RÉSUMÉ
Programmable genome-engineering technologies, such as CRISPR (clustered regularly interspaced short palindromic repeats) nucleases and massively parallel CRISPR screens that capitalize on this programmability, have transformed biomedical science. These screens connect genes and noncoding genome elements to disease-relevant phenotypes, but until recently have been limited to individual phenotypes such as growth or fluorescent reporters of gene expression. By pairing massively parallel screens with high-dimensional profiling of single-cell types/states, we can now measure how individual genetic perturbations or combinations of perturbations impact the cellular transcriptome, proteome, and epigenome. We review technologies that pair CRISPR screens with single-cell multiomics and the unique opportunities afforded by extending pooled screens using deep multimodal phenotyping.
Sujet(s)
Systèmes CRISPR-Cas , Édition de gène , Édition de gène/méthodes , Génome , Dépistage génétique , Analyse sur cellule unique/méthodes , Clustered regularly interspaced short palindromic repeatsRÉSUMÉ
Odorant binding proteins (Obps) are expressed at extremely high levels in the antennae of insects, and have long been believed essential for carrying hydrophobic odorants to odor receptors. Previously we found that when one functional type of olfactory sensillum in Drosophila was depleted of its sole abundant Obp, it retained a robust olfactory response (Larter et al., 2016). Here we have deleted all the Obp genes that are abundantly expressed in the antennal basiconic sensilla. All of six tested sensillum types responded robustly to odors of widely diverse chemical or temporal structure. One mutant gave a greater physiological and behavioral response to an odorant that affects oviposition. Our results support a model in which many sensilla can respond to odorants in the absence of Obps, and many Obps are not essential for olfactory response, but that some Obps can modulate olfactory physiology and the behavior that it drives.
Sujet(s)
Drosophila/physiologie , Récepteurs olfactifs/déficit , Sensilles/physiologie , Odorat , Animaux , Comportement animal , Drosophila/génétique , Odorisants , Perception olfactive , Récepteurs olfactifs/métabolismeRÉSUMÉ
The Drosophila wing was proposed to be a taste organ more than 35 years ago, but there has been remarkably little study of its role in chemoreception. We carry out a differential RNA-seq analysis of a row of sensilla on the anterior wing margin and find expression of many genes associated with pheromone and chemical perception. To ask whether these sensilla might receive pheromonal input, we devised a dye-transfer paradigm and found that large, hydrophobic molecules comparable to pheromones can be transferred from one fly to the wing margin of another. One gene, Ionotropic receptor (IR)52a, is coexpressed in neurons of these sensilla with fruitless, a marker of sexual circuitry; IR52a is also expressed in legs. Mutation of IR52a and optogenetic silencing of IR52a+ neurons decrease levels of male sexual behavior. Optogenetic activation of IR52a+ neurons induces males to show courtship toward other males and, remarkably, toward females of another species. Surprisingly, IR52a is also required in females for normal sexual behavior. Optogenetic activation of IR52a+ neurons in mated females induces copulation, which normally occurs at very low levels. Unlike other chemoreceptors that act in males to inhibit male-male interactions and promote male-female interactions, IR52a acts in both males and females, and can promote male-male as well as male-female interactions. Moreover, IR52a+ neurons can override the circuitry that normally suppresses sexual behavior toward unproductive targets. Circuit mapping and Ca2+ imaging using the trans-Tango system reveals second-order projections of IR52a+ neurons in the subesophageal zone (SEZ), some of which are sexually dimorphic. Optogenetic activation of IR52a+ neurons in the wing activates second-order projections in the SEZ. Taken together, this study provides a molecular description of the chemosensory sensilla of a greatly understudied taste organ and defines a gene that regulates the sexual circuitry of the fly.
