscopy were used to monitor the structure of the photoreceptors. WhennorpA ^H44 flies were maintained exclusively in the dark, no changes in structure or rhodopsin concentration were observed. When maintained on a 12 h light-12 h dark cycle, structural changes were first observed at 6 days of age for flies maintained at 24 °C or at 12 days of age for flies maintained at 19 °C. When the light-dark cycle was initiated after 10 days in the dark there was a more rapid loss of rhodopsin concentration and pseudopupil. The data suggest that even in the dark, although no obvious changes in structure or rhodopsin concentration were observed, certain processes that support these components had been affected.NorpA ^P12 , an allele that exhibits small receptor potential amplitudes, also displayed age- and light-dependent photoreceptor degeneration and decreases in rhodopsin concentration, whereas no degeneration or decreases in rhodopsin were observed innorpA ^P16 , an allele that exhibits receptor potential amplitudes similar to those of wild-type. The data suggest that the processes that affect phototransduction, such as the phosphatidylinositol cycle, have a long-term role in the maintenance of rhodopsin concentration and photoreceptor integrity.Abbreviation PI phosphatidylinositol     

Impaired Mitochondrial Energy Production Causes Light-Induced Photoreceptor Degeneration Independent of Oxidative Stress

Two insults often underlie a variety of eye diseases including glaucoma, optic atrophy, and retinal degeneration—defects in mitochondrial function and aberrant Rhodopsin trafficking. Although mitochondrial defects are often associated with oxidative stress, they have not been linked to Rhodopsin trafficking. In an unbiased forward genetic screen designed to isolate mutations that cause photoreceptor degeneration, we identified mutations in a nuclear-encoded mitochondrial gene, ppr, a homolog of human LRPPRC. We found that ppr is required for protection against light-induced degeneration. Its function is essential to maintain membrane depolarization of the photoreceptors upon repetitive light exposure, and an impaired phototransduction cascade in ppr mutants results in excessive Rhodopsin1 endocytosis. Moreover, loss of ppr results in a reduction in mitochondrial RNAs, reduced electron transport chain activity, and reduced ATP levels. Oxidative stress, however, is not induced. We propose that the reduced ATP level in ppr mutants underlies the phototransduction defect, leading to increased Rhodopsin1 endocytosis during light exposure, causing photoreceptor degeneration independent of oxidative stress. This hypothesis is bolstered by characterization of two other genes isolated in the screen, pyruvate dehydrogenase and citrate synthase. Their loss also causes a light-induced degeneration, excessive Rhodopsin1 endocytosis and reduced ATP without concurrent oxidative stress, unlike many other mutations in mitochondrial genes that are associated with elevated oxidative stress and light-independent photoreceptor demise.     

Molecular genetics of retinal degeneration: A Drosophila perspective

Inherited retinal degeneration in Drosophila has been explored for insights into similar processes in humans. Based on the mechanisms, I divide these mutations in Drosophila into three classes. The first consists of genes that control the specialization of photoreceptor cells including the morphogenesis of visual organelles (rhabdomeres) that house the visual signaling proteins. The second class contains genes that regulate the activity or level of the major rhodopsin, Rh1, which is the light sensor and also provides a structural role for the maintenance of rhabdomeres. Some mutations in Rh1 (NinaE) are dominant due to constitutive activity or folding defects, like autosomal dominant retinitis pigmentosa (ADRP) in humans. The third class consists of genes that control the Ca²⁺ influx directly or indirectly by promoting the turnover of the second messenger and regeneration of PIP₂, or mediate the Ca²⁺-dependent regulation of the visual response. These gene products are critical for the increase in cytosolic Ca²⁺ following light stimulation to initiate negative regulatory events. Here I will focus on the signaling mechanisms underlying the degeneration in norpA, and in ADRP-type NinaE mutants that produce misfolded Rh1. Accumulation of misfolded Rh1 in the ER triggers the unfolded protein response (UPR), while endosomal accumulation of activated Rh1 may initiate autophagy in norpA. Both autophagy and the UPR are beneficial for relieving defective endosomal trafficking and the ER stress, respectively. However, when photoreceptors fail to cope with the persistence of these stresses, a cell death program is activated leading to retinal degeneration.     

