RESUMO
Actomyosin contraction shapes the Drosophila eye's panoramic view. The convex curvature of the retinal epithelium, organized in â¼800 close-packed ommatidia, depends upon a fourfold condensation of the retinal floor mediated by contraction of actin stress fibers in the endfeet of interommatidial cells (IOCs). How these tensile forces are coordinated is not known. Here, we discover a previously unobserved phenomenon: Ca2+ waves regularly propagate across the IOC network in pupal and adult eyes. Genetic evidence demonstrates that IOC waves are independent of phototransduction, but require the inositol 1,4,5-triphosphate receptor (IP3R), suggesting that these waves are mediated by Ca2+ releases from endoplasmic reticulum stores. Removal of IP3R disrupts stress fibers in IOC endfeet and increases the basal retinal surface by â¼40%, linking IOC waves to facilitation of stress fiber contraction and floor morphogenesis. Furthermore, IP3R loss disrupts the organization of a collagen IV network underneath the IOC endfeet, implicating the extracellular matrix and its interaction with stress fibers in eye morphogenesis. We propose that coordinated cytosolic Ca2+ increases in IOC waves promote stress fiber contractions, ensuring an organized application of the planar tensile forces that condense the retinal floor. This article has an associated 'The people behind the papers' interview.
Assuntos
Actinas/genética , Cálcio/metabolismo , Morfogênese/genética , Fibras de Estresse/genética , Citoesqueleto de Actina/genética , Actinas/metabolismo , Actomiosina/genética , Actomiosina/metabolismo , Animais , Sinalização do Cálcio/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Retículo Endoplasmático/genética , Pupa , Retina/crescimento & desenvolvimento , Retina/metabolismoRESUMO
The endoplasmic reticulum (ER) serves virtually all aspects of cell physiology and, by pathways that are incompletely understood, is dynamically remodeled to meet changing cell needs. Inositol-requiring enzyme 1 (Ire1), a conserved core protein of the unfolded protein response (UPR), participates in ER remodeling and is particularly required during the differentiation of cells devoted to intense secretory activity, so-called 'professional' secretory cells. Here, we characterize the role of Ire1 in ER differentiation in the developing Drosophila compound eye photoreceptors (R cells). As part of normal development, R cells take a turn as professional secretory cells with a massive secretory effort that builds the photosensitive membrane organelle, the rhabdomere. We find rough ER sheets proliferate as rhabdomere biogenesis culminates, and Ire1 is required for normal ER differentiation. Ire1 is active early in R cell development and is required in anticipation of peak biosynthesis. Without Ire1, the amount of rough ER sheets is strongly reduced and the extensive cortical ER network at the rhabdomere base, the subrhabdomere cisterna (SRC), fails. Instead, ER proliferates in persistent and ribosome-poor tubular tangles. A phase of Ire1 activity early in R cell development thus shapes dynamic ER.
Assuntos
Diferenciação Celular , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/citologia , Retículo Endoplasmático/fisiologia , Endorribonucleases/fisiologia , Células Fotorreceptoras de Invertebrados/fisiologia , Animais , Olho Composto de Artrópodes/citologia , Olho Composto de Artrópodes/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Retículo Endoplasmático/ultraestrutura , Feminino , Masculino , Morfogênese , Células Fotorreceptoras de Invertebrados/ultraestruturaRESUMO
The prototypical transient receptor potential (TRP) channel is the major light-sensitive, and Ca(2+)-permeable channel in the microvillar photoreceptors of Drosophila. TRP channels are activated following hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] by the key effector enzyme phospholipase C (PLC). Mutants lacking TRP channels undergo light-dependent retinal degeneration, as a consequence of the reduced Ca(2+) influx. It has been proposed that degeneration is caused by defects in the Ca(2+)-dependent visual pigment cycle, which result in accumulation of toxic phosphorylated metarhodopsin-arrestin complexes (MPP-Arr2). Here we show that two interventions, which prevent accumulation of MPP-Arr2, namely rearing under red light or eliminating the C-terminal rhodopsin phosphorylation sites, failed to rescue degeneration in trp mutants. Instead, degeneration in trp mutants reared under red light was rescued by mutation of PLC. Degeneration correlated closely with the light-induced depletion of PtdIns(4,5)P2 that occurs in trp mutants due to failure of Ca(2+)-dependent inhibition of PLC. Severe retinal degeneration was also induced in the dark in otherwise wild-type flies by overexpression of a bacterial PtdInsPn phosphatase (SigD) to deplete PtdIns(4,5)P2. In degenerating trp photoreceptors, phosphorylated Moesin, a PtdIns(4,5)P2-regulated membrane-cytoskeleton linker essential for normal microvillar morphology, was found to delocalize from the rhabdomere and there was extensive microvillar actin depolymerisation. The results suggest that compromised light-induced Ca(2+) influx, due to loss of TRP channels, leads to PtdIns(4,5)P2 depletion, resulting in dephosphorylation of Moesin, actin depolymerisation and disintegration of photoreceptor structure.
