Differential localizations of and requirements for the two Drosophila ninaC kinase/myosins in photoreceptor cells

The ninaC gene encodes two retinal specific proteins (p132 and p174) consisting of a protein kinase domain joined to a domain homologous to the head region of the myosin heavy chain. The putative myosin domain of p174 is linked at the COOH-terminus to a tail which has some similarities to myosin-I tails. In the current report, we demonstrate that the ninaC mutation results in light- and age-dependent retinal degeneration. We also show that ninaC flies display an electrophysiological phenotype before any discernible retinal degeneration indicating that the electrophysiological defect is the primary effect of the mutation. This suggests that ninaC has a role in phototransduction and that the retinal degeneration is a secondary effect resulting from the defect in phototransduction. To examine the requirements for the individual ninaC isoforms, mutant alleles were generated which express only p132 or p174. Elimination of p174 resulted in a ninaC phenotype as strong as the null allele; however, elimination of p132 had little if any effect. As a first step in investigating the basis for the difference in requirements for p174 and p132 we performed immuno-localization at the electron microscopic level and found that the two isoforms display different subcellular distributions in the photoreceptor cells. The p132 protein is restricted primarily to the cytoplasm and p174 to the rhabdomeres, the microvillar structure which is the site of action of many of the steps in phototransduction. This suggests that the p174 myosin-I type tail is the domain responsible for association with the rhabdomeres and that the substrate for the p174 putative kinase may be a rhabdomeric protein important in photo-transduction.     

Calmodulin binding to Drosophila NinaC required for termination of phototransduction.

The ninaC locus encodes two unconventional myosins, p132 and p174, consisting of fused protein kinase and myosin head domains expressed in Drosophila photoreceptor cells. NinaC are the major calmodulin-binding proteins in the retina and the NinaC-calmodulin interaction is required for the normal subcellular localization of calmodulin as well as for normal photo-transduction. In the current report, we present evidence for two calmodulin-binding sites in NinaC, C1 and C2, which have different in vitro binding properties. C1 was found to be common to both p132 and p174 while C2 was unique to p174. To address the requirements for calmodulin binding at each site in vivo, we generated transgenic flies expressing ninaC genes deleted for either C1 or C2. We found that the spatial localization of calmodulin depended on binding to both C1 and C2. Furthermore, mutation of either site resulted in a defective photoresponse. A prolonged depolarization afterpotential (PDA) was elicited at lower light intensities than necessary to produce a PDA in wild-type flies. These results suggest that calmodulin binding to both C1 and C2 is required in vivo for termination of phototransduction.     

Light-dependent translocation of visual arrestin regulated by the NINAC myosin III

The rhodopsin regulatory protein, visual arrestin, undergoes light-dependent trafficking in mammalian and Drosophila photoreceptor cells, though the mechanisms underlying these movements are poorly understood. In Drosophila, the movement of the visual arrestin, Arr2, functions in long-term adaptation and is dependent on interaction with phosphoinositides (PIs). However, the basis for the requirement for PIs for light-dependent shuttling was unclear. Here, we demonstrated that the dynamic trafficking of Arr2 into the phototransducing compartment, the rhabdomere, required the eye-enriched myosin III, NINAC. We showed that defects in ninaC resulted in a long-term adaptation phenotype similar to that which occurred in arr2 mutants. The interaction between Arr2 and NINAC was PI dependent and NINAC bound directly to PIs. These data demonstrate that the light-dependent translocation of Arr2 into the rhabdomeres requires PI-mediated interactions between Arr2 and the NINAC myosin III.     

Mbt,a Drosophila PAK protein,combines with Cdc42 to regulate photoreceptor cell morphogenesis

The Drosophila gene mushroom bodies tiny (mbt) encodes a putative p21-activated kinase (PAK), a family of proteins that has been implicated in a multitude of cellular processes including regulation of the cytoskeleton, cell polarisation, control of MAPK signalling cascades and apoptosis. The mutant phenotype of mbt is characterised by fewer neurones in the brain and the eye, indicating a role of the protein in cell proliferation, differentiation or survival. We show that mutations in mbt interfere with photoreceptor cell morphogenesis. Mbt specifically localises at adherens junctions of the developing photoreceptor cells. A structure-function analysis of the Mbt protein in vitro and in vivo revealed that the Mbt kinase domain and the GTPase binding domain, which specifically interacts with GTP-loaded Cdc42, are important for Mbt function. Besides regulation of kinase activity, another important function of Cdc42 is to recruit Mbt to adherens junctions. We propose a role for Mbt as a downstream effector of Cdc42 in photoreceptor cell morphogenesis.     