Modulation of the dihydropyridine-sensitive calcium channels in Drosophila by a phospholipase C-mediated pathway

Disruption of phospholipase C-β (PLC) by the norpA mutations of Drosophila renders flies blind by affecting the light-evoked photoreceptor potential. We report here that the norpA-coded PLC modulates the 1,4-dihydropyridine (DHP)-sensitive Ca²⁺ channels in larval muscles. The DHP-sensitive current was reduced in the norpA mutants. Application of 1 μM phorbol 12-myristate 13-acetate (TPA) and 1 μM phorbol 12,13-didecanoate (PDD), activators of protein kinase C (PKC), rescued the current in the mutant fibers without significantly affecting the normal current. 4α-phorbol 12,13-didecanoate (4αPDD), an inactive analog of PDD, did not affect either the normal or the mutant current. One micromolar bisindolylmaleimide (BIM), an inhibitor of PKC, reduced the current in the normal fibers without affecting the mutant current. 300 μM sn-1,2-dioctanoyl-glycerol (DOG), an analog of diacylglycerol (DAG), increased the current in the mutant fibers. These experiments suggest that the DHP-sensitive Ca²⁺ channels in Drosophila may be modulated by the PLC-DAG-PKC pathway, and that the same PLC isozyme which is involved in phototransduction in the adult flies may also modulate muscle Ca²⁺ channels in the larval stage of development. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 265–275, 1997     

The rdgB gene in drosophila: Retinal degeneration in different mutant alleles and inhibition of degeneration by norpA

We used histology, optical measurements of photoreceptor integrity and electrophysiology to characterize rdgB mutants of Drosophila which have hereditary retinal degeneration. We studied different mutant alleles of rdgB which had been reported to have differing receptor involvement in degeneration. However, we found similar profiles of degeneration: compound eye receptors R1-6 degenerate structurally over one week in a diurnal lighting cycle while R7/8 are preserved. We confirmed earlier findings that norpA transduction mutants inhibit degeneration in rdgB. Our micrographs show degeneration in the lamina (first synaptic neuropil) but none in the medulla (second synaptic neuropil).     

Drosophila fabp is required for light-dependent Rhodopsin-1 clearance and photoreceptor survival

Rhodopsins are light-detecting proteins coupled with retinal chromophores essential for visual function. Coincidentally, dysfunctional Rhodopsin homeostasis underlies retinal degeneration in humans and model organisms. Drosophila ninaE^G69D mutant is one such example, where the encoded Rh1 protein imposes endoplasmic reticulum (ER) stress and causes light-dependent retinal degeneration. The underlying reason for such light-dependency remains unknown. Here, we report that Drosophila fatty acid binding protein (fabp) is a gene induced in ninaE^G69D/+ photoreceptors, and regulates light-dependent Rhodopsin-1 (Rh1) protein clearance and photoreceptor survival. Specifically, our photoreceptor-specific gene expression profiling study in ninaE^G69D/+ flies revealed increased expression of fabp together with other genes that control light-dependent Rh1 protein degradation. fabp induction in ninaE^G69D photoreceptors required vitamin A and its transporter genes. In flies reared under light, loss of fabp caused an accumulation of Rh1 proteins in cytoplasmic vesicles. The increase in Rh1 levels under these conditions was dependent on Arrestin2 that mediates feedback inhibition of light-activated Rh1. fabp mutants exhibited light-dependent retinal degeneration, a phenotype also found in other mutants that block light-induced Rh1 degradation. These observations reveal a previously unrecognized link between light-dependent Rh1 proteostasis and the ER-stress imposing ninaE^G69D mutant that cause retinal degeneration.     

Cross species analysis of Prominin reveals a conserved cellular role in invertebrate and vertebrate photoreceptor cells

The two fundamental types of photoreceptor cells have evolved unique structures to expand the apical membrane to accommodate the phototransduction machinery, exemplified by the cilia-based outer segment of the vertebrate photoreceptor cell and the microvilli-based rhabdomere of the invertebrate photoreceptor. The morphogenesis of these compartments is integral for photoreceptor cell integrity and function. However, little is known about the elementary cellular and molecular mechanisms required to generate these compartments. Here we investigate whether a conserved cellular mechanism exists to create the phototransduction compartments by examining the functional role of a photoreceptor protein common to both rhabdomeric and ciliated photoreceptor cells, Prominin. First and foremost we demonstrate that the physiological role of Prominin is conserved between rhabdomeric and ciliated photoreceptor cells. Human Prominin1 is not only capable of rescuing the corresponding rhabdomeric Drosophila prominin mutation but also demonstrates a conserved genetic interaction with a second photoreceptor protein Eyes Shut. Furthermore, we demonstrate the Prominin homologs in vertebrate and invertebrate photoreceptors require the same structural features and post-translational modifications for function. Moreover, expression of mutant human Prominin1, associated with autosomal dominant retinal degeneration, in rhabdomeric photoreceptor cells disrupts morphogenesis in ways paralleling retinal degeneration seen in ciliated photoreceptors. Taken together, our results suggest the existence of an ancestral Prominin-directed cellular mechanism to create and model the apical membranes of the two fundamental types of photoreceptor cells into their respective phototransduction compartments.     