Assuntos
Fosfatidilinositol 4,5-Difosfato/metabolismo , Degeneração Retiniana/fisiopatologia , Actinas/genética , Actinas/metabolismo , Animais , Drosophila , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Degeneração Retiniana/genética , Rodopsina/metabolismo , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/metabolismo , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismoRESUMO
Drosophila compound eye morphogenesis transforms a simple epithelium into an approximate hollow hemisphere comprised of â¼700 ommatidia, packed as tapering hexagonal prisms between a rigid external array of cuticular lenses and a parallel, rigid internal floor, the fenestrated membrane (FM). Critical to vision, photosensory rhabdomeres are sprung between these two surfaces, grading their length and shape accurately across the eye and aligning them to the optical axis. Using fluorescently tagged collagen and laminin, we show that that the FM assembles sequentially, emerging in the larval eye disc in the wake of the morphogenetic furrow as the original collagen-containing basement membrane (BM) separates from the epithelial floor and is replaced by a new, laminin-rich BM, which advances around axon bundles of newly differentiated photoreceptors as they exit the retina, forming fenestrae in this new, laminin-rich BM. In mid-pupal development, the interommatidial cells (IOCs) autonomously deposit collagen at fenestrae, forming rigid, tension-resisting grommets. In turn, stress fibers assemble in the IOC basal endfeet, where they contact grommets at anchorages mediated by integrin linked kinase (ILK). The hexagonal network of IOC endfeet tiling the retinal floor couples nearest-neighbor grommets into a supracellular tri-axial tension network. Late in pupal development, IOC stress fiber contraction folds pliable BM into a hexagonal grid of collagen-stiffened ridges, concomitantly decreasing the area of convex FM and applying essential morphogenetic longitudinal tension to rapidly growing rhabdomeres. Together, our results reveal an orderly program of sequential assembly and activation of a supramolecular tensile network that governs Drosophila retinal morphogenesis.
Assuntos
Drosophila melanogaster , Animais , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Colágeno/metabolismo , Larva , Retina/crescimento & desenvolvimento , Retina/metabolismoRESUMO
Sensory neuron terminal differentiation tasks apical secretory transport with delivery of abundant biosynthetic traffic to the growing sensory membrane. We recently showed Drosophila Rab11 is essential for rhodopsin transport in developing photoreceptors and asked here if myosin V and the Drosophila Rab11 interacting protein, dRip11, also participate in secretory transport. Reduction of either protein impaired rhodopsin transport, stunting rhabdomere growth and promoting accumulation of cytoplasmic rhodopsin. MyoV-reduced photoreceptors also developed ectopic rhabdomeres inappropriately located in basolateral membrane, indicating a role for MyoV in photoreceptor polarity. Binary yeast two hybrids and in vitro protein-protein interaction predict a ternary complex assembled by independent dRip11 and MyoV binding to Rab11. We propose this complex delivers morphogenic secretory traffic along polarized actin filaments of the subcortical terminal web to the exocytic plasma membrane target, the rhabdomere base. A protein trio conserved across eukaryotes thus mediates normal, in vivo sensory neuron morphogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Diferenciação Celular/fisiologia , Proteínas de Drosophila/fisiologia , Proteínas de Membrana/fisiologia , Miosina Tipo V/fisiologia , Células Fotorreceptoras/citologia , Proteínas rab de Ligação ao GTP/fisiologia , Actinas/metabolismo , Animais , Animais Geneticamente Modificados , Membrana Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Exocitose/fisiologia , Células Fotorreceptoras/metabolismoRESUMO
Approximately 40 years ago, an elegant automatic-gain control was revealed in compound eye photoreceptors: In bright light, an assembly of small pigment granules migrates to the cytoplasmic face of the photosensitive membrane organelle, the rhabdomere, where they attenuate waveguide propagation along the rhabdomere. This migration results in a "longitudinal pupil" that reduces rhodopsin exposure by a factor of 0.8 log units. Light-induced elevation of cytosolic free Ca(2+) triggers the migration of pigment granules, and pigment granules fail to migrate in a mutant deficient in photoactivated TRP calcium channels. However, the mechanism that moves photoreceptor pigment granules remains elusive. Are the granules actively pulled toward the rhabdomere upon light, or are they instead actively pulled into the cytoplasm in the absence of light? Here we show that Ca(2+)-activated Myosin V (MyoV) pulls pigment granules to the rhabdomere. Thus, one of MyoV's several functions is also as a sensory-adaptation motor. In vitro, Ca(2+) both activates and inhibits MyoV motility; in vivo, its role is undetermined. This first demonstration of an in vivo role for Ca(2+) in MyoV activity shows that in Drosophila photoreceptors, Ca(2+) stimulates MyoV motility.
