RESUMEN
Drosophila retinal degeneration C (RDGC) is the founding member of the PPEF family of protein phosphatases. RDGC mediates dephosphorylation of the visual pigment rhodopsin and the TRP ion channel. From the rdgC locus, three protein isoforms, termed RDGC-S, -M, and -L, with different N-termini are generated. Due to fatty acylation, RDGC-M and -L are attached to the plasma membrane while RDGC-S is soluble. To assign physiological roles to these RDGC isoforms, we constructed flies that express various combinations of RDGC protein isoforms. Expression of the RDGC-L isoform alone did not fully prevent rhodopsin hyperphosphorylation and resulted in impaired photoreceptor physiology and in decelerated TRP dephosphorylation at Ser936. However, expression of RDGC-L alone as well as RDGC-S/M was sufficient to prevent degeneration of photoreceptor cells which is a hallmark of the rdgC null mutant. Membrane-attached RDGC-M displayed higher activity of TRP dephosphorylation than the soluble isoform RDGC-S. Taken together, in vivo activities of RDGC splice variants are controlled by their N-termini.
Asunto(s)
Empalme Alternativo , Proteínas de Unión al Calcio , Proteínas de Drosophila , Proteínas de la Membrana , Fosfoproteínas Fosfatasas , Células Fotorreceptoras de Invertebrados/enzimología , Acilación , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Dominios Proteicos , Rodopsina/genética , Rodopsina/metabolismoRESUMEN
Polarized epithelia develop distinct cell surface domains, with the apical membrane acquiring characteristic morphological features such as microvilli. Cell polarization is driven by polarity determinants including the evolutionarily conserved partitioning-defective (PAR) proteins that are separated into distinct cortical domains. PAR protein segregation is thought to be a consequence of asymmetric actomyosin contractions. The mechanism of activation of apically polarized actomyosin contractility is unknown. Here we show that the Cdc42 effector MRCK activates myosin-II at the apical pole to segregate aPKC-Par6 from junctional Par3, defining the apical domain. Apically polarized MRCK-activated actomyosin contractility is reinforced by cooperation with aPKC-Par6 downregulating antagonistic RhoA-driven junctional actomyosin contractility, and drives polarization of cytosolic brush border determinants and apical morphogenesis. MRCK-activated polarized actomyosin contractility is required for apical differentiation and morphogenesis in vertebrate epithelia and Drosophila photoreceptors. Our results identify an apical origin of actomyosin-driven morphogenesis that couples cytoskeletal reorganization to PAR polarity signalling.
Asunto(s)
Membrana Celular/enzimología , Polaridad Celular , Células Epiteliales/enzimología , Proteína Quinasa de Distrofia Miotónica/metabolismo , Actomiosina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Animales Modificados Genéticamente , Células CACO-2 , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular , Perros , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/enzimología , Genotipo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Células de Riñón Canino Madin Darby , Proteínas de la Membrana/metabolismo , Morfogénesis , Miosina Tipo II/metabolismo , Proteína Quinasa de Distrofia Miotónica/genética , Fenotipo , Células Fotorreceptoras de Invertebrados/enzimología , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , Transducción de Señal , Factores de Tiempo , Transfección , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Mitochondria undergo frequent morphological changes through fission and fusion. Mutations in core members of the mitochondrial fission/fusion machinery are responsible for severe neurodegenerative diseases. However, the mitochondrial fission/fusion mechanisms are poorly understood. We found that the loss of a mitochondrial protein encoding gene, mitoguardin (miga), leads to mitochondrial defects and neurodegeneration in fly eyes. Mammals express two orthologs of miga: Miga1 and Miga2. Both MIGA1 and MIGA2 form homotypic and heterotypic complexes on the outer membrane of the mitochondria. Loss of MIGA results in fragmented mitochondria, whereas overexpression of MIGA leads to clustering and fusion of mitochondria in both fly and mammalian cells. MIGA proteins function downstream of mitofusin and interact with MitoPLD to stabilize MitoPLD and facilitate MitoPLD dimer formation. Therefore, we propose that MIGA proteins promote mitochondrial fusion by regulating mitochondrial phospholipid metabolism via MitoPLD.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/enzimología , Dinámicas Mitocondriales , Proteínas Mitocondriales/metabolismo , Neuronas/enzimología , Fosfolipasa D/metabolismo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Endorribonucleasas , Femenino , Genotipo , Células HEK293 , Células HeLa , Homeostasis , Humanos , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Mitocondrias/patología , Membranas Mitocondriales/enzimología , Proteínas Mitocondriales/genética , Mutación , Células 3T3 NIH , Neuronas/patología , Fenotipo , Fosfolipasa D/genética , Células Fotorreceptoras de Invertebrados/enzimología , Multimerización de Proteína , Interferencia de ARN , TransfecciónRESUMEN