Drosophila Fatty Acid Taste Signals through the PLC Pathway in Sugar-Sensing Neurons

Taste is the primary sensory system for detecting food quality and palatability. Drosophila detects five distinct taste modalities that include sweet, bitter, salt, water, and the taste of carbonation. Of these, sweet-sensing neurons appear to have utility for the detection of nutritionally rich food while bitter-sensing neurons signal toxicity and confer repulsion. Growing evidence in mammals suggests that taste for fatty acids (FAs) signals the presence of dietary lipids and promotes feeding. While flies appear to be attracted to fatty acids, the neural basis for fatty acid detection and attraction are unclear. Here, we demonstrate that a range of FAs are detected by the fly gustatory system and elicit a robust feeding response. Flies lacking olfactory organs respond robustly to FAs, confirming that FA attraction is mediated through the gustatory system. Furthermore, flies detect FAs independent of pH, suggesting the molecular basis for FA taste is not due to acidity. We show that low and medium concentrations of FAs serve as an appetitive signal and they are detected exclusively through the same subset of neurons that sense appetitive sweet substances, including most sugars. In mammals, taste perception of sweet and bitter substances is dependent on phospholipase C (PLC) signaling in specialized taste buds. We find that flies mutant for norpA, a Drosophila ortholog of PLC, fail to respond to FAs. Intriguingly, norpA mutants respond normally to other tastants, including sucrose and yeast. The defect of norpA mutants can be rescued by selectively restoring norpA expression in sweet-sensing neurons, corroborating that FAs signal through sweet-sensing neurons, and suggesting PLC signaling in the gustatory system is specifically involved in FA taste. Taken together, these findings reveal that PLC function in Drosophila sweet-sensing neurons is a conserved molecular signaling pathway that confers attraction to fatty acids.     

Use of Drosophila mutants to investigate the effect of disuse on the maintenance of muscle

The effect of continued muscular inactivity and prolonged paralysis on the structure and function of muscles was investigated in Drosophila melanogaster. A number of flightless mutants was examined to see whether their flight muscles degenerated as a result of disuse. No sign of progressive deterioration was observed in any of these mutants. Further, by producing mosaic flies in which part of the body expressed the temperature-sensitive paralytic mutation shibire^ST139, reversible local paralysis was obtained, and maintained for prolonged periods. Flies in which parts of the leg or flight musculature had been paralysed for several days were examined; no effect of such inactivity on the structure and function of the muscles was observed in any of the flies. These results indicate that in Drosophila continued muscular inactivity does not result in extensive degeneration of the musculature.     

Circadian expression of the presynaptic active zone protein bruchpilot in the lamina of Drosophila melanogaster

In the fly's visual system, the morphology of cells and the number of synapses change during the day. In the present study we show that in the first optic neuropil (lamina) of Drosophila melanogaster, a presynaptic active zone protein Bruchpilot (BRP) exhibits a circadian rhythm in abundance. In day/night (or light/dark, LD) conditions the level of BRP increases two times, in the morning and in the evening. The same pattern of changes in the BRP level was detected in whole brain homogenates, thus indicating that the majority of synapses in the brain peaks twice during the day. However, these two peaks in BRP abundance, measured as the fluorescence intensity of immunolabeling, seem to be regulated differently. The peak in the morning is predominantly regulated by light and involves the transduction pathway in the retina photoreceptors. This peak is present neither in wild‐type Canton‐S flies in constant darkness (DD), nor in norpA⁷ phototransduction mutant in LD. However, it also depends on the clock gene per, because it is abolished in the per⁰ arrhythmic mutant. In turn, the peak of BRP in the evening is endogenously regulated by an input from the pacemaker located in the brain. This peak is present in Canton‐S flies in DD, as well as in the norpA⁷ mutant in LD, but is absent in per⁰¹, tim,⁰¹ and cry⁰¹ mutants in LD. In addition both peaks seem to depend on clock gene‐expressing photoreceptors and glial cells of the visual system. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