Sujet(s)
Protéines de Drosophila/métabolisme , Drosophila melanogaster/métabolisme , Récepteurs aux phéromones/métabolisme , Sensilles/métabolisme , Ailes d'animaux/métabolisme , Animaux , Protéines de Drosophila/génétique , Femelle , Extinction de l'expression des gènes , Interactions hydrophobes et hydrophiles , Canaux ioniques régulés par des ligands/génétique , Canaux ioniques régulés par des ligands/métabolisme , Mâle , Neurones/métabolisme , Optogénétique , Caractères sexuels , Comportement sexuel chez les animaux/physiologie , Goût/physiologieRÉSUMÉ
Dipteran or "true" flies occupy nearly every terrestrial habitat, and have evolved to feed upon a wide variety of sources including fruit, pollen, decomposing animal matter, and even vertebrate blood. Here we analyze the molecular, genetic and cellular basis of odor response in the tsetse fly Glossina morsitans, which feeds on the blood of humans and their livestock, and is a vector of deadly trypanosomes. The G. morsitans antenna contains specialized subtypes of sensilla, some of which line a sensory pit not found in the fruit fly Drosophila. We characterize distinct patterns of G. morsitans Odor receptor (GmmOr) gene expression in the antenna. We devise a new version of the "empty neuron" heterologous expression system, and use it to functionally express several GmmOrs in a mutant olfactory receptor neuron (ORN) of Drosophila. GmmOr35 responds to 1-hexen-3-ol, an odorant found in human emanations, and also alpha-pinene, a compound produced by malarial parasites. Another receptor, GmmOr9, which is expressed in the sensory pit, responds to acetone, 2-butanone and 2-propanol. We confirm by electrophysiological recording that neurons of the sensory pit respond to these odorants. Acetone and 2-butanone are strong attractants long used in the field to trap tsetse. We find that 2-propanol is also an attractant for both G. morsitans and the related species G. fuscipes, a major vector of African sleeping sickness. The results identify 2-propanol as a candidate for an environmentally friendly and practical tsetse attractant. Taken together, this work characterizes the olfactory system of a highly distinct kind of fly, and it provides an approach to identifying new agents for controlling the fly and the devastating diseases that it carries.
Sujet(s)
Récepteurs olfactifs/génétique , Phéromones sexuelles/génétique , Odorat/génétique , Maladie du sommeil/génétique , Propan-2-ol/composition chimique , Animaux , Drosophila melanogaster/génétique , Drosophila melanogaster/parasitologie , Humains , Huiles/composition chimique , Neurorécepteurs olfactifs/métabolisme , Neurorécepteurs olfactifs/parasitologie , Phéromones sexuelles/composition chimique , Trypanosoma/génétique , Trypanosoma/pathogénicité , Maladie du sommeil/parasitologie , Mouches tsé-tsé/génétique , Mouches tsé-tsé/pathogénicitéRÉSUMÉ
Hygrosensation is an essential sensory modality that is used to find sources of moisture. Hygroreception allows animals to avoid desiccation, an existential threat that is increasing with climate change. Humidity response, however, remains poorly understood. Here we find that humidity-detecting sensilla in the Drosophila antenna express and rely on a small protein, Obp59a. Mutants lacking this protein are defective in three hygrosensory behaviors, one operating over seconds, one over minutes, and one over hours. Remarkably, loss of Obp59a and humidity response leads to an increase in desiccation resistance. Obp59a is an exceptionally well-conserved, highly localized, and abundantly expressed member of a large family of secreted proteins. Antennal Obps have long been believed to transport hydrophobic odorants, and a role in hygroreception was unexpected. The results enhance our understanding of hygroreception, Obp function, and desiccation resistance, a process that is critical to insect survival.
Sujet(s)
Protéines de transport/génétique , Protéines de Drosophila/génétique , Drosophila melanogaster/génétique , Humidité , Animaux , Changement climatique , Drosophila melanogaster/physiologie , Récepteurs olfactifs/génétique , Récepteurs olfactifs/physiologie , Sensilles/physiologieRÉSUMÉ
The term 'odorant-binding proteins (Obps)' is used to refer to a large family of insect proteins that are exceptional in their number, abundance and diversity. The name derives from the expression of many family members in the olfactory system of insects and their ability to bind odorants in vitro. However, an increasing body of evidence reveals a much broader role for this family of proteins. Recent results also provoke interesting questions about their mechanisms of action, both within and outside the olfactory system. Here we describe the identification of the first Obps and some cardinal properties of these proteins. We then consider their function, discussing both the prevailing orthodoxy and the increasing grounds for heterodox views. We then examine these proteins from a broader perspective and consider some intriguing questions in need of answers.