Crumbs regulates polarity and prevents light-induced degeneration of the simple eyes of Drosophila, the ocelli

The evolutionary conserved transmembrane protein Crumbs (Crb) regulates morphogenesis of photoreceptor cells in the compound eye of Drosophila and prevents light-dependent retinal degeneration. Here we examine the role of Crb in the ocelli, the simple eyes of Drosophila. We show that Crb is expressed in ocellar photoreceptor cells, where it defines a stalk membrane apical to the adherens junctions, similar as in photoreceptor cells of the compound eyes. Loss of function of crb disrupts polarity of ocellar photoreceptor cells, and results in mislocalisation of adherens junction proteins. This phenotype is more severe than that observed in mutant photoreceptor cells of the compound eye, and resembles more that of embryonic epithelia lacking crb. Similar as in compound eyes, crb protects ocellar photoreceptors from light induced degeneration, a function that depends on the extracellular portion of the Crb protein. Our data demonstrate that the function of crb in photoreceptor development and homeostasis is conserved in compound eyes and ocelli and underscores the evolutionarily relationship between these visual sense organs of Drosophila. The data will be discussed with respect to the difference in apico-basal organisation of these two cell types.     

An opsin gene that is expressed only in the R7 photoreceptor cell of Drosophila.

We have used two techniques to isolate and characterize eye-specific genes from Drosophila melanogaster. First, we identified genes whose expression is limited to eyes, photoreceptor cells, or R7 photoreceptor cells by differential screening with [32P]cDNAs derived from the heads of mutant flies that have reduced amounts of these tissues and cells (Microcephalus, glass3, and sevenless, respectively). Secondly, we identified opsin genes by hybridization with synthetic [32P]oligonucleotides that encode domains that have been conserved between some opsin genes. We found seven clones that contain genes expressed only in the eye or optic lobes of Drosophila; three are expressed only in photoreceptor cells. One is expressed only in R7 photoreceptor cells and hybridizes to some of the previously mentioned oligonucleotides. The complete DNA sequence of the R7-specific opsin gene and its 5' and 3' flanking regions was determined. It is quite different from other known Drosophila opsin genes, in that it is not interrupted by introns and shares only 37-38% amino acid identity with the proteins encoded by these genes. The predicted protein structure contains many characteristics that are common to all rhodopsins, and the sequence differences help to identify four domains of the rhodopsin molecule that have been conserved in evolution.     

The Ste20 kinase misshapen regulates both photoreceptor axon targeting and dorsal closure, acting downstream of distinct signals

We have previously shown that the Ste20 kinase encoded by misshapen (msn) functions upstream of the c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase module in Drosophila. msn is required to activate the Drosophila JNK, Basket (Bsk), to promote dorsal closure of the embryo. A mammalian homolog of Msn, Nck interacting kinase, interacts with the SH3 domains of the SH2-SH3 adapter protein Nck. We now show that Msn likewise interacts with Dreadlocks (Dock), the Drosophila homolog of Nck. dock is required for the correct targeting of photoreceptor axons. We have performed a structure-function analysis of Msn in vivo in Drosophila in order to elucidate the mechanism whereby Msn regulates JNK and to determine whether msn, like dock, is required for the correct targeting of photoreceptor axons. We show that Msn requires both a functional kinase and a C-terminal regulatory domain to activate JNK in vivo in Drosophila. A mutation in a PXXP motif on Msn that prevents it from binding to the SH3 domains of Dock does not affect its ability to rescue the dorsal closure defect in msn embryos, suggesting that Dock is not an upstream regulator of msn in dorsal closure. Larvae with only this mutated form of Msn show a marked disruption in photoreceptor axon targeting, implicating an SH3 domain protein in this process; however, an activated form of Msn is not sufficient to rescue the dock mutant phenotype. Mosaic analysis reveals that msn expression is required in photoreceptors in order for their axons to project correctly. The data presented here genetically link msn to two distinct biological events, dorsal closure and photoreceptor axon pathfinding, and thus provide the first evidence that Ste20 kinases of the germinal center kinase family play a role in axonal pathfinding. The ability of Msn to interact with distinct classes of adapter molecules in dorsal closure and photoreceptor axon pathfinding may provide the flexibility that allows it to link to distinct upstream signaling systems.     

The cyclophilin homolog ninaA is a tissue-specific integral membrane protein required for the proper synthesis of a subset of Drosophila rhodopsins.

Mutations in the Drosophila ninaA gene cause dramatic reductions in rhodopsin levels, leading to impaired visual function. The ninaA protein is a homolog of peptidyl-prolyl cis-trans isomerases. We find that ninaA is unique among this family of proteins in that it is an integral membrane protein, and it is expressed in a cell type-specific manner. We have used transgenic animals misexpressing different rhodopsins in the major class of photoreceptor cells to demonstrate that ninaA is required for normal function by two homologous rhodopsins, but not by a less conserved member of the Drosophila rhodopsin gene family. This demonstrates in vivo substrate specificity in a cyclophilin-like molecule. We also show that vertebrate retina contains a ninaA-related protein and that ninaA is a member of a gene family in Drosophila. These data offer insights into the in vivo role of this important family of proteins.     

Drosophila Amphiphysin is implicated in protein localization and membrane morphogenesis but not in synaptic vesicle endocytosis.