The Phosphatidylinositol Transfer Protein Domain of Drosophila Retinal Degeneration B Protein Is Essential for Photoreceptor Cell Survival and Recovery from Light Stimulation

The Drosophila retinal degeneration B (rdgB) gene encodes an integral membrane protein involved in phototransduction and prevention of retinal degeneration. RdgB represents a nonclassical phosphatidylinositol transfer protein (PITP) as all other known PITPs are soluble polypeptides. Our data demonstrate roles for RdgB in proper termination of the phototransduction light response and dark recovery of the photoreceptor cells. Expression of RdgB''s PITP domain as a soluble protein (RdgB-PITP) in rdgB² mutant flies is sufficient to completely restore the wild-type electrophysiological light response and prevent the degeneration. However, introduction of the T59E mutation, which does not affect RdgB-PITP''s phosphatidylinositol (PI) and phosphatidycholine (PC) transfer in vitro, into the soluble (RdgB-PITP-T59E) or full-length (RdgB-T59E) proteins eliminated rescue of retinal degeneration in rdgB² flies, while the light response was partially maintained. Substitution of the rat brain PITPα, a classical PI transfer protein, for RdgB''s PITP domain (PITPα or PITPα-RdgB chimeric protein) neither restored the light response nor maintained retinal integrity when expressed in rdgB² flies. Therefore, the complete repertoire of essential RdgB functions resides in RdgB''s PITP domain, but other PITPs possessing PI and/or PC transfer activity in vitro cannot supplant RdgB function in vivo. Expression of either RdgB-T59E or PITPα-RdgB in rdgB ⁺ flies produced a dominant retinal degeneration phenotype. Whereas RdgB-T59E functioned in a dominant manner to significantly reduce steady-state levels of rhodopsin, PITPα-RdgB was defective in the ability to recover from prolonged light stimulation and caused photoreceptor degeneration through an unknown mechanism. This in vivo analysis of PITP function in a metazoan system provides further insights into the links between PITP dysfunction and an inherited disease in a higher eukaryote.The Drosophila retinal degeneration B protein (RdgB)¹ plays a critical role in the fly photoreceptor cell. The rdgB mutant phenotype is characterized by retinal degeneration whose onset, while discernible in dark-reared flies, is greatly accelerated by raising the flies in light (Harris and Stark, 1977; Stark et al., 1983). Typically, rdgB mutant flies begin to exhibit the morphological hallmarks of photoreceptor cell degeneration several days after eclosion (Harris and Stark, 1977; Stark et al., 1983). In addition, these mutant flies exhibit an abnormal light response, as recorded by the rapid deterioration of the electroretinogram (ERG), shortly after the fly''s initial exposure to light. This ERG defect is manifested before any obvious physical signs of retinal degeneration (Harris and Stark, 1977), which suggests that the defect in the light response may precipitate the course of retinal degeneration.In the photoreceptor cell, RdgB localizes to both the axon and the subrhabdomeric cisternae (SRC) (Vihtelic et al., 1993; Suzuki and Hirosawa, 1994). The SRC is an extension of the endoplasmic reticulum that functions both as an intracellular Ca²⁺ store and a compartment through which rhodopsin traffics en route to the rhabdomere (Walz, 1982; Matsumoto-Suzuki et al., 1989; Suzuki and Hiosawa, 1991). Thus, RdgB is the first identified protein required for visual transduction that is not localized in the photoreceptor rhabdomere. Genetic epistasis analyses suggest RdgB functions downstream of both rhodopsin and phospholipase C (PLC) in the visual transduction cascade as both the ninaE (encoding the opsin expressed in photoreceptor cells R1-6 [O''Tousa et al., 1985; Zuker et al., 1985]) and norpA (encoding phospholipase C [Bloomquist et al., 1988]) mutations suppress the rdgB-dependent, light-enhanced retinal degeneration (Harris and Stark, 1977; Stark et al., 1983). Consistent with this view, constitutive activation of the Drosophila G protein transducin analogue (DGq), either by application of nonhydrolyzable GTP analogues or by expression of a constitutively activated Gα subunit (Dgq1), effects a rapid degeneration of rdgB retinas in the absence of light (Rubinstein et al., 1989; Lee et al., 1994). RdgB apparently functions downstream of the