Sujet(s)
Récepteurs olfactifs/composition chimique , Récepteurs olfactifs/métabolisme , Animaux , Protéines d'insecte/composition chimique , Protéines d'insecte/génétique , Protéines d'insecte/métabolisme , Insectes/génétique , Insectes/métabolisme , Modèles moléculaires , Famille multigénique , Structure secondaire des protéines , Récepteurs olfactifs/génétiqueRÉSUMÉ
Superparasitism is a common phenomenon in mosquito-parasitic mermithid nematodes. Multiple nematodes are needed in a single host to produce males. Host selection behavior and intraspecific competition among Romanomermis iyengari and Strelkovimermis spiculatus were investigated against their host, Culex pipiens pipiens in laboratory experiments. In a choice assay between previously infected and uninfected host larvae, infectious preparasites of both nematode species could distinguish not only between infected and uninfected hosts, but even between different parasite loads in showing a strong preference for uninfected hosts or hosts with a low parasite load. Host heart rate declined briefly immediately after parasitism. Superparasitism resulted in increased parasite mortality. Scramble competition within mosquito larvae for limited host nutrients, coupled with a skewed sex ratio favoring males, is assumed to lead to parasite population decline and subsequently toward host-parasite population equilibrium. The ability of mermithid preparasites to accurately assess parasite load likely plays an important role in host population dynamics and regulation.
RÉSUMÉ
The neural control of sugar consumption is critical for normal metabolism. In contrast to sugar-sensing taste neurons that promote consumption, we identify a taste neuron that limits sucrose consumption in Drosophila. Silencing of the neuron increases sucrose feeding; optogenetic activation decreases it. The feeding inhibition depends on the IR60b receptor, as shown by behavioral analysis and Ca2+ imaging of an IR60b mutant. The IR60b phenotype shows a high degree of chemical specificity when tested with a broad panel of tastants. An automated analysis of feeding behavior in freely moving flies shows that IR60b limits the duration of individual feeding bouts. This receptor and neuron provide the molecular and cellular underpinnings of a new element in the circuit logic of feeding regulation. We propose a dynamic model in which sucrose acts via IR60b to activate a circuit that inhibits feeding and prevents overconsumption.
Sujet(s)
Protéines de Drosophila/génétique , Drosophila melanogaster/effets des médicaments et des substances chimiques , Consommation alimentaire/physiologie , Comportement alimentaire/physiologie , Neurones/effets des médicaments et des substances chimiques , Récepteurs couplés aux protéines G/génétique , Saccharose/pharmacologie , Animaux , Calcium/métabolisme , Diholoside/métabolisme , Diholoside/pharmacologie , Protéines de Drosophila/métabolisme , Drosophila melanogaster/physiologie , Rétrocontrôle physiologique , Expression des gènes , Glycérol/métabolisme , Glycérol/pharmacologie , Oses/métabolisme , Oses/pharmacologie , Mutation , Neurones/cytologie , Neurones/métabolisme , Imagerie optique , Récepteurs couplés aux protéines G/métabolisme , Saccharose/métabolisme , Goût/physiologie , Perception du goût/physiologieRÉSUMÉ
Symbiotic bacteria assist in maintaining homeostasis of the animal immune system. However, the molecular mechanisms that underlie symbiont-mediated host immunity are largely unknown. Tsetse flies (Glossina spp.) house maternally transmitted symbionts that regulate the development and function of their host's immune system. Herein we demonstrate that the obligate mutualist, Wigglesworthia, up-regulates expression of odorant binding protein six in the gut of intrauterine tsetse larvae. This process is necessary and sufficient to induce systemic expression of the hematopoietic RUNX transcription factor lozenge and the subsequent production of crystal cells, which actuate the melanotic immune response in adult tsetse. Larval Drosophila's indigenous microbiota, which is acquired from the environment, regulates an orthologous hematopoietic pathway in their host. These findings provide insight into the molecular mechanisms that underlie enteric symbiont-stimulated systemic immune system development, and indicate that these processes are evolutionarily conserved despite the divergent nature of host-symbiont interactions in these model systems.
Sujet(s)
Hématopoïèse , Protéines d'insecte/métabolisme , Mouches tsé-tsé/microbiologie , Mouches tsé-tsé/physiologie , Régulation positive , Wigglesworthia/immunologie , Wigglesworthia/physiologie , Animaux , Drosophila , Larve/microbiologie , Larve/physiologieRÉSUMÉ
Odorant binding proteins (Obps) are remarkable in their number, diversity, and abundance, yet their role in olfactory coding remains unclear. They are widely believed to be required for transporting hydrophobic odorants through an aqueous lymph to odorant receptors. We construct a map of the Drosophila antenna, in which the abundant Obps are mapped to olfactory sensilla with defined functions. The results lay a foundation for an incisive analysis of Obp function. The map identifies a sensillum type that contains a single abundant Obp, Obp28a. Surprisingly, deletion of the sole abundant Obp in these sensilla does not reduce the magnitude of their olfactory responses. The results suggest that this Obp is not required for odorant transport and that this sensillum does not require an abundant Obp. The results further suggest a novel role for this Obp in buffering changes in the odor environment, perhaps providing a molecular form of gain control.