The development of a functional organ requires coordinated programs of cell fate specification, terminal differentiation and morphogenesis. Whereas signaling mechanisms that specify individual cell fates are well documented, little is known about the pathways and molecules that maintain these fates stably as normal development proceeds or how their dysregulation may contribute to altered cell states in diseases such as cancer. In Drosophila, the tyrosine kinase Abelson (Abl) interfaces with multiple signaling pathways to direct epithelial and neuronal morphogenesis during embryonic and retinal development. Here we show that Abl is required for photoreceptor cell fate maintenance, as Abl mutant photoreceptors lose neuronal markers during late pupal stages but do not re-enter a proliferative state or undergo apoptosis. Failure to maintain the differentiated state correlates with impaired trafficking of the Notch receptor and ectopic Notch signaling, and can be suppressed by reducing the genetic dose of Notch or of its downstream transcriptional effector Suppressor of Hairless. Together, these data reveal a novel mechanism for maintaining the terminally differentiated state of Drosophila photoreceptors and suggest that neuronal fates in the fly retina retain plasticity late into development. Given the general evolutionary conservation of developmental signaling mechanisms, Abl-mediated regulation of Notch could be broadly relevant to cell fate maintenance and reprogramming during normal development, regeneration and oncogenic transformation.
Asunto(s)
Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiología , Drosophila melanogaster/metabolismo , Endocitosis/fisiología , Neuronas/citología , Neuronas/metabolismo , Células Fotorreceptoras de Invertebrados/fisiología , Proteínas Tirosina Quinasas/fisiología , Receptores Notch/metabolismo , Transducción de Señal/fisiología , Animales , Apoptosis/genética , Diferenciación Celular/genética , Proliferación Celular , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Endocitosis/genética , Neuronas/enzimología , Células Fotorreceptoras de Invertebrados/enzimología , Transducción de Señal/genéticaRESUMEN
In Drosophila photoreceptors Ca(2+)-permeable channels TRP and TRPL are the targets of phototransduction, occurring in photosensitive microvilli and mediated by a phospholipase C (PLC) pathway. Using a novel Drosophila brain slice preparation, we studied the distribution and physiological properties of TRP and TRPL in the lamina of the visual system. Immunohistochemical images revealed considerable expression in photoreceptors axons at the lamina. Other phototransduction proteins are also present, mainly PLC and protein kinase C, while rhodopsin is absent. The voltage-dependent Ca(2+) channel cacophony is also present there. Measurements in the lamina with the Ca(2+) fluorescent protein G-CaMP ectopically expressed in photoreceptors, revealed depolarization-induced Ca(2+) increments mediated by cacophony. Additional Ca(2+) influx depends on TRP and TRPL, apparently functioning as store-operated channels. Single synaptic boutons resolved in the lamina by FM4-64 fluorescence revealed that vesicle exocytosis depends on cacophony, TRP and TRPL. In the PLC mutant norpA bouton labeling was also impaired, implicating an additional modulation by this enzyme. Internal Ca(2+) also contributes to exocytosis, since this process was reduced after Ca(2+)-store depletion. Therefore, several Ca(2+) pathways participate in photoreceptor neurotransmitter release: one is activated by depolarization and involves cacophony; this is complemented by internal Ca(2+) release and the activation of TRP and TRPL coupled to Ca(2+) depletion of internal reservoirs. PLC may regulate the last two processes. TRP and TRPL would participate in two different functions in distant cellular regions, where they are opened by different mechanisms. This work sheds new light on the mechanism of neurotransmitter release in tonic synapses of non-spiking neurons.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Exocitosis , Células Fotorreceptoras de Invertebrados/citología , Canales de Potencial de Receptor Transitorio/metabolismo , Animales , Axones/enzimología , Señalización del Calcio , Drosophila melanogaster/enzimología , Fluorescencia , Espacio Intracelular/metabolismo , Fototransducción , Modelos Biológicos , Células Fotorreceptoras de Invertebrados/enzimología , Vesículas Sinápticas/metabolismo , Fosfolipasas de Tipo C/metabolismo , Vías Visuales/citologíaRESUMEN
Autophagy plays an important role in cellular survival by resupplying cells with nutrients during starvation or by clearing misfolded proteins and damaged organelles and thereby preventing degenerative diseases. Conversely, the autophagic process is also recognized as a cellular death mechanism. The circumstances that determine whether autophagy has a beneficial or a detrimental role in cellular survival are currently unclear. We recently showed that autophagy induction is detrimental in neurons that lack a functional AMPK enzyme (AMP-activated protein kinase) and that suffer from severe metabolic stress. We further demonstrated that autophagy and AMPK are interconnected in a negative feedback loop that prevents excessive and destructive stimulation of the autophagic process. Finally, we uncovered a new survival mechanism in AMPK-deficient neurons--cell cannibalism.