Assuntos
Cálcio/metabolismo , Drosophila/metabolismo , Miosina Tipo V/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Pigmentos Biológicos/metabolismo , Animais , Grânulos Citoplasmáticos/metabolismo , Luz , Pupila/fisiologiaRESUMO
BACKGROUND: Arrestins are pivotal, multifunctional organizers of cell responses to GPCR stimulation, including cell survival and cell death. In Drosophila norpA and rdgC mutants, endocytosis of abnormally stable complexes of rhodopsin (Rh1) and fly photoreceptor Arrestin2 (Arr2) triggers cell death, implicating Rh1/Arr2-bearing endosomes in pro-cell death signaling, potentially via arrestin-mediated GPCR activation of effector kinase pathways. In order to further investigate arrestin function in photoreceptor physiology and survival, we studied Arr2's partner photoreceptor arrestin, Arr1, in developing and adult Drosophila compound eyes. RESULTS: We report that Arr1, but not Arr2, is essential for normal, light-induced rhodopsin endocytosis. Also distinct from Arr2, Arr1 is essential for light-independent photoreceptor survival. Photoreceptor cell death caused by loss of Arr1 is strongly suppressed by coordinate loss of Arr2. We further find that Rh1 C-terminal phosphorylation is essential for light-induced endocytosis and also for translocation of Arr1, but not Arr2, from dark-adapted photoreceptor cytoplasm to photosensory membrane rhabdomeres. In contrast to a previous report, we do not find a requirement for photoreceptor myosin kinase NINAC in Arr1 or Arr2 translocation. CONCLUSIONS: The two Drosophila photoreceptor arrestins mediate distinct and essential cell pathways downstream of rhodopsin activation. We propose that Arr1 mediates an endocytotic cell-survival activity, scavenging phosphorylated rhodopsin and thereby countering toxic Arr2/Rh1 accumulation; elimination of toxic Arr2/Rh1 in double mutants could thus rescue arr1 mutant photoreceptor degeneration.
Assuntos
Arrestinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/fisiologia , Endocitose/fisiologia , Células Fotorreceptoras de Invertebrados/fisiologia , Rodopsina/metabolismo , Animais , Arrestinas/fisiologia , Western Blotting , Sobrevivência Celular/fisiologia , Proteínas de Drosophila/fisiologia , Imuno-Histoquímica , Microscopia Eletrônica , Fosforilação , Células Fotorreceptoras de Invertebrados/ultraestrutura , Ligação ProteicaRESUMO
Lipid peroxides are generated by oxidative stress in cells, and contribute to ageing and neurodegenerative disease. The eye is at special risk for lipid peroxidation because photoreceptors possess amplified sensory membranes rich in peroxidation-susceptible polyunsaturated fatty acids. Light-induced lipid peroxidation in the retina contributes to retinal degeneration, and lipid peroxidation has been implicated in the progression of age-associated ocular diseases such as age-related macular degeneration (AMD). Here, we show that exposing Drosophila melanogaster to strong blue light induces oxidative stress including lipid peroxidation that results in retinal degeneration. Surprisingly, very young flies are resilient to this acute light stress, suggesting they possess endogenous neuroprotective mechanisms. While lipophilic antioxidants partially suppressed blue light-induced retinal degeneration in older flies, we find that overexpression of cytochrome b5 (Cyt-b5) completely suppressed both blue light-induced lipid peroxidation and retinal degeneration. Our data identify Cyt-b5 as a neuroprotective factor that targets light-induced oxidative damage, particularly lipid peroxidation. Cyt-b5 may function via supporting antioxidant recycling, thereby providing a strategy to prevent oxidative stress in ageing photoreceptors that would be synergistic with dietary antioxidant supplementation.
