RESUMO
SignificanceTo move efficiently, animals must continuously work out their x,y,z positions with respect to real-world objects, and many animals have a pair of eyes to achieve this. How photoreceptors actively sample the eyes' optical image disparity is not understood because this fundamental information-limiting step has not been investigated in vivo over the eyes' whole sampling matrix. This integrative multiscale study will advance our current understanding of stereopsis from static image disparity comparison to a morphodynamic active sampling theory. It shows how photomechanical photoreceptor microsaccades enable Drosophila superresolution three-dimensional vision and proposes neural computations for accurately predicting these flies' depth-perception dynamics, limits, and visual behaviors.
Assuntos
Percepção de Profundidade , Drosophila , Animais , Olho , Disparidade Visual , Visão OcularRESUMO
Drosophila TRP is a calcium-permeable cation channel essential for fly visual signal transduction. During phototransduction, Ca2+ mediates both positive and negative feedback regulation on TRP channel activity, possibly via binding to calmodulin (CaM). However, the molecular mechanism underlying Ca2+ modulated CaM/TRP interaction is poorly understood. Here, we discover an unexpected, Ca2+-dependent binding mode between CaM and TRP. The TRP tail contains two CaM binding sites (CBS1 and CBS2) separated by an â¼70-residue linker. CBS1 binds to the CaM N-lobe and CBS2 recognizes the CaM C-lobe. Structural studies reveal the lobe-specific binding of CaM to CBS1&2. Mutations introduced in both CBS1 and CBS2 eliminated CaM binding in full-length TRP, but surprisingly had no effect on the response to light under physiological conditions, suggesting alternative mechanisms governing Ca2+-mediated feedback on the channel activity. Finally, we discover that TRPC4, the closest mammalian paralog of Drosophila TRP, adopts a similar CaM binding mode.
Assuntos
Calmodulina/química , Proteínas de Drosophila/química , Canais de Potencial de Receptor Transitório/química , Animais , Sítios de Ligação , Cálcio/metabolismo , Calmodulina/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Células HEK293 , Humanos , Camundongos , Mutação , Ligação Proteica , Canais de Cátion TRPC/química , Canais de Cátion TRPC/metabolismo , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
The daily changes of light and dark exemplify a prominent cue for the synchronization of circadian clocks with the environment. The match between external and internal time is crucial for the fitness of organisms, and desynchronization has been linked to numerous physical and mental health problems. Organisms therefore developed complex and not fully understood mechanisms to synchronize their circadian clock to light. In mammals and in Drosophila, both the visual system and non-image-forming photoreceptors contribute to circadian clock resetting. In Drosophila, light-dependent degradation of the clock protein TIMELESS by the blue light photoreceptor Cryptochrome is considered the main mechanism for clock synchronization, although the visual system also contributes. To better understand the visual system contribution, we generated a genetic variant exhibiting extremely slow phototransduction kinetics, yet normal sensitivity. In this variant, the visual system is able to contribute its full share to circadian clock entrainment, both with regard to behavioral and molecular light synchronization. This function depends on an alternative phospholipase C-ß enzyme, encoded by PLC21C, presumably playing a dedicated role in clock resetting. We show that this pathway requires the ubiquitin ligase CULLIN-3, possibly mediating CRY-independent degradation of TIMELESS during light:dark cycles. Our results suggest that the PLC21C-mediated contribution to circadian clock entrainment operates on a drastically slower timescale compared with fast, norpA-dependent visual phototransduction. Our findings are therefore consistent with the general idea that the visual system samples light over prolonged periods of time (h) in order to reliably synchronize their internal clocks with the external time.