Asunto(s)
Autofagia , Citofagocitosis , Neuronas/citología , Proteínas Quinasas Activadas por AMP/deficiencia , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Supervivencia Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/enzimología , Neuronas/enzimología , Células Fotorreceptoras de Invertebrados/citología , Células Fotorreceptoras de Invertebrados/enzimología , Estrés FisiológicoRESUMEN
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.
Asunto(s)
Drosophila melanogaster/enzimología , Drosophila melanogaster/crecimiento & desarrollo , Ojo/enzimología , Ojo/crecimiento & desarrollo , Transducción de Señal , Ubiquitina Tiolesterasa/metabolismo , Envejecimiento/metabolismo , Animales , Caspasa 3/metabolismo , Muerte Celular , Diferenciación Celular , Proliferación Celular , Regulación hacia Abajo , Drosophila melanogaster/citología , Drosophila melanogaster/ultraestructura , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ojo/citología , Ojo/ultraestructura , Discos Imaginales/citología , Discos Imaginales/enzimología , Sistema de Señalización de MAP Quinasas , Mitosis , Fenotipo , Células Fotorreceptoras de Invertebrados/citología , Células Fotorreceptoras de Invertebrados/enzimologíaRESUMEN
In mammalian rods and cones, light activation of the visual pigments leads to release of the chromophore, which is then recycled through a multistep enzymatic pathway, referred to as the visual or retinoid cycle. In invertebrates such as Drosophila, a visual cycle was thought not to exist since the rhodopsins are bistable photopigments, which consist of a chromophore that normally stays bound to the opsin following light activation. Nevertheless, we recently described a visual cycle in Drosophila that serves to recycle the free chromophore that is released following light-induced internalization of rhodopsin, and a retinol dehydrogenase (RDH) that catalyzes the first step of the pathway. Here, we describe the identification of a putative RDH, referred to as RDHB (retinol dehydrogenase B), which functions in the visual cycle and in de novo synthesis of the chromophore. RDHB was expressed in the retinal pigment cells (RPCs), where it promoted the final enzymatic reaction necessary for the production of the chromophore. Mutation of rdhB caused moderate light-dependent degeneration of the phototransducing compartment of the photoreceptor cells-the rhabdomeres, reminiscent of the effects of mutations in some human RDH genes. Since the first and last steps in the visual cycle take place in the RPCs, it appears that these cells are the sites of action for this entire enzymatic pathway in Drosophila.
Asunto(s)
Oxidorreductasas de Alcohol/biosíntesis , Proteínas de Drosophila/biosíntesis , Células Fotorreceptoras de Invertebrados/enzimología , Epitelio Pigmentado de la Retina/enzimología , Transducción de Señal/fisiología , Animales , Animales Modificados Genéticamente , Drosophila melanogaster , Femenino , Técnicas de Inactivación de Genes , Masculino , Degeneración Retiniana/enzimología , Degeneración Retiniana/patología , Epitelio Pigmentado de la Retina/patología , Pigmentos Retinianos/biosíntesisRESUMEN
The photolyase/cryptochrome family of proteins are FAD-containing flavoproteins which carry out blue-light-dependent functions including DNA repair, plant growth and development, and regulation of the circadian clock. In addition to FAD, many members of the family contain a second chromophore which functions as a photo-antenna, harvesting light and transferring the excitation energy to FAD and thus increasing the efficiency of the system. The second chromophore is methenyltetrahydrofolate (MTHF) in most photolyases characterized to date and FAD, FMN, or 5-deazariboflavin in others. To date, no second chromophore has been identified in cryptochromes. Drosophila contains three members of the cryptochrome/photolyase family: cyclobutane pyrimidine dimer (CPD) photolyase, (6-4) photoproduct photolyase, and cryptochrome. We developed an expression system capable of incorporating all known second chromophores into the cognate cryptochrome/photolyase family members. Using this system, we demonstrate that Drosophila CPD photolyase and (6-4) photolyase employ 5-deazariboflavin as their second chromophore, but Drosophila cryptochrome, which is evolutionarily closer to (6-4) photolyase than the CPD photolyase, lacks a second chromophore.