inaC-encoded protein kinase C (PKC) because: (a) application of phorbol ester to rdgB mutant retinas, which presumably activates the inaC-encoded PKC, stimulates retinal degeneration in the absence of light (Minke et al., 1990); and (b) the rdgB retinal degeneration is weakly suppressed by the inaC mutation (Smith et al., 1991). Thus, the available evidence identifies an execution point for RdgB downstream of PKC in the visual transduction cascade.RdgB is a 116-kD membrane polypeptide with six potential transmembrane domains (Vihtelic et al., 1991). Additionally, the amino-terminal 281 RdgB residues share 42% amino acid identity with the rat brain phosphatidylinositol (PI) transfer protein α isoform (PITPα) (Vihtelic et al., 1993). Whereas PITPs are operationally defined by their ability to catalyze the transfer of either PI or phosphatidylcholine (PC) monomers between membrane bilayers in vitro (Bankaitis et al., 1990; Cleves et al., 1991; Wirtz, 1991), how the phospholipid transfer activity pertains to in vivo function is less clear. The yeast PITP (Sec14p) uses its PI and PC binding activities in two independent, yet complementary, ways that serve to preserve a Golgi pool of diacylglycerol that is critical for the biogenesis of Golgi-derived secretory vesicles (Kearns et al., 1997). Reconstitution studies suggest that mammalian PITPs play important roles in PLC-mediated inositol signaling, ATP-dependent, Ca²⁺-activated secretion, and constitutive secretion from the trans-Golgi network (Hay and Martin, 1993, 1995; Thomas et al., 1993, 1995; Ohashi et al., 1995). However, because the PITP requirement for these processes is generally satisfied by any PITP (even those lacking any primary sequence identity), the physiological relevance of these PITP involvements remains to be determined (Skinner et al., 1993; Cunningham et al., 1995; Ohashi et al., 1995; Alb et al., 1996). The recent finding that the mouse vibrator mutation represents a hypomorphic mutation in the pitpn gene, which encodes PITPα, indicates that PITP function is important to neuronal function (Hamilton et al., 1997). RdgB''s PITP domain (when expressed as a soluble protein in Escherichia coli) is able to effect intermembrane transfer of PI in vitro (Vihtelic et al., 1993). Unlike all previously characterized PITPs, which are 32–35-kD soluble proteins (Bankaitis et al., 1989; Cleves et al., 1991; Wirtz, 1991), RdgB is a large integral membrane protein. In spite of postulated in vivo activities for PITPs, the function of RdgB in the photoreceptor cell remains unknown. Recently, vertebrate orthologues of the rdgB gene were identified in mice, bovines, and humans (Chang et al., 1997). Expression of the mouse rdgB cDNA in rdgB² null mutant flies resulted in the elimination of the retinal degeneration and complete restoration of the wild-type ERG light response (Chang et al., 1997). Thus, the Drosophila RdgB protein defines a new class of functionally equivalent transmembrane PITPs.In this work, we analyzed RdgB''s involvement in the Drosophila phototransduction cascade and the mechanism by which it prevents the onset of retinal degeneration. This represents the first in vivo analysis of the transmembrane PITP class, and we report several novel and unanticipated aspects of RdgB function. We demonstrate that the complete repertoire of RdgB functions essential for normal phototransduction reside in the PITP domain. Expression of this domain as a soluble polypeptide fully complements the rdgB² null allele. Yet, other PITPs that possess PI and/ or PC transfer activities in vitro cannot substitute for RdgB in the photoreceptor cell. Whereas the recessive rdgB² null mutation demonstrates an essential role for RdgB in proper termination of the ERG light response and dark recovery of the photoreceptor cell, one novel dominant rdgB mutation affects the maintenance of steady- state rhodopsin levels in photoreceptor cells. Another dominant rdgB mutation induces retinal degeneration and compromises the rapid regeneration of a wild-type ERG light-response amplitude subsequent to multiple or prolonged light exposure. Taken together, these data indicate an underlying complexity to the mechanism of RdgB function and its role in the photoreceptor cell that is not easily reconciled with a simple role in potentiating signal transduction via phosphoinositide-driven signaling pathways.     

