The fruit fly Drosophila melanogaster was used to examine the mode of action of the novel insecticide and acaricide nodulisporic acid. Flies resistant to nodulisporic acid were selected by stepwise increasing the dose of drug in the culture media. The resistant strain, glc 1 , is at least 20-fold resistant to nodulisporic acid and 3-fold cross-resistant to the parasiticide ivermectin, and exhibited decreased brood size, decreased locomotion, and bang sensitivity. Binding assays using glc 1 head membranes showed a marked decrease in the affinity for nodulisporic acid and ivermectin. A combination of genetics and sequencing identified a proline to serine mutation (P299S) in the gene coding for the glutamategated chloride channel subunit DmGluCl␣. To examine the effect of this mutation on the biophysical properties of DmGluCl␣ channels, it was introduced into a recombinant DmGluCl␣, and RNA encoding wild-type and mutant subunits was injected into Xenopus oocytes. Nodulisporic acid directly activated wild-type and mutant DmGluCl␣ channels. However, mutant channels were Ϸ10-fold less sensitive to activation by nodulisporic acid, as well as ivermectin and the endogenous ligand glutamate, providing direct evidence that nodulisporic acid and ivermectin act on DmGluCl␣ channels.
The performance of a task is often assumed to be a prerequisite for the learning of many tasks, including the associative conditioning of courtship in the fruit fly, Drosophila melanogaster. Transgenic flies specifically inhibited for the enzyme protein kinase C dissociate the acquisition of learning and memory from performance of the task. They fail to show immediate suppression of courtship but nonetheless develop normal memory of it.
Genome editing using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated nuclease (Cas9) enables specific genetic modifications, including deletions, insertions, and substitutions in numerous organisms, such as the fruit fly Drosophila melanogaster. One challenge of the CRISPR/Cas9 system can be the laborious and time-consuming screening required to find CRISPR-induced modifications due to a lack of an obvious phenotype and low frequency after editing. Here we apply the successful co-CRISPR technique in Drosophila to simultaneously target a gene of interest and a marker gene, ebony, which is a recessive gene that produces dark body color and has the further advantage of not being a commonly used transgenic marker. We found that Drosophila broods containing higher numbers of CRISPR-induced ebony mutations (“jackpot” lines) are significantly enriched for indel events in a separate gene of interest, while broods with few or no ebony offspring showed few mutations in the gene of interest. Using two different PAM sites in our gene of interest, we report that ∼61% (52–70%) of flies from the ebony-enriched broods had an indel in DNA near either PAM site. Furthermore, this marker mutation system may be useful in detecting the less frequent homology-directed repair events, all of which occurred in the ebony-enriched broods. By focusing on the broods with a significant number of ebony flies, successful identification of CRISPR-induced events is much faster and more efficient. The co-CRISPR technique we present significantly improves the screening efficiency in identification of genome-editing events in Drosophila.
Actively dividing cells, including some cancers, rely on aerobic glycolysis rather than oxidative phosphorylation to generate energy, a phenomenon termed the Warburg effect. Constitutive activation of the Hypoxia Inducible Factor (HIF-1), a transcription factor known for mediating an adaptive response to oxygen deprivation (hypoxia), is a hallmark of the Warburg effect. HIF-1 is thought to promote glycolysis and suppress oxidative phosphorylation. Here, we instead show that HIF-1 can promote gluconeogenesis. Using a multiomics approach, we reveal the genomic, transcriptomic, and metabolomic landscapes regulated by constitutively active HIF-1 in C. elegans. We use RNA-seq and ChIP-seq under aerobic conditions to analyze mutants lacking EGL-9, a key negative regulator of HIF-1. We integrate these approaches to identify over two hundred genes directly and functionally upregulated by HIF-1, including the PEP carboxykinase PCK-1, a rate-limiting mediator of gluconeogenesis. This activation of PCK-1 by HIF-1 promotes survival in response to both oxidative and hypoxic stress. Our work identifies functional direct targets of HIF-1 in vivo, comprehensively describing the metabolome induced by HIF-1 activation in an organism.