Asunto(s)
Criptocromos/química , Desoxirribodipirimidina Fotoliasa/química , Proteínas de Drosophila/química , Células Fotorreceptoras de Invertebrados/enzimología , Animales , Proteínas de Arabidopsis/química , Baculoviridae , Catálisis , Proteínas de Drosophila/genética , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Células Fotorreceptoras de Invertebrados/virología , ATPasas de Translocación de Protón/química , Dímeros de Pirimidina/química , Esteroide Hidroxilasas/químicaRESUMEN
We have created a Drosophila model of lysozyme amyloidosis to investigate the in vivo behavior of disease-associated variants. To achieve this objective, wild-type (WT) protein and the amyloidogenic variants F57I and D67H were expressed in Drosophila melanogaster using the UAS-gal4 system and both the ubiquitous and retinal expression drivers Act5C-gal4 and gmr-gal4. The nontransgenic w(1118) Drosophila line was used as a control throughout. We utilized ELISA experiments to probe lysozyme protein levels, scanning electron microscopy for eye phenotype classification, and immunohistochemistry to detect the unfolded protein response (UPR) activation. We observed that expressing the destabilized F57I and D67H lysozymes triggers UPR activation, resulting in degradation of these variants, whereas the WT lysozyme is secreted into the fly hemolymph. Indeed, the level of WT was up to 17 times more abundant than the variant proteins. In addition, the F57I variant gave rise to a significant disruption of the eye development, and this correlated to pronounced UPR activation. These results support the concept that the onset of familial amyloid disease is linked to an inability of the UPR to degrade completely the amyloidogenic lysozymes prior to secretion, resulting in secretion of these destabilized variants, thereby leading to deposition and associated organ damage.
Asunto(s)
Amiloidosis/enzimología , Anomalías del Ojo/enzimología , Muramidasa/metabolismo , Respuesta de Proteína Desplegada/fisiología , Amiloidosis/patología , Animales , Animales Modificados Genéticamente , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Proteínas de Drosophila/genética , Drosophila melanogaster , Estrés del Retículo Endoplásmico/fisiología , Anomalías del Ojo/genética , Anomalías del Ojo/patología , Femenino , Proteínas Fluorescentes Verdes/genética , Hemolinfa/enzimología , Humanos , Masculino , Metamorfosis Biológica/fisiología , Microscopía Electrónica de Rastreo , Muramidasa/genética , Células Fotorreceptoras de Invertebrados/enzimología , Células Fotorreceptoras de Invertebrados/patología , Células Fotorreceptoras de Invertebrados/ultraestructura , SolubilidadRESUMEN
The vesicular adenosine triphosphatase (v-ATPase) is a proton pump that acidifies intracellular compartments. In addition, mutations in components of the membrane-bound v-ATPase V0 sector cause acidification-independent defects in yeast, worm, fly, zebrafish, and mouse. In this study, we present a dual function for the neuron-specific V0 subunit a1 orthologue v100 in Drosophila melanogaster. A v100 mutant that selectively disrupts proton translocation rescues a previously characterized synaptic vesicle fusion defect and vesicle fusion with early endosomes. Correspondingly, V100 selectively interacts with syntaxins on the respective target membranes, and neither synaptic vesicles nor early endosomes require v100 for their acidification. In contrast, V100 is required for acidification once endosomes mature into degradative compartments. As a consequence of the complete loss of this neuronal degradation mechanism, photoreceptors undergo slow neurodegeneration, whereas selective rescue of the acidification-independent function accelerates cell death by increasing accumulations in degradation-incompetent compartments. We propose that V100 exerts a temporally integrated dual function that increases neuronal degradative capacity.