Sujet(s)
Antennes des arthropodes/composition chimique , Drosophila/composition chimique , Récepteurs olfactifs/analyse , Récepteurs olfactifs/classification , Sensilles/composition chimique , Animaux , Antennes des arthropodes/physiologie , Drosophila/physiologie , Analyse de profil d'expression de gènes , Hybridation fluorescente in situ , Récepteurs olfactifs/génétique , Récepteurs olfactifs/isolement et purification , Sensilles/physiologieRÉSUMÉ
Oxygenic photosynthesis efficiency at increasing solar flux is limited by light-induced damage (photoinhibition) of Photosystem II (PSII), primarily targeting the D1 reaction center subunit. Some cyanobacteria contain two natural isoforms of D1 that function better under low light (D1:1) or high light (D1:2). Herein, rates and yields of photoassembly of the Mn4CaO5 water-oxidizing complex (WOC) from the free inorganic cofactors (Mn(2+), Ca(2+), water, electron acceptor) and apo-WOC-PSII are shown to differ significantly: D1:1 apo-WOC-PSII exhibits a 2.3-fold faster rate-limiting step of photoassembly and up to seven-fold faster rate to the first light-stable Mn(3+) intermediate, IM1*, but with a much higher rate of photoinhibition than D1:2. Conversely, D1:2 apo-WOC-PSII assembles slower but has up to seven-fold higher yield, achieved by a higher quantum yield of charge separation and slower photoinhibition rate. These results confirm and extend previous observations of the two holoenzymes: D1:2-PSII has a greater quantum yield of primary charge separation, faster [P680 (+) Q A (-) ] charge recombination and less photoinhibition that results in a slower rate and higher yield of photoassembly of its apo-WOC-PSII complex. In contrast, D1:1-PSII has a lower quantum yield of primary charge separation, a slower [P680 (+) Q A (-) ] charge recombination rate, and faster photoinhibition that together result in higher rate but lower yield of photoassembly at higher light intensities. Cyanobacterial PSII reaction centers that contain the high- and low-light D1 isoforms can tailor performance to optimize photosynthesis at varying light conditions, with similar consequences on their photoassembly kinetics and yield. These different efficiencies of photoassembly versus photoinhibition impose differential costs for biosynthesis as a function of light intensity.
Sujet(s)
Chlamydomonas reinhardtii/physiologie , Oxygène/métabolisme , Photosynthèse/effets des radiations , Complexe protéique du photosystème II/métabolisme , Eau/métabolisme , Chlamydomonas reinhardtii/effets des radiations , Lumière , Oxydoréduction , Complexe protéique du photosystème II/effets des radiations , Isoformes de protéinesRÉSUMÉ
The normal pathway of electron transfer on the electron-acceptor side of photosystem II (PSII) involves electron transfer from quinone A, QA, to quinone B, QB. It is possible to redirect electrons from QA(-) to water-soluble Co(III) complexes, which opens a new avenue for harvesting electrons from water oxidation by immobilization of PSII on electrode surfaces. Herein, the kinetics of electron transfer from QA(-) to [Co(III)(terpy)2](3+) (terpy = 2,2';6',2â³-terpyridine) are investigated with a spectrophotometric assay revealing that the reaction follows Michaelis-Menten saturation kinetics, is inhibited by cations, and is not affected by variation of the QA reduction potential. A negatively charged site on the stromal surface of the PSII protein complex, composed of glutamic acid residues near QA, is hypothesized to bind cations, especially divalent cations. The cations are proposed to tune the redox properties of QA through electrostatic interactions. These observations may thus explain the molecular basis of the effect of divalent cations like Ca(2+), Sr(2+), Mg(2+), and Zn(2+) on the redox properties of the quinones in PSII, which has previously been attributed to long-range conformational changes propagated from divalent cations binding to the Ca(II)-binding site in the oxygen-evolving complex on the lumenal side of the PSII complex.