Amphiphysin family members are implicated in synaptic vesicle endocytosis, actin localization and one isoform is an autoantigen in neurological autoimmune disorder; however, there has been no genetic analysis of Amphiphysin function in higher eukaryotes. We show that Drosophila Amphiphysin is localized to actin-rich membrane domains in many cell types, including apical epithelial membranes, the intricately folded apical rhabdomere membranes of photoreceptor neurons and the postsynaptic density of glutamatergic neuromuscular junctions. Flies that lack all Amphiphysin function are viable, lack any observable endocytic defects, but have abnormal localization of the postsynaptic proteins Discs large, Lethal giant larvae and Scribble, altered synaptic physiology, and behavioral defects. Misexpression of Amphiphysin outside its normal membrane domain in photoreceptor neurons results in striking morphological defects. The strong misexpression phenotype coupled with the mild mutant and lack of phenotypes suggests that Amphiphysin acts redundantly with other proteins to organize specialized membrane domains within a diverse array of cell types.     

Multiple domains in the Crumbs Homolog 2a (Crb2a) protein are required for regulating rod photoreceptor size

Background  

Vertebrate retinal photoreceptors are morphologically complex cells that have two apical regions, the inner segment and the outer segment. The outer segment is a modified cilium and is continuously regenerated throughout life. The molecular and cellular mechanisms that underlie vertebrate photoreceptor morphogenesis and the maintenance of the outer segment are largely unknown. The Crumbs (Crb) complex is a key regulator of apical membrane identity and size in epithelia and in Drosophila photoreceptors. Mutations in the human gene CRUMBS HOMOLOG 1 (CRB1) are associated with early and severe vision loss. Drosophila Crumbs and vertebrate Crb1 and Crumbs homolog 2 (Crb2) proteins are structurally similar, all are single pass transmembrane proteins with a large extracellular domain containing multiple laminin- and EGF-like repeats and a small intracellular domain containing a FERM-binding domain and a PDZ-binding domain. In order to begin to understand the role of the Crb family of proteins in vertebrate photoreceptors we generated stable transgenic zebrafish in which rod photoreceptors overexpress full-length Crb2a protein and several other Crb2a constructs engineered to lack specific domains.     

Overexpression of ubiquitin carboxyl terminal hydrolase impairs multiple pathways during eye development in Drosophila melanogaster

UCH-L1 (ubiquitin carboxyl terminal hydrolase L1) is well known as an enzyme that hydrolyzes polyubiquitin at its C-terminal to release ubiquitin monomers. Although the overexpression of UCH-L1 inhibits proteasome activity in cultured cells, its biological significance in living organisms has not been clarified in detail. Here, we utilized Drosophila as a model system to examine the effects of the overexpression of dUCH, a Drosophila homologue of UCH-L1, on development. Overexpression in the eye imaginal discs induced a rough eye phenotype in the adult, at least partly resulting from the induction of caspase-dependent apoptosis followed by compensatory proliferation. Genetic crosses with enhancer trap lines marking the photoreceptor cells also revealed that the overexpression of dUCH specifically impaired R7 photoreceptor cell differentiation with a reduction in activated extracellular-signal-regulated kinase signals. Furthermore, the dUCH-induced rough eye phenotype was rescued by co-expression of the sevenless gene or the Draf gene, a downstream component of the mitogen-activated protein kinase (MAPK) cascade. These results indicate that the overexpression of dUCH impairs R7 photoreceptor cell differentiation by down-regulating the MAPK pathway. Interestingly, this process appears to be independent of its pro-apoptotic function.     

Star is required in a subset of photoreceptor cells in the developing Drosophila retina and displays dosage sensitive interactions with rough.

We report that mutations at the Star locus act as dominant enhancers of the eye phenotype displayed by flies carrying a null allele of rough. Our analysis of double mutants at different stages of eye development suggests that this phenotype results from defects in the early stages of photoreceptor cell differentiation in the eye imaginal disc. Complete loss of Star function during retinal development, analyzed in mosaic animals, results in cell death, visible as scars in the adult eye. The requirement for wild-type Star function, however, is confined to only a subset of photoreceptor cells, R8, R2, and R5, which are the first three cells to differentiate neurally in the developing retina. These results suggest an essential role for the Star gene in the initial events of ommatidial cluster formation during the development of the Drosophila compound eye.     

Evolution of double MutT/Nudix domain-containing proteins: similar domain architectures from independent gene duplication-fusion events

The MutT/Nudix superfamily proteins repair DNA damage and play a role in human health and disease. In this study, we examined two different cases of double MutT/Nudix domain-containing proteins from eukaryotes and prokaryotes. Firstly, these double domain proteins were discovered in Drosophila, but only single Nudix domain proteins were found in other animals. The phylogenetic tree was constructed based on the protein sequence of Nudix_N and Nudix_C from Drosophila, and Nudix from other animals. The phylogenetic analysis suggested that the double Nudix domain proteins might have undergone a gene duplication-speciation-fusion process. Secondly, two genes of the MutT family, DR0004 and DR0329, were fused by two mutT gene segments and formed double MutT domain protein genes in Deinococcus radiodurans. The evolutionary tree of bacterial MutT proteins suggested that the double MutT domain proteins in D. radiodurans probably resulted from a gene duplication-fusion event after speciation. Gene duplication-fusion is a basic and important gene innovation mechanism for the evolution of double MutT/Nudix domain proteins. Independent gene duplication-fusion events resuited in similar domain architectures of different double MutT/Nudix domain proteins.