Asunto(s)
Drosophila melanogaster/metabolismo , Endosomas/metabolismo , Lisosomas/metabolismo , Células Fotorreceptoras de Invertebrados/citología , Células Fotorreceptoras de Invertebrados/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Adenosina Trifosfatasas , Animales , Autofagia/genética , Supervivencia Celular/genética , Vesículas Citoplasmáticas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/enzimología , Electrorretinografía , Concentración de Iones de Hidrógeno , Macrólidos/farmacología , Glicoproteínas de Membrana/metabolismo , Potenciales de la Membrana/genética , Modelos Neurológicos , Mutación/fisiología , Degeneración Nerviosa/genética , Degeneración Nerviosa/fisiopatología , Células Fotorreceptoras de Invertebrados/efectos de los fármacos , Células Fotorreceptoras de Invertebrados/enzimología , Unión Proteica/fisiología , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas Recombinantes/metabolismo , Transmisión Sináptica/genética , Sinaptosomas/metabolismo , Sintaxina 16/metabolismo , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , ATPasas de Translocación de Protón Vacuolares/genéticaRESUMEN
Receptor protein tyrosine phosphatases (RPTPs) control many aspects of nervous system development. At the Drosophila neuromuscular junction (NMJ), regulation of synapse growth and maturation by the RPTP LAR depends on catalytic phosphatase activity and on the extracellular ligands Syndecan and Dally-like. We show here that the function of LAR in controlling R7 photoreceptor axon targeting in the visual system differs in several respects. The extracellular domain of LAR important for this process is distinct from the domains known to bind Syndecan and Dally-like, suggesting the involvement of a different ligand. R7 targeting does not require LAR phosphatase activity, but instead depends on the phosphatase activity of another RPTP, PTP69D. In addition, a mutation that prevents dimerization of the intracellular domain of LAR interferes with its ability to promote R7 targeting, although it does not disrupt phosphatase activity or neuromuscular synapse growth. We propose that LAR function in R7 is independent of its phosphatase activity, but requires structural features that allow dimerization and may promote the assembly of downstream effectors.
Asunto(s)
Axones/fisiología , Proteínas de Drosophila/fisiología , Drosophila melanogaster/crecimiento & desarrollo , Unión Neuromuscular/crecimiento & desarrollo , Células Fotorreceptoras de Invertebrados/fisiología , Proteínas Tirosina Fosfatasas Similares a Receptores/fisiología , Animales , Axones/enzimología , Proteínas de Drosophila/genética , Drosophila melanogaster/enzimología , Unión Neuromuscular/enzimología , Células Fotorreceptoras de Invertebrados/enzimología , Proteínas Tirosina Fosfatasas Similares a Receptores/genética , Transducción de SeñalRESUMEN
The serine-threonine kinase LKB1 regulates cell polarity from Caenorhabditis elegans to man. Loss of lkb1 leads to a cancer predisposition, known as Peutz-Jeghers Syndrome. Biochemical analysis indicates that LKB1 can phosphorylate and activate a family of AMPK- like kinases, however, the precise contribution of these kinases to the establishment and maintenance of cell polarity is still unclear. Recent studies propose that LKB1 acts primarily through the AMP kinase to establish and/or maintain cell polarity. To determine whether this simple model of how LKB1 regulates cell polarity has relevance to complex tissues, we examined lkb1 mutants in the Drosophila eye. We show that adherens junctions expand and apical, junctional, and basolateral domains mix in lkb1 mutants. Surprisingly, we find LKB1 does not act primarily through AMPK to regulate cell polarity in the retina. Unlike lkb1 mutants, ampk retinas do not show elongated rhabdomeres or expansion of apical and junctional markers into the basolateral domain. In addition, nutrient deprivation does not reveal a more dramatic polarity phenotype in lkb1 photoreceptors. These data suggest that AMPK is not the primary target of LKB1 during eye development. Instead, we find that a number of other AMPK-like kinase, such as SIK, NUAK, Par-1, KP78a, and KP78b show phenotypes similar to weak lkb1 loss of function in the eye. These data suggest that in complex tissues, LKB1 acts on an array of targets to regulate cell polarity.