Assuntos
Relógios Circadianos/genética , Proteínas Culina/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Locomoção/fisiologia , Fosfolipases/genética , Visão Ocular , Vias Visuais , Animais , Proteínas Culina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Masculino , Fosfolipases/metabolismoRESUMO
Phototransduction in Drosophila is mediated by phospholipase C (PLC) and Ca2+-permeable TRP channels, but the function of endoplasmic reticulum (ER) Ca2+ stores in this important model for Ca2+ signaling remains obscure. We therefore expressed a low affinity Ca2+ indicator (ER-GCaMP6-150) in the ER, and measured its fluorescence both in dissociated ommatidia and in vivo from intact flies of both sexes. Blue excitation light induced a rapid (tau â¼0.8 s), PLC-dependent decrease in fluorescence, representing depletion of ER Ca2+ stores, followed by a slower decay, typically reaching â¼50% of initial dark-adapted levels, with significant depletion occurring under natural levels of illumination. The ER stores refilled in the dark within 100-200 s. Both rapid and slow store depletion were largely unaffected in InsP3 receptor mutants, but were much reduced in trp mutants. Strikingly, rapid (but not slow) depletion of ER stores was blocked by removing external Na+ and in mutants of the Na+/Ca2+ exchanger, CalX, which we immuno-localized to ER membranes in addition to its established localization in the plasma membrane. Conversely, overexpression of calx greatly enhanced rapid depletion. These results indicate that rapid store depletion is mediated by Na+/Ca2+ exchange across the ER membrane induced by Na+ influx via the light-sensitive channels. Although too slow to be involved in channel activation, this Na+/Ca2+ exchange-dependent release explains the decades-old observation of a light-induced rise in cytosolic Ca2+ in photoreceptors exposed to Ca2+-free solutions.SIGNIFICANCE STATEMENT Phototransduction in Drosophila is mediated by phospholipase C, which activates TRP cation channels by an unknown mechanism. Despite much speculation, it is unknown whether endoplasmic reticulum (ER) Ca2+ stores play any role. We therefore engineered flies expressing a genetically encoded Ca2+ indicator in the photoreceptor ER. Although NCX Na+/Ca2+ exchangers are classically believed to operate only at the plasma membrane, we demonstrate a rapid light-induced depletion of ER Ca2+ stores mediated by Na+/Ca2+ exchange across the ER membrane. This NCX-dependent release was too slow to be involved in channel activation, but explains the decades-old observation of a light-induced rise in cytosolic Ca2+ in photoreceptors bathed in Ca2+-free solutions.
Assuntos
Antiporters/metabolismo , Cálcio/metabolismo , Proteínas de Drosophila/metabolismo , Retículo Endoplasmático/metabolismo , Transdução de Sinal Luminoso/fisiologia , Células Fotorreceptoras de Invertebrados/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Animais , Animais Geneticamente Modificados , Antiporters/genética , Sinalização do Cálcio/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster , Feminino , Masculino , Trocador de Sódio e Cálcio/genéticaRESUMO
Ca2+-activated K+ channels (BK and SK) are ubiquitous in synaptic circuits, but their role in network adaptation and sensory perception remains largely unknown. Using electrophysiological and behavioral assays and biophysical modeling, we discover how visual information transfer in mutants lacking the BK channel (dSlo- ), SK channel (dSK- ), or both (dSK- ;; dSlo- ) is shaped in the female fruit fly (Drosophila melanogaster) R1-R6 photoreceptor-LMC circuits (R-LMC-R system) through synaptic feedforward-feedback interactions and reduced R1-R6 Shaker and Shab K+ conductances. This homeostatic compensation is specific for each mutant, leading to distinctive adaptive dynamics. We show how these dynamics inescapably increase the energy cost of information and promote the mutants' distorted motion perception, determining the true price and limits of chronic homeostatic compensation in an in vivo genetic animal model. These results reveal why Ca2+-activated K+ channels reduce network excitability (energetics), improving neural adaptability for transmitting and perceiving sensory information.SIGNIFICANCE STATEMENT In this study, we directly link in vivo and ex vivo experiments with detailed stochastically operating biophysical models to extract new mechanistic knowledge of how Drosophila photoreceptor-interneuron-photoreceptor (R-LMC-R) circuitry homeostatically retains its information sampling and transmission capacity against chronic perturbations in its ion-channel composition, and what is the cost of this compensation and its impact on optomotor behavior. We anticipate that this novel approach will provide a useful template to other model organisms and computational neuroscience, in general, in dissecting fundamental mechanisms of homeostatic compensation and deepening our understanding of how biological neural networks work.