Inactivation of VCP/ter94 Suppresses Retinal Pathology Caused by Misfolded Rhodopsin in Drosophila

The most common Rhodopsin (Rh) mutation associated with autosomal dominant retinitis pigmentosa (ADRP) in North America is the substitution of proline 23 by histidine (Rh^P23H). Unlike the wild-type Rh, mutant Rh^P23H exhibits folding defects and forms intracellular aggregates. The mechanisms responsible for the recognition and clearance of misfolded Rh^P23H and their relevance to photoreceptor neuron (PN) degeneration are poorly understood. Folding-deficient membrane proteins are subjected to Endoplasmic Reticulum (ER) quality control, and we have recently shown that Rh^P23H is a substrate of the ER–associated degradation (ERAD) effector VCP/ter94, a chaperone that extracts misfolded proteins from the ER (a process called retrotranslocation) and facilitates their proteasomal degradation. Here, we used Drosophila, in which Rh1^P37H (the equivalent of mammalian Rh^P23H) is expressed in PNs, and found that the endogenous Rh1 is required for Rh1^P37H toxicity. Genetic inactivation of VCP increased the levels of misfolded Rh1^P37H and further activated the Ire1/Xbp1 ER stress pathway in the Rh1^P37H retina. Despite this, Rh1^P37H flies with decreased VCP function displayed a potent suppression of retinal degeneration and blindness, indicating that VCP activity promotes neurodegeneration in the Rh1^P37H retina. Pharmacological treatment of Rh1^P37H flies with the VCP/ERAD inhibitor Eeyarestatin I or with the proteasome inhibitor MG132 also led to a strong suppression of retinal degeneration. Collectively, our findings raise the possibility that excessive retrotranslocation and/or degradation of visual pigment is a primary cause of PN degeneration.     

Characterization of Leber Congenital Amaurosis-associated NMNAT1 Mutants

Leber congenital amaurosis 9 (LCA9) is an autosomal recessive retinal degeneration condition caused by mutations in the NAD⁺ biosynthetic enzyme NMNAT1. This condition leads to early blindness but no other consistent deficits have been reported in patients with NMNAT1 mutations despite its central role in metabolism and ubiquitous expression. To study how these mutations affect NMNAT1 function and ultimately lead to the retinal degeneration phenotype, we performed detailed analysis of LCA-associated NMNAT1 mutants, including the expression, nuclear localization, enzymatic activity, secondary structure, oligomerization, and promotion of axonal and cellular integrity in response to injury. In many assays, most mutants produced results similar to wild type NMNAT1. Indeed, NAD⁺ synthetic activity is unlikely to be a primary mechanism underlying retinal degeneration as most LCA-associated NMNAT1 mutants had normal enzymatic activity. In contrast, the secondary structure of many NMNAT1 mutants was relatively less stable as they lost enzymatic activity after heat shock, whereas wild type NMNAT1 retains significant activity after this stress. These results suggest that LCA-associated NMNAT1 mutants are more vulnerable to stressful conditions that lead to protein unfolding, a potential contributor to the retinal degeneration observed in this syndrome.     

Transduction in photoreceptors with bistable pigments: Intermediate processes

The prolonged depolarizing after potential (PDA) in the R1–6 receptors of the fly was used to isolate intermediate processes in phototransduction which are not manifested directly in the voltage response. It is first demonstrated that a pigment shift by light from metarhodopsin to rhodopsin in four species of the flies: Drosophila, Calliphora, Chrysomya and Musca induces an independent antagonistic process to the PDA, which is manifested in a strong inhibitory effect on PDA induction and is called the anti-PDA.By using mutants of Drosophila the existence of processes underlying the PDA were examined. The norpA ^H52and the trp mutant were used in which the voltage response of the photoreceptors could be reversibly abolished by elavated temperature and long intense light respectively. It is shown that the excitatory process underlying the PDA could be induced and depressed in conditions that block the voltage response of the photoreceptors, thus indicating the existance of intermediate processes which link the pigment activation by light to the PDA voltage response.Based on material presented at the European Neurosciences Meeting, Florence, September 1978     

Autophagy-mediated catabolism of visual transduction proteins prevents retinal degeneration