RESUMO
A programmable optical stimulator for Drosophila eyes is presented. The target application of the stimulator is to induce retinal degeneration in fly photoreceptor cells by exposing them to light in a controlled manner. The goal of this work is to obtain a reproducible system for studying age-related changes in susceptibility to environmental ocular stress. The stimulator uses light emitting diodes and an embedded computer to control illuminance, color (blue or red) and duration in two independent chambers. Further, the stimulator is equipped with per-chamber light and temperature sensors and a fan to monitor light intensity and to control temperature. An ON/OFF temperature control implemented on the embedded computer keeps the temperature from reaching levels that will induce the heat shock stress response in the flies. A custom enclosure was fabricated to house the electronic components of the stimulator. The enclosure provides a light-impermeable environment that allows air flow and lets users easily load and unload fly vials. Characterization results show that the fabricated stimulator can produce light at illuminances ranging from 0 to 16000 lux and power density levels from 0 to 7.2 mW/cm2 for blue light. For red light the maximum illuminance is 8000 lux which corresponds to a power density of 3.54 mW/cm2. The fans and the ON/OFF temperature control are able to keep the temperature inside the chambers below 28.17°C. Experiments with white-eye male flies were performed to assess the ability of the fabricated simulator to induce blue light-dependent retinal degeneration. Retinal degeneration is observed in flies exposed to 8 hours of blue light at 7949 lux. Flies in a control experiment with no light exposure show no retinal degeneration. Flies exposed to red light for the similar duration and light intensity (8 hours and 7994 lux) do not show retinal degeneration either. Hence, the fabricated stimulator can be used to create environmental ocular stress using blue light.
RESUMO
Drosophila photoreceptors (R cells) are an extreme instance of sensory membrane amplification via apical microvilli, a widely deployed and deeply conserved operation of polarized epithelial cells. Developmental rotation of R cell apices aligns rhabdomere microvilli across the optical axis and enables enormous membrane expansion in a new, proximal distal dimension. R cell ectoplasm, the specialized cortical cytoplasm abutting the rhabdomere is likewise enormously amplified. Ectoplasm is dominated by the actin-rich terminal web, a conserved operational domain of the ancient vesicle-transport motor, Myosin V. R cells harness Myosin V to move two distinct cargoes, the biosynthetic traffic that builds the rhabdomere during development, and the migration of pigment granules that mediates the adaptive "longitudinal pupil" in adults, using two distinct Rab proteins. Ectoplasm further shapes a distinct cortical endosome compartment, the subrhabdomeral cisterna (SRC), vital to normal cell function. Reticulon, a protein that promotes endomembrane curvature, marks the SRC. R cell visual arrestin 2 (Arr2) is predominantly cytoplasmic in dark-adapted photoreceptors but on illumination it translocates to the rhabdomere, where it quenches ongoing photosignaling by binding to activated metarhodopsin. Arr2 translocation is "powered" by diffusion; a motor is not required to move Arr2 and ectoplasm does not obstruct its rapid diffusion to the rhabdomere.