Assuntos
Retroalimentação Fisiológica , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Potenciais Sinápticos , Percepção Visual , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Feminino , Interneurônios/metabolismo , Interneurônios/fisiologia , Modelos Neurológicos , Células Fotorreceptoras de Invertebrados/fisiologia , Canais de Potássio Shab/metabolismo , Superfamília Shaker de Canais de Potássio/metabolismo , Vias Visuais/metabolismo , Vias Visuais/fisiologiaRESUMO
Cell and organelle membranes consist of a complex mixture of phospholipids (PLs) that determine their size, shape, and function. Phosphatidylcholine (PC) is the most abundant phospholipid in eukaryotic membranes, yet how cells sense and regulate its levels in vivo remains unclear. Here we show that PCYT1A, the rate-limiting enzyme of PC synthesis, is intranuclear and re-locates to the nuclear membrane in response to the need for membrane PL synthesis in yeast, fly, and mammalian cells. By aligning imaging with lipidomic analysis and data-driven modeling, we demonstrate that yeast PCYT1A membrane association correlates with membrane stored curvature elastic stress estimates. Furthermore, this process occurs inside the nucleus, although nuclear localization signal mutants can compensate for the loss of endogenous PCYT1A in yeast and in fly photoreceptors. These data suggest an ancient mechanism by which nucleoplasmic PCYT1A senses surface PL packing defects on the inner nuclear membrane to control PC homeostasis.
Assuntos
Membrana Celular/química , Núcleo Celular/química , Colina-Fosfato Citidililtransferase/metabolismo , Elasticidade , Membrana Nuclear/química , Fosfatidilcolinas/metabolismo , Estresse Fisiológico , Animais , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Colina-Fosfato Citidililtransferase/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Homeostase , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismoRESUMO
The daily light-dark cycles represent a key signal for synchronizing circadian clocks. Both insects and mammals possess dedicated "circadian" photoreceptors but also utilize the visual system for clock resetting. In Drosophila, circadian clock resetting is achieved by the blue-light photoreceptor cryptochrome (CRY), which is expressed within subsets of the brain clock neurons. In addition, rhodopsin-expressing photoreceptor cells contribute to light synchronization. Light resets the molecular clock by CRY-dependent degradation of the clock protein Timeless (TIM), although in specific subsets of key circadian pacemaker neurons, including the small ventral lateral neurons (s-LNvs), TIM and Period (PER) oscillations can be synchronized by light independent of CRY and canonical visual Rhodopsin phototransduction. Here, we show that at least three of the seven Drosophila rhodopsins can utilize an alternative transduction mechanism involving the same α-subunit of the heterotrimeric G protein operating in canonical visual phototransduction (Gq). Surprisingly, in mutants lacking the canonical phospholipase C-ß (PLC-ß) encoded by the no receptor potential A (norpA) gene, we uncovered a novel transduction pathway using a different PLC-ß encoded by the Plc21C gene. This novel pathway is important for behavioral clock resetting to semi-natural light-dark cycles and mediates light-dependent molecular synchronization within the s-LNv clock neurons. The same pathway appears to be responsible for norpA-independent light responses in the compound eye. We show that Rhodopsin 5 (Rh5) and Rh6, present in the R8 subset of retinal photoreceptor cells, drive both the long-term circadian and rapid light responses in the eye.