Asunto(s)
Uniones Adherentes/metabolismo , Polaridad Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Ojo/crecimiento & desarrollo , Proteínas Quinasas/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Proteínas Quinasas Activadas por AMP/metabolismo , Uniones Adherentes/genética , Animales , Polaridad Celular/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Ojo/ultraestructura , Mutación , Células Fotorreceptoras de Invertebrados/enzimología , Células Fotorreceptoras de Invertebrados/ultraestructura , Proteínas Quinasas/genética , Pupa/enzimología , Pupa/genética , Pupa/crecimiento & desarrollo , Retina/crecimiento & desarrollo , Retina/ultraestructuraRESUMEN
The Drosophila mutant tan (t) shows reciprocal pigmentation defects compared with the ebony (e) mutant. Visual phenotypes, however, are similar in both flies: Electroretinogram (ERG) recordings lack "on" and "off" transients, an indication of impaired synaptic transmission to postsynaptic cells L1 and L2. Cloning of tan revealed transcription of the gene in the retina, apparently in photoreceptor cells. We expressed Tan in Escherichia coli and confirmed by Western blotting and mass spectroscopic analyses that Tan is expressed as preprotein, followed by proteolytic cleavage into two subunits at a conserved --Gly--Cys-- motif like its fungal ortholog isopenicillin-N N-acyltransferase (IAT). Tan thus belongs to the large family of cysteine peptidases. To discriminate expression of Tan and Ebony in retina and optic neuropils, we raised antisera against specific Tan peptides. Testing for colocalization with GMR-driven n-Syb-GFP labeling revealed that Tan expression is confined to the photoreceptor cells R1-R8. A close proximity of Tan and Ebony expression is evident in lamina cartridges, where three epithelial glia cells envelop the six photoreceptor terminals R1-R6. In the medulla, R7/R8 axonal terminals appeared lined up side by side with glial extensions. This local proximity supports a model for Drosophila visual synaptic transmission in which Tan and Ebony interact biochemically in a putative histamine inactivation and recycling pathway in Drosophila.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Cisteína Endopeptidasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/enzimología , Proteínas del Tejido Nervioso/metabolismo , Células Fotorreceptoras de Invertebrados/enzimología , Animales , Neuroglía/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Transducción de Señal/fisiología , Transmisión Sináptica/fisiologíaRESUMEN
Guanine nucleotides are key players in mediating growth-cone signaling during neural development. The supply of cellular guanine nucleotides in animals can be achieved via the de novo synthesis and salvage pathways. The de novo synthesis of guanine nucleotides is required for lymphocyte proliferation in animals. Whether the de novo synthesis pathway is essential for any other cellular processes, however, remains unknown. In a search for genes required for the establishment of neuronal connectivity in the fly visual system, we identify the burgundy (bur) gene as an essential player in photoreceptor axon guidance. The bur gene encodes the only GMP synthetase in Drosophila that catalyzes the final reaction of de novo GMP synthesis. Loss of bur causes severe defects in axonal fasciculation, retinotopy, and growth-cone morphology, but does not affect photoreceptor differentiation or retinal patterning. Similar defects were observed when the raspberry (ras) gene, encoding for inosine monophosphate dehydrogenase catalyzing the IMP-to-XMP conversion in GMP de novo synthesis, was mutated. Our study thus provides the first in vivo evidence to support an essential and specific role for de novo synthesis of guanine nucleotides in axon guidance.