Assuntos
Micropartículas Derivadas de Células/metabolismo , Citoplasma/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Membrana/metabolismo , Células Fotorreceptoras de Invertebrados/citologia , Animais , Arrestinas/metabolismo , Drosophila , Microvilosidades/metabolismo , Miosina Tipo V/metabolismo , Células Fotorreceptoras de Invertebrados/ultraestruturaRESUMO
Upon illumination, visual arrestin translocates from photoreceptor cell bodies to rhodopsin and membrane-rich photosensory compartments, vertebrate outer segments or invertebrate rhabdomeres, where it quenches activated rhodopsin. Both the mechanism and function of arrestin translocation are unresolved and controversial. In dark-adapted photoreceptors of the fruitfly Drosophila, confocal immunocytochemistry shows arrestin (Arr2) associated with distributed photoreceptor endomembranes. Immunocytochemistry and live imaging of GFP-tagged Arr2 demonstrate rapid reversible translocation to stimulated rhabdomeres in stoichiometric proportion to rhodopsin photoisomerization. Translocation is very rapid in normal photoreceptors (time constant <10 s) and can also be resolved in the time course of electroretinogram recordings. Genetic elimination of key phototransduction proteins, including phospholipase C (PLC), Gq, and the light-sensitive Ca2+-permeable TRP channels, slows translocation by 10- to 100-fold. Our results indicate that Arr2 translocation in Drosophila photoreceptors is driven by diffusion, but profoundly accelerated by phototransduction and Ca2+ influx.
Assuntos
Arrestinas/metabolismo , Proteínas de Drosophila/metabolismo , Transdução de Sinal Luminoso/fisiologia , Células Fotorreceptoras de Invertebrados/fisiologia , Rodopsina/metabolismo , Animais , Animais Geneticamente Modificados , Arrestinas/genética , Cálcio/metabolismo , Adaptação à Escuridão/genética , Drosophila , Proteínas de Drosophila/genética , Eletrorretinografia/métodos , Endocitose/fisiologia , Regulação da Expressão Gênica/efeitos da radiação , Proteínas de Fluorescência Verde/genética , Isomerismo , Luz , Transdução de Sinal Luminoso/genética , Mutação/genética , Fosfolipase C beta/genética , Transporte Proteico/fisiologia , Análise Espectral , Fatores de Tempo , Canais de Potencial de Receptor Transitório/genéticaRESUMO
Phototransduction in flies is the fastest known G protein-coupled signaling cascade, but how this performance is achieved remains unclear. Here, we investigate the mechanism and role of rhodopsin inactivation. We determined the lifetime of activated rhodopsin (metarhodopsin = M( *)) in whole-cell recordings from Drosophila photoreceptors by measuring the time window within which inactivating M( *) by photoreisomerization to rhodopsin could suppress responses to prior illumination. M( *) was inactivated rapidly (tau approximately 20 ms) under control conditions, but approximately 10-fold more slowly in Ca2+-free solutions. This pronounced Ca2+ dependence of M( *) inactivation was unaffected by mutations affecting phosphorylation of rhodopsin or arrestin but was abolished in mutants of calmodulin (CaM) or the CaM-binding myosin III, NINAC. This suggests a mechanism whereby Ca2+ influx acting via CaM and NINAC accelerates the binding of arrestin to M( *). Our results indicate that this strategy promotes quantum efficiency, temporal resolution, and fidelity of visual signaling.
Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Rodopsina/metabolismo , Visão Ocular/fisiologia , Fatores Etários , Animais , Animais Geneticamente Modificados , Arrestina/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Mutação/fisiologia , Cadeias Pesadas de Miosina/metabolismo , Técnicas de Patch-Clamp , Estimulação Luminosa/métodos , Células Fotorreceptoras de Invertebrados/efeitos dos fármacos , Células Fotorreceptoras de Invertebrados/fisiologia , Visão Ocular/efeitos dos fármacosRESUMO
In developing Drosophila photoreceptors, rhodopsin is trafficked to the rhabdomere, a specialized domain within the apical membrane surface. Rab11, a small GTPase implicated in membrane traffic, immunolocalizes to the trans-Golgi network, cytoplasmic vesicles and tubules, and the base of rhabdomeres. One hour after release from the endoplasmic reticulum, rhodopsin colocalizes with Rab11 in vesicles at the base of the rhabdomere. When Rab11 activity is reduced by three different genetic procedures, rhabdomere morphogenesis is inhibited and rhodopsin-bearing vesicles proliferate within the cytosol. Rab11 activity is also essential for development of MVB endosomal compartments; this is probably a secondary consequence of impaired rhabdomere development. Furthermore, Rab11 is required for transport of TRP, another rhabdomeric protein, and for development of specialized membrane structures within Garland cells. These results establish a role for Rab11 in the post-Golgi transport of rhodopsin and of other proteins to the rhabdomeric membranes of photoreceptors, and in analogous transport processes in other cells.