Assuntos
Relógios Circadianos/fisiologia , Drosophila melanogaster/fisiologia , Transdução de Sinal Luminoso/fisiologia , Células Fotorreceptoras de Invertebrados/fisiologia , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Luz , Transdução de Sinal Luminoso/genética , Rodopsina/metabolismoRESUMO
Phototransduction in Drosophila is mediated by phospholipase C-dependent hydrolysis of PIP2-, and is an important model for phosphoinositide signalling. Although generally assumed to operate by generic machinery conserved from yeast to mammals, some key elements of the phosphoinositide cycle have yet to be identified in Drosophila photoreceptors. Here, we used transgenic flies expressing fluorescently tagged probes (P4M and TbR332H), which allow in vivo quantitative measurements of PI4P and PIP2 dynamics in photoreceptors of intact living flies. Using mutants and RNA interference for candidate genes potentially involved in phosphoinositide turnover, we identified Drosophila PI4KIIIα (CG10260) as the PI4-kinase responsible for PI4P synthesis in the photoreceptor membrane. Our results also indicate that PI4KIIIα activity requires rbo (the Drosophila orthologue of Efr3) and CG8325 (orthologue of YPP1), both of which are implicated as scaffolding proteins necessary for PI4KIIIα activity in yeast and mammals. However, our evidence indicates that the recently reported central role of dPIP5K59B (CG3682) in PIP2 synthesis in the rhabdomeres should be re-evaluated; although PIP2 resynthesis was suppressed by RNAi directed against dPIP5K59B, little or no defect was detected in a reportedly null mutant (dPIP5K18 ).
Assuntos
Fosfatidilinositóis/genética , Células Fotorreceptoras/metabolismo , Animais , Drosophila , Fosfatidilinositóis/metabolismoRESUMO
Heterotrimeric G proteins play central roles in many signaling pathways, including the phototransduction cascade in animals. However, the degree of involvement of the G protein subunit Gαq is not clear since animals with previously reported strong loss-of-function mutations remain responsive to light stimuli. We recovered a new allele of Gαq in Drosophila that abolishes light response in a conventional electroretinogram assay, and reduces sensitivity in whole-cell recordings of dissociated cells by at least five orders of magnitude. In addition, mutant eyes demonstrate a rapid rate of degeneration in the presence of light. Our new allele is likely the strongest hypomorph described to date. Interestingly, the mutant protein is produced in the eyes but carries a single amino acid change of a conserved hydrophobic residue that has been assigned to the interface of interaction between Gαq and its downstream effector, PLC. Our study has thus uncovered possibly the first point mutation that specifically affects this interaction in vivo.
Assuntos
Proteínas de Drosophila/deficiência , Drosophila melanogaster/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/deficiência , Mutação Puntual , Degeneração Retiniana/genética , Visão Ocular , Alelos , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Eletrorretinografia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica , Luz , Ligação Proteica , Retina/metabolismo , Retina/patologia , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismoRESUMO
Small fly eyes should not see fine image details. Because flies exhibit saccadic visual behaviors and their compound eyes have relatively few ommatidia (sampling points), their photoreceptors would be expected to generate blurry and coarse retinal images of the world. Here we demonstrate that Drosophila see the world far better than predicted from the classic theories. By using electrophysiological, optical and behavioral assays, we found that R1-R6 photoreceptors' encoding capacity in time is maximized to fast high-contrast bursts, which resemble their light input during saccadic behaviors. Whilst over space, R1-R6s resolve moving objects at saccadic speeds beyond the predicted motion-blur-limit. Our results show how refractory phototransduction and rapid photomechanical photoreceptor contractions jointly sharpen retinal images of moving objects in space-time, enabling hyperacute vision, and explain how such microsaccadic information sampling exceeds the compound eyes' optical limits. These discoveries elucidate how acuity depends upon photoreceptor function and eye movements.