Asunto(s)
Axones/fisiología , Drosophila/fisiología , Guanosina Monofosfato/biosíntesis , Secuencia de Aminoácidos , Animales , Axones/enzimología , Axones/metabolismo , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Ligasas de Carbono-Nitrógeno/genética , Ligasas de Carbono-Nitrógeno/fisiología , Diferenciación Celular/genética , Drosophila/genética , Drosophila/crecimiento & desarrollo , Evolución Molecular , Larva/enzimología , Larva/crecimiento & desarrollo , Larva/metabolismo , Datos de Secuencia Molecular , Mutación , Lóbulo Óptico de Animales no Mamíferos/enzimología , Lóbulo Óptico de Animales no Mamíferos/crecimiento & desarrollo , Células Fotorreceptoras de Invertebrados/enzimología , Células Fotorreceptoras de Invertebrados/metabolismo , Células Fotorreceptoras de Invertebrados/fisiología , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/fisiologíaRESUMEN
In vivo light-induced and basal hydrolysis of phosphatidyl inositol 4,5-bisphosphate (PIP2) by phospholipase C (PLC) were monitored in Drosophila photoreceptors using genetically targeted PIP2-sensitive ion channels (Kir2.1) as electrophysiological biosensors for PIP2. In cells loaded via patch pipettes with varying concentrations of Ca2+ buffered by 4 mM free BAPTA, light-induced PLC activity, showed an apparent bell-shaped dependence on free Ca2+ (maximum at "100 nM", approximately 10-fold inhibition at <10nM or approximately 1 microM). However, experiments where the total BAPTA concentration was varied whilst free [Ca2+] was maintained constant indicated that inhibition of PLC at higher (>100 nM) nominal Ca2+ concentrations was independent of Ca2+ and due to inhibition by BAPTA itself (IC50 approximately 8 mM). Di-bromo BAPTA (DBB) was yet more potent at inhibiting PLC activity (IC50 approximately 1mM). Both BAPTA and DBB also appeared to induce a modest, but less severe inhibition of basal PLC activity. By contrast, EGTA, failed to inhibit PLC activity when pre-loaded with Ca2+, but like BAPTA, inhibited both basal and light-induced PLC activity when introduced without Ca2+. The results indicate that both BAPTA and DBB inhibit PLC activity independently of their role as Ca2+ chelators, whilst non-physiologically low (<100 nM) levels of Ca2+ suppress both basal and light-induced PLC activity.
Asunto(s)
Proteínas de Drosophila/antagonistas & inhibidores , Drosophila melanogaster/enzimología , Ácido Egtácico/análogos & derivados , Células Fotorreceptoras de Invertebrados/efectos de los fármacos , Células Fotorreceptoras de Invertebrados/enzimología , Fosfolipasas de Tipo C/antagonistas & inhibidores , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/efectos de los fármacos , Ácido Egtácico/farmacología , Técnicas de Placa-Clamp , Células Fotorreceptoras de Invertebrados/citología , Fosfolipasas de Tipo C/metabolismoRESUMEN
Drosophila melanogaster phototransduction proceeds via a phospholipase C (PLC)-triggered cascade of phosphatidylinositol (PI) lipid modifications, many steps of which remain undefined. We describe the involvement of the lipid phosphatidic acid and the enzyme that generates it, phospholipase D (Pld), in this process. Pld(null) flies exhibit decreased light sensitivity as well as a heightened susceptibility to retinal degeneration. Pld overexpression rescues flies lacking PLC from light-induced, metarhodopsin-mediated degeneration and restores visual signaling in flies lacking the PI transfer protein, which is a key player in the replenishment of the PI 4,5-bisphosphate (PIP2) substrate used by PLC to transduce light stimuli into neurological signals. Altogether, these findings suggest that Pld facilitates phototransduction by maintaining adequate levels of PIP2 and by protecting the visual system from metarhodopsin-induced, low light degeneration.
Asunto(s)
Drosophila melanogaster/enzimología , Fosfolipasa D/metabolismo , Fosfolípidos/metabolismo , Células Fotorreceptoras de Invertebrados/enzimología , Retina/enzimología , Visión Ocular/fisiología , Animales , Drosophila melanogaster/ultraestructura , Luz/efectos adversos , Proteínas de la Membrana/metabolismo , Mutación/fisiología , Ácidos Fosfatidicos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipasa D/genética , Proteínas de Transferencia de Fosfolípidos/metabolismo , Células Fotorreceptoras de Invertebrados/ultraestructura , Retina/ultraestructura , Degeneración Retiniana/enzimología , Degeneración Retiniana/genética , Degeneración Retiniana/fisiopatología , Rodopsina/metabolismo , Rodopsina/efectos de la radiación , Fosfolipasas de Tipo C/metabolismoRESUMEN
The rolling blackout (rbo) gene encodes an integral plasma membrane lipase required for Drosophila phototransduction. Photoreceptors are enriched for the RBO protein, and temperature-sensitive rbo mutants show reversible elimination of phototransduction within minutes, demonstrating an acute requirement for the protein. The block is activity dependent, indicating that the action of RBO is use dependent. Conditional rbo mutants show activity-dependent depletion of diacylglycerol and concomitant accumulation of phosphatidylinositol phosphate and phosphatidylinositol 4,5-bisphosphate within minutes of induction, suggesting rapid downregulation of phospholipase C (PLC) activity. The RBO requirement identifies an essential regulatory step in G-protein-coupled, PLC-dependent inositol lipid signaling mediating activation of TRP and TRPL channels during phototransduction.