Assuntos
Drosophila/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Rodopsina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Animais Geneticamente Modificados , Canais de Cálcio/biossíntese , Canais de Cálcio/genética , Drosophila/genética , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/genética , Endocitose/fisiologia , Imuno-Histoquímica , Microscopia Eletrônica , Transporte Proteico/fisiologia , Canais de Potencial de Receptor TransitórioRESUMO
Starting with a mutation impacting photoreceptor morphogenesis, we identify here a Drosophila gene, eyes closed (eyc), as a fly homolog of p47, a protein co-factor of the p97 ATPase implicated in membrane fusion. Temporal misexpression of Eyc during rhabdomere extension early in pupal life results in inappropriate retention of normally transient adhesions between developing rhabdomeres. Later Eyc misexpression results in endoplasmic reticulum proliferation and inhibits rhodopsin transport to the developing photosensitive membrane. Loss of Eyc function results in a lethal failure of nuclear envelope assembly in early zygotic divisions. Phenotypes resulting from eyc mutations provide the first in vivo evidence for a role for p47 in membrane biogenesis.
Assuntos
Proteínas de Transporte/genética , Proteínas de Drosophila/genética , Drosophila/genética , Proteínas de Membrana/genética , Células Fotorreceptoras de Invertebrados/embriologia , Células Fotorreceptoras de Invertebrados/fisiologia , Proteínas de Transporte Vesicular , Sequência de Aminoácidos , Animais , Drosophila/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos , Dados de Sequência Molecular , Morfogênese/genética , Mutação , Homologia de Sequência de Aminoácidos , Proteínas de Ligação a Fator Solúvel Sensível a N-EtilmaleimidaRESUMO
Ezrin-Radixin-Moesin (ERM) family proteins organize heterogeneous sub-plasma membrane protein scaffolds that shape membranes and their physiology. In Drosophila oocytes and imaginal discs, epithelial organization, fundamental to development and physiology, is devastated by the loss of Moesin. Here, we show that Moesin is crucial for Drosophila photoreceptor morphogenesis. Beyond its requirement for retinal epithelium integrity, Moesin is essential for the proper assembly of the apical membrane skeleton that builds the photosensitive membrane, the rhabdomere. Moesin localizes to the rhabdomere base, a dynamic locus of cytoskeletal reorganization and membrane traffic. Downregulation of Moesin through RNAi or genetic loss of function profoundly disrupts the membrane cytoskeleton and apical membrane organization. We find normal levels and distribution of Moesin in photoreceptors of a Moesin mutant previously regarded as protein null, suggesting alternative interpretations for studies using this allele. Our results show an essential structural role for Moesin in photoreceptor morphology.
Assuntos
Drosophila melanogaster/embriologia , Proteínas dos Microfilamentos/metabolismo , Células Fotorreceptoras de Invertebrados/embriologia , Animais , Citoesqueleto/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Imunofluorescência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/genética , Microscopia Confocal , Células Fotorreceptoras de Invertebrados/metabolismo , Interferência de RNARESUMO
The apical transmembrane protein Crumbs is a central regulator of epithelial apical-basal polarity in Drosophila. Loss-of-function mutations in the human homologue of Crumbs, CRB1 (RP12), cause recessive retinal dystrophies, including retinitis pigmentosa. Here we show that Crumbs and CRB1 localize to corresponding subdomains of the photoreceptor apical plasma membrane: the stalk of the Drosophila photoreceptor and the inner segment of mammalian photoreceptors. These subdomains support the morphogenesis and orientation of the photosensitive membrane organelles: rhabdomeres and outer segments, respectively. Drosophila Crumbs is required to maintain zonula adherens integrity during the rapid apical membrane expansion that builds the rhabdomere. Crumbs also regulates stalk development by stabilizing the membrane-associated spectrin cytoskeleton, a function mechanistically distinct from its role in epithelial apical-basal polarity. We propose that Crumbs is a central component of a molecular scaffold that controls zonula adherens assembly and defines the stalk as an apical membrane subdomain. Defects in such scaffolds may contribute to human CRB1-related retinal dystrophies.