Assuntos
Drosophila melanogaster/fisiologia , Movimentos Oculares/fisiologia , Estimulação Luminosa , Visão Ocular/fisiologia , Acuidade Visual/fisiologia , Animais , Simulação por Computador , Drosophila melanogaster/ultraestrutura , Fixação Ocular/fisiologia , Modelos Neurológicos , Movimento , Fótons , Células Fotorreceptoras de Invertebrados/metabolismo , Células Fotorreceptoras de Invertebrados/ultraestrutura , Retina/fisiologiaRESUMO
Drosophila phototransduction is mediated by phospholipase C, leading to activation of transient receptor potential (TRP) and TRP-like (TRPL) channels by mechanisms that are unresolved. A role for InsP3 receptors (IP3Rs) had been excluded because IP3R mutants (itpr) appeared to have normal light responses; however, this was recently challenged by Kohn et al. ("Functional cooperation between the IP3 receptor and phospholipase C secures the high sensitivity to light of Drosophila photoreceptors in vivo," Journal of Neuroscience 35:2530), who reported defects in phototransduction after IP3R-RNAi knockdown. They concluded that InsP3-induced Ca2+ release plays a critical role in facilitating channel activation, and that previous failure to detect IP3R phenotypes resulted from trace Ca2+ in electrodes substituting for InsP3-induced Ca2+ release. In an attempt to confirm this, we performed electroretinograms, whole-cell recordings, and GCaMP6f Ca2+ imaging from both IP3R-RNAi flies and itpr-null mutants. Like Kohn et al., we used GMRGal4 to drive expression of UAS-IP3R-RNAi, but we also used controls expressing GMRGal4 alone. We describe several GMRGal4 phenotypes suggestive of compromised development, including reductions in sensitivity, dark noise, potassium currents, and cell size and capacitance, as well as extreme variations in sensitivity between cells. However, we found no effect of IP3R RNAi or mutation on photoreceptor responses or Ca2+ signals, indicating that the IP3R plays little or no role in Drosophila phototransduction.
Assuntos
Proteínas de Drosophila/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Transdução de Sinal Luminoso/fisiologia , Fatores de Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Expressão Gênica , Receptores de Inositol 1,4,5-Trifosfato/genética , Potenciais da Membrana/fisiologia , Mutação , Técnicas de Patch-Clamp , Fenótipo , Fosfolipase C beta/genética , Fosfolipase C beta/metabolismo , Estimulação Luminosa , Interferência de RNA , Retina/metabolismo , Retina/patologia , Técnicas de Cultura de TecidosRESUMO
Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e. spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.
Assuntos
Drosophila melanogaster/fisiologia , Luz , Técnicas de Patch-Clamp/métodos , Células Fotorreceptoras de Invertebrados/fisiologia , Retina/fisiologia , Visão Ocular/fisiologia , Animais , Eletrofisiologia , Potenciais da Membrana/fisiologia , Fenômenos Fisiológicos Oculares , Técnicas de Patch-Clamp/instrumentação , Retina/citologia , Canais de Potencial de Receptor Transitório/fisiologiaRESUMO
Drosophila phototransduction is mediated by phospholipase C leading to activation of cation channels (TRP and TRPL) in the 30000 microvilli forming the light-absorbing rhabdomere. The channels mediate massive Ca2+ influx in response to light, but whether Ca2+ is released from internal stores remains controversial. We generated flies expressing GCaMP6f in their photoreceptors and measured Ca2+ signals from dissociated cells, as well as in vivo by imaging rhabdomeres in intact flies. In response to brief flashes, GCaMP6f signals had latencies of 10-25ms, reached 50% Fmax with â¼1200 effectively absorbed photons and saturated (ΔF/F0â¼10-20) with 10000-30000 photons. In Ca2+ free bath, smaller (ΔF/F0 â¼4), long latency (â¼200ms) light-induced Ca2+ rises were still detectable. These were unaffected in InsP3 receptor mutants, but virtually eliminated when Na+ was also omitted from the bath, or in trpl;trp mutants lacking light-sensitive channels. Ca2+ free rises were also eliminated in Na+/Ca2+ exchanger mutants, but greatly accelerated in flies over-expressing the exchanger. These results show that Ca2+ free rises are strictly dependent on Na+ influx and activity of the exchanger, suggesting they reflect re-equilibration of Na+/Ca2+ exchange across plasma or intracellular membranes following massive Na+ influx. Any tiny Ca2+ free rise remaining without exchanger activity was equivalent to <10nM (ΔF/F0 â¼0.1), and unlikely to play any role in phototransduction.