Signal transduction of the conserved transforming growth factor-β (TGFβ) family signaling pathway functions through two distinct serine/threonine transmembrane receptors, the type I and type II receptors. Endocytosis orchestrates the assembly of signaling complexes by coordinating the entry of receptors with their downstream signaling mediators. Recently, we showed that the C. elegans type I bone morphogenetic protein (BMP) receptor SMA-6, part of the TGFβ family, is recycled through the retromer complex while the type II receptor, DAF-4 is recycled in a retromer-independent, ARF-6 dependent manner. From genetic screens in C. elegans aimed at identifying new modifiers of BMP signaling, we reported on SMA-10, a conserved LRIG (leucine-rich and immunoglobulin-like domains) transmembrane protein. It is a positive regulator of BMP signaling that binds to the SMA-6 receptor. Here we show that the loss of sma-10 leads to aberrant endocytic trafficking of SMA-6, resulting in its accumulation in distinct intracellular endosomes including the early endosome, multivesicular bodies (MVB), and the late endosome with a reduction in signaling strength. Our studies show that trafficking defects caused by the loss of sma-10 are not universal, but affect only a limited set of receptors. Likewise, in Drosophila, we find that the fly homolog of sma-10, lambik (lbk), reduces signaling strength of the BMP pathway, consistent with its function in C. elegans and suggesting evolutionary conservation of function. Loss of sma-10 results in reduced ubiquitination of the type I receptor SMA-6, suggesting a possible mechanism for its regulation of BMP signaling.
Embryonic neurons were cultured from transgenic Drosophila melanogaster expressing a highly specific pseudosubstrate inhibitor of protein kinase C (PKC). Flies homozygous for this transgene, which is under the control of the yeast UAS promoter, were crossed to flies homozygous for the yeast heat shock inducible transcription factor GAL 4. Following heat shock, the progeny express the pseudosubstrate inhibitor at high levels. This strategy, which has the advantage of avoiding the non-specific effects of drugs, was used to study the role of PKC in process growth of cultured, differentiating neuroblasts. An external gold particle labeling procedure using a cell surface antigen expressed by mature neurons and processes was used to visualize neuronal processes directly in the scanning electron microscope. We observed that cell cultures expressing a low concentration of the pseudosubstrate inhibitor showed a significant decrease in the number of type I and II processes as compared to control cultures, while the proportions of neuroblasts, ganglion mother cells (GMCs), and mature neurons in the clusters were little affected.
Decapentaplegic (Dpp), the Drosophila homolog of the vertebrate bone morphogenetic protein (BMP2/4), is crucial for patterning and growth in many developmental contexts. The Dpp pathway is regulated at many different levels to exquisitely control its activity. We show that bantam (ban), a microRNA, modulates Dpp signaling activity. Over expression of ban decreases phosphorylated Mothers against decapentaplegic (Mad) levels and negatively affects Dpp pathway transcriptional target genes, while null mutant clones of ban upregulate the pathway. We provide evidence that dpp upregulates ban in the wing imaginal disc, and attenuation of Dpp signaling results in a reduction of ban expression, showing that they function in a feedback loop. Furthermore, we show that this feedback loop is important for maintaining anterior-posterior compartment boundary stability in the wing disc through regulation of optomotor blind (omb), a known target of the pathway. Our results support a model that ban functions with dpp in a negative feedback loop.
Transgenic Drosophila strains expressing an inhibitory peptide of Ca2+/calmodulin dependent protein kinase II (CaM kinase), or a constitutively activated CaM kinase, show altered neuronal process morphology compared to wild type in scanning electron microscopy (SEM) of cultured mature neurons from embryonic neuroblasts. We observed significantly enhanced process growth in cells with inhibited enzyme, and reduced process growth in cells with activated enzyme, suggesting that active CaM kinase is involved in the inhibition of neurite growth during development. The subcellular distribution of CaM kinase in wild type neuronal cultures was determined using a gold particle labeling procedure which allowed the mapping of the enzyme directly in the scanning electron microscope (SEM). Before neuronal contact there was little labeling of processes, but after connections had been made the processes were heavily labeled. Our results suggest that the major transport of CaM kinase to the terminals does not occur until after or during the formation of neuronal connections when a functional synapse might be formed. Taken together, these results suggest a target-dependent transport of the enzyme along processes and an inhibitory role for CaM kinase on neurite branching.
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