Asunto(s)
Hidrolasas de Éster Carboxílico/metabolismo , Diglicéridos/metabolismo , Proteínas de Drosophila/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipasas/metabolismo , Visión Ocular/genética , Secuencia de Aminoácidos/genética , Animales , Animales Modificados Genéticamente , Secuencia de Bases/genética , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/aislamiento & purificación , Membrana Celular/enzimología , Mapeo Cromosómico , ADN Complementario/análisis , ADN Complementario/genética , Regulación hacia Abajo/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/aislamiento & purificación , Drosophila melanogaster , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Regulación de la Expresión Génica/genética , Potenciales de la Membrana/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Mutación/genética , Fosfolipasas/genética , Fosfolipasas/aislamiento & purificación , Células Fotorreceptoras de Invertebrados/enzimología , Temperatura , Fosfolipasas de Tipo C/metabolismoRESUMEN
The phosphatidylinositol 4,5-bisphosphate (PIP(2))-sensitive inward rectifier channel Kir2.1 was expressed in Drosophila photoreceptors and used to monitor in vivo PIP(2) levels. Since the wild-type (WT) Kir2.1 channel appeared to be saturated by the prevailing PIP(2) concentration, we made a single amino acid substitution (R228Q), which reduced the effective affinity for PIP(2) and yielded channels generating currents proportional to the PIP(2) levels relevant for phototransduction. To isolate Kir2.1 currents, recordings were made from mutants lacking both classes of light-sensitive transient receptor potential channels (TRP and TRPL). Light resulted in the effective depletion of PIP(2) by phospholipase C (PLC) in approximately three or four microvilli per absorbed photon at rates exceeding approximately 150% of total microvillar phosphoinositides per second. PIP(2) was resynthesized with a half-time of approximately 50 s. When PIP(2) resynthesis was prevented by depriving the cell of ATP, the Kir current spontaneously decayed at maximal rates representing a loss of approximately 40% loss of total PIP(2) per minute. This loss was attributed primarily to basal PLC activity, because it was greatly decreased in norpA mutants lacking PLC. We tried to confirm this by using the PLC inhibitor U73122; however, this was found to act as a novel inhibitor of the Kir2.1 channel. PIP(2) levels were reduced approximately 5-fold in the diacylglycerol kinase mutant (rdgA), but basal PLC activity was still pronounced, consistent with the suggestion that raised diacylglycerol levels are responsible for the constitutive TRP channel activity characteristic of this mutant.
Asunto(s)
Drosophila melanogaster/fisiología , Fosfatidilinositol 4,5-Difosfato/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Fosfolipasas de Tipo C/metabolismo , Animales , Drosophila melanogaster/genética , Activación Enzimática , Marcación de Gen , Luz , Técnicas de Placa-Clamp , Células Fotorreceptoras de Invertebrados/citología , Células Fotorreceptoras de Invertebrados/enzimología , Células Fotorreceptoras de Invertebrados/fisiología , Mutación Puntual , Canales de Potasio de Rectificación Interna/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Fosfolipasas de Tipo C/genéticaRESUMEN
ERK MAP kinase plays a key role in relaying extracellular signals to transcriptional regulation. As different activity levels or the different duration of ERK activity can elicit distinct responses in one and the same cell, ERK has to be under strict positive and negative control. Although numerous genes acting positively in the ERK signaling pathway have been recovered in genetic screens, mutations in genes encoding negative ERK regulators appear underrepresented. We therefore sought to genetically characterize the dual-specificity phosphatase DMKP3. First, we established a novel assay to elucidate the substrate preferences of eukaryotic phosphatases in vivo and thereby confirmed the specificity of DMKP3 as an ERK phosphatase. The Dmkp3 overexpression phenotype characterized in this assay permitted us to isolate Dmkp3 null mutations. By genetic analysis we show that DMKP3 and the tyrosine phosphatase PTP-ER perform partially redundant functions on the same substrate, ERK. DMKP3 functions autonomously in a subset of photoreceptor progenitor cells in eye imaginal discs. In addition, DMKP3 function appears to be required in surrounding non-neuronal cells for ommatidial patterning and photoreceptor differentiation.