Assuntos
Sinalização do Cálcio , Proteínas de Drosophila/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Microscopia de Fluorescência , Células Fotorreceptoras de Invertebrados/citologia , Fosfolipases Tipo C/genéticaRESUMO
More than five decades ago it was postulated that sensory neurons detect and selectively enhance behaviourally relevant features of natural signals. Although we now know that sensory neurons are tuned to efficiently encode natural stimuli, until now it was not clear what statistical features of the stimuli they encode and how. Here we reverse-engineer the neural code of Drosophila photoreceptors and show for the first time that photoreceptors exploit nonlinear dynamics to selectively enhance and encode phase-related features of temporal stimuli, such as local phase congruency, which are invariant to changes in illumination and contrast. We demonstrate that to mitigate for the inherent sensitivity to noise of the local phase congruency measure, the nonlinear coding mechanisms of the fly photoreceptors are tuned to suppress random phase signals, which explains why photoreceptor responses to naturalistic stimuli are significantly different from their responses to white noise stimuli.
Assuntos
Drosophila , Células Fotorreceptoras de Invertebrados/fisiologia , Algoritmos , Animais , Simulação por Computador , Fenômenos Eletrofisiológicos , Modelos Teóricos , Estimulação LuminosaRESUMO
Synaptic feedback from interneurons to photoreceptors can help to optimize visual information flow by balancing its allocation on retinal pathways under changing light conditions. But little is known about how this critical network operation is regulated dynamically. Here, we investigate this question by comparing signaling properties and performance of wild-type Drosophila R1-R6 photoreceptors to those of the hdc (JK910) mutant, which lacks the neurotransmitter histamine and therefore cannot transmit information to interneurons. Recordings show that hdc (JK910) photoreceptors sample similar amounts of information from naturalistic stimulation to wild-type photoreceptors, but this information is packaged in smaller responses, especially under bright illumination. Analyses reveal how these altered dynamics primarily resulted from network overload that affected hdc (JK910) photoreceptors in two ways. First, the missing inhibitory histamine input to interneurons almost certainly depolarized them irrevocably, which in turn increased their excitatory feedback to hdc (JK910) R1-R6s. This tonic excitation depolarized the photoreceptors to artificially high potentials, reducing their operational range. Second, rescuing histamine input to interneurons in hdc (JK910) mutant also restored their normal phasic feedback modulation to R1-R6s, causing photoreceptor output to accentuate dynamic intensity differences at bright illumination, similar to the wild-type. These results provide mechanistic explanations of how synaptic feedback connections optimize information packaging in photoreceptor output and novel insight into the operation and design of dynamic network regulation of sensory neurons.
Assuntos
Proteínas de Drosophila/genética , Histamina/deficiência , Mutação/genética , Células Fotorreceptoras de Invertebrados/fisiologia , Vias Visuais/fisiologia , Animais , Animais Geneticamente Modificados , Cegueira/genética , Cegueira/patologia , Adaptação à Escuridão/genética , Modelos Animais de Doenças , Drosophila , Estimulação Elétrica , Eletrorretinografia , Feminino , Análise de Fourier , Potenciais da Membrana , Microscopia Eletrônica de Transmissão , Técnicas de Patch-Clamp , Estimulação Luminosa , Células Fotorreceptoras de Invertebrados/ultraestruturaRESUMO
In order to monitor phosphoinositide turnover during phospholipase C (PLC)-mediated Drosophila phototransduction, fluorescently tagged lipid probes were expressed in photoreceptors and imaged both in dissociated cells, and in eyes of intact living flies. Of six probes tested, Tb(R332H) (a mutant of the Tubby protein pleckstrin homology domain) was judged the best reporter for phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2], and the P4M domain from Legionella SidM for phosphatidylinositol 4-phosphate (PtdIns4P). Using accurately calibrated illumination, we found that only â¼50% of PtdIns(4,5)P2 and very little PtdIns4P were depleted by full daylight intensities in wild-type flies, but both were severely depleted by â¼100-fold dimmer intensities in mutants lacking Ca(2+)-permeable transient receptor potential (TRP) channels or protein kinase C (PKC). Resynthesis of PtdIns4P (t½ â¼12â s) was faster than PtdIns(4,5)P2 (t½ â¼40â s), but both were greatly slowed in mutants of DAG kinase (rdgA) or PtdIns transfer protein (rdgB). The results indicate that Ca(2+)- and PKC-dependent inhibition of PLC is required for enabling photoreceptors to maintain phosphoinositide levels despite high rates of hydrolysis by PLC, and suggest that phosphorylation of PtdIns4P to PtdIns(4,5)P2 is the rate-limiting step of the cycle.