Autophagy is a lysosomal degradation pathway critical to preventing the accumulation of cytotoxic proteins. Deletion of the essential autophagy gene Atg5 from the rod photoreceptors of the retina (atg5^Δrod mouse) results in the accumulation of the phototransduction protein transducin and the degeneration of these neurons. The purpose of this study is to test the hypothesis that autophagic degradation of visual transduction proteins prevents retinal degeneration. Targeted deletion of both Gnat1 (a gene encoding the α subunit of the heterotrimeric G-protein transducin) and Atg5 in the rod photoreceptors resulted in a significantly decreased rate of rod cell degeneration as compared to the atg5^Δrod mouse retina, and considerable preservation of photoreceptors. Supporting this we used a novel technique to immunoprecipitate green fluorescent protein (GFP)-tagged autophagosomes from the retinas of the GFP-LC3 mice and demonstrated that the visual transduction proteins transducin and ARR/arrestin are associated with autophagosome-specific proteins. Altogether, this study shows that degradation of phototransduction proteins by autophagy is necessary to prevent retinal degeneration. In addition, we demonstrate a simple and easily reproducible immunoisolation technique for enrichment of autophagosomes from the GFP-LC3 mouse retina, providing a novel application to the study of autophagosome contents across different organs and specific cell types in vivo.     

A forward genetic screen reveals essential and non-essential RNAi factors in Paramecium tetraurelia

In most eukaryotes, small RNA-mediated gene silencing pathways form complex interacting networks. In the ciliate Paramecium tetraurelia, at least two RNA interference (RNAi) mechanisms coexist, involving distinct but overlapping sets of protein factors and producing different types of short interfering RNAs (siRNAs). One is specifically triggered by high-copy transgenes, and the other by feeding cells with double-stranded RNA (dsRNA)-producing bacteria. In this study, we designed a forward genetic screen for mutants deficient in dsRNA-induced silencing, and a powerful method to identify the relevant mutations by whole-genome sequencing. We present a set of 47 mutant alleles for five genes, revealing two previously unknown RNAi factors: a novel Paramecium-specific protein (Pds1) and a Cid1-like nucleotidyl transferase. Analyses of allelic diversity distinguish non-essential and essential genes and suggest that the screen is saturated for non-essential, single-copy genes. We show that non-essential genes are specifically involved in dsRNA-induced RNAi while essential ones are also involved in transgene-induced RNAi. One of the latter, the RNA-dependent RNA polymerase RDR2, is further shown to be required for all known types of siRNAs, as well as for sexual reproduction. These results open the way for the dissection of the genetic complexity, interconnection, mechanisms and natural functions of RNAi pathways in P. tetraurelia.     

Mutants of satellite bacteriophage P4 that are defective in the suppression of transcriptional polarity

The satellite bacteriophage P4 relies on a helper such as P2 to supply the gene products necessary for virion assembly and cell lysis (Six, 1975). P4 has the unique capacity to activate the late genes of P2 by a mechanism that differs from the one normally used by P2 itself. This process has been termed transactivation (Calendar et al., 1977). In addition, P4 is able to suppress the strong polarity associated with certain P2 amber mutations. The isolation of P4 mutants solely defective in polarity suppression (psu^?) demonstrates that the ability of P4 to suppress polarity is non-essential for P4 growth. In particular, polarity suppression plays no essential role in either transactivation or head size determination. The product of the P4 psu gene has been identified as a 19,900 M_r P4 late protein.     

Knockout of DLIC1 leads to retinal cone degeneration via disturbing Rab8 transport in zebrafish

Retinal photoreceptors execute phototransduction functions and require an efficient system for the transport of materials (e.g. proteins and lipids) from inner segments to outer segments. Cytoplasmic dynein 1 is a minus-end-directed microtubule motor and participates in cargo transport in the cytoplasm. However, the roles of dynein 1 motor in photoreceptor cargo transport and retinal development are still ambiguous. In our present study, the light intermediate chain protein DLIC1 (encoded by dync1li1), links activating adaptors to bind diverse cargos in the dynein 1 motor, was depleted using CRISPR-Cas9 technology in zebrafish. The dync1li1^?/? zebrafish displayed progressive degeneration of retinal cone photoreceptors, especially blue cones. The retinal rods were not affected in dync1li1^?/? zebrafish. Knockout of DLIC1 resulted in abnormal expression and localization of cone opsins in dync1li1^?/? retinas. TUNEL staining suggested that apoptosis was induced after aberrant accumulation of cone opsins in photoreceptors of dync1li1^?/? zebrafish. Instead of Rab11 transport, Rab8 transport was disturbed in dync1li1^?/? retinas. Our data demonstrate that DLIC1 is required for function maintenance and survival of cone photoreceptors, and hint at an essential role of the cytoplasmic dynein 1 motor in photoreceptor cargo transport.