Assuntos
Fosfatidilinositol 4,5-Difosfato/metabolismo , Células Fotorreceptoras de Invertebrados/citologia , Células Fotorreceptoras de Invertebrados/metabolismo , Animais , Cálcio/metabolismo , Diacilglicerol Quinase/genética , Diacilglicerol Quinase/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosfatidilinositol 4,5-Difosfato/genética , Proteína Quinase C/genética , Proteína Quinase C/metabolismoRESUMO
Drosophila phototransduction is mediated via a G-protein-coupled PLC cascade. Recent evidence, including the demonstration that light evokes rapid contractions of the photoreceptors, suggested that the light-sensitive channels (TRP and TRPL) may be mechanically gated, together with protons released by PLC-mediated PIP2 hydrolysis. If mechanical gating is involved we predicted that the response to light should be influenced by altering the physical properties of the membrane. To achieve this, we used diet to manipulate the degree of saturation of membrane phospholipids. In flies reared on a yeast diet, lacking polyunsaturated fatty acids (PUFAs), mass spectrometry showed that the proportion of polyunsaturated phospholipids was sevenfold reduced (from 38 to â¼5%) but rescued by adding a single species of PUFA (linolenic or linoleic acid) to the diet. Photoreceptors from yeast-reared flies showed a 2- to 3-fold increase in latency and time to peak of the light response, without affecting quantum bump waveform. In the absence of Ca(2+) influx or in trp mutants expressing only TRPL channels, sensitivity to light was reduced up to â¼10-fold by the yeast diet, and essentially abolished in hypomorphic G-protein mutants (Gαq). PLC activity appeared little affected by the yeast diet; however, light-induced contractions measured by atomic force microscopy or the activation of ectopic mechanosensitive gramicidin channels were also slowed â¼2-fold. The results are consistent with mechanosensitive gating and provide a striking example of how dietary fatty acids can profoundly influence sensory performance in a classical G-protein-coupled signaling cascade.
Assuntos
Membrana Celular/fisiologia , Drosophila melanogaster/fisiologia , Transdução de Sinal Luminoso/fisiologia , Fosfolipídeos/fisiologia , Animais , Membrana Celular/metabolismo , Dieta , Ativação do Canal Iônico/fisiologia , Luz , Metabolismo dos Lipídeos/fisiologia , Fosfolipídeos/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Rodopsina/metabolismo , Razão Sinal-Ruído , Trocador de Sódio e Cálcio/metabolismo , Fosfolipases Tipo C/metabolismoRESUMO
Phototransduction in Drosophila's microvillar photoreceptors is mediated by phospholipase C (PLC) resulting in activation of two distinct Ca(2+)-permeable channels, TRP and TRPL. Here we review recent evidence on the unresolved mechanism of their activation, including the hypothesis that the channels are mechanically activated by physical effects of PIP2 depletion on the membrane, in combination with protons released by PLC. We also review molecularly explicit models indicating how Ca(2+)-dependent positive and negative feedback along with the ultracompartmentalization provided by the microvillar design can account for the ability of fly photoreceptors to respond to single photons 10-100× more rapidly than vertebrate rods, yet still signal under full sunlight.