Acetyl-CoA-mediated autoacetylation of fatty acid synthase as a metabolic switch of de novo lipogenesis in Drosophila

De novo lipogenesis is a highly regulated metabolic process, which is known to be activated through transcriptional regulation of lipogenic genes, including fatty acid synthase (FASN). Unexpectedly, we find that the expression of FASN protein remains unchanged durin

Three microtubule severing enzymes contribute to the “Pacman-flux” machinery that moves chromosomes

Chromosomes move toward mitotic spindle poles by a Pacman-flux mechanism linked to microtubule depolymerization: chromosomes actively depolymerize attached microtubule plus ends (Pacman) while being reeled in to spindle poles by the continual poleward flow of tubulin subunits driven by minus-end depolymerization (flux). We report that Pacman-flux in Drosophila melanogaster incorporates the activities of three different microtubule severing enzymes, Spastin, Fidgetin, and Katanin. Spastin and Fidgetin are utilized to stimulate microtubule minus-end depolymerization and flux. Both proteins concentrate at centrosomes, where they catalyze the turnover of γ-tubulin, consistent with the hypothesis that they exert their influence by releasing stabilizing γ-tubulin ring complexes from minus ends. In contrast, Katanin appears to function primarily on anaphase chromosomes, where it stimulates microtubule plus-end depolymerization and Pacman-based chromatid motility. Collectively, these findings reveal novel and significant roles for microtubule severing within the spindle and broaden our understanding of the molecular machinery used to move chromosomes

Neuronal expression in Drosophila of an evolutionarily conserved metallophosphodiesterase reveals pleiotropic roles in longevity and odorant response

Evolutionarily conserved genes often play critical roles in organismal physiology. Here, we describe multiple roles of a previously uncharacterized Class III metallophosphodiesterase in Drosophila, an ortholog of the MPPED1 and MPPED2 proteins expressed in the mammalian brain. dMpped, the product of CG16717, hydrolyzed phosphodiester substrates including cAMP and cGMP in a metal-dependent manner. dMpped is expressed during development and in the adult fly. RNA-seq analysis of dMppedKO flies revealed misregulation of innate immune pathways. dMppedKO flies showed a reduced lifespan, which could be restored in Dredd hypomorphs, indicating that excessive production of antimicrobial peptides contributed to reduced longevity. Elevated levels of cAMP and cGMP in the brain of dMppedKO flies was restored on neuronal expression of dMpped, with a concomitant reduction in levels of antimicrobial peptides and restoration of normal life span. We observed that dMpped is expressed in the antennal lobe in the fly brain. dMppedKO flies showed defective specific attractant perception and desiccation sensitivity, correlated with the overexpression of Obp28 and Obp59 in knock-out flies. Importantly, neuronal expression of mammalian MPPED2 restored lifespan in dMppedKO flies. This is the first description of the pleiotropic roles of an evolutionarily conserved metallophosphodiesterase that may moonlight in diverse signaling pathways in an organism

IFT88 transports Gucy2d, a guanylyl cyclase, to maintain sensory cilia function in Drosophila

Cilia are involved in a plethora of motility and sensory-related functions. Ciliary defects cause several ciliopathies, some of which with late-onset, suggesting cilia are actively maintained. While much is known about cilia assembly, little is understood about the mechanisms of their maintenance. Given that intraflagellar transport (IFT) is essential for cilium assembly, we investigated the role of one of its main players, IFT88, in ciliary maintenance. We show that DmIFT88, the Drosophila melanogaster orthologue of IFT88, continues to move along fully formed sensory cilia, and that its acute knockdown in the ciliated neurons of the adult affects sensory behaviour. We further identify DmGucy2d, the Drosophila guanylyl cyclase 2d, as a DmIFT88 cargo, whose loss also leads to defects in sensory behaviour maintenance. DmIFT88 binds to the intracellular part of DmGucy2d, a highly, evolutionarily conserved and mutated in several degenerative retina diseases, taking the cyclase into the cilia. Our results offer a novel mechanism for the maintenance of sensory cilia function and its potential role in human diseases

Of Single Nucleotides and Single Cells: Charting the Genotype-Phenotype Map at High Resolution Using \u3ci\u3eDrosophila melanogaster\u3c/i\u3e

Understanding the mechanisms by which genetic variation brings about phenotypic variation is essential for understanding variation in complex traits. Drosophila melanogaster is a powerful model organism for such studies. Flies are easy to raise in the laboratory under controlled genetic and environmental conditions and many genetic tools are widely available. To chart the genotype-phenotype map, we need to study how individual genetic variants contribute to phenotypic variation, as well as how environmental perturbations influence gene expression. Regarding the former, I generated single nucleotide substitutions in Obp56h in a common genetic background. Obp56h, a member of the Odorant binding protein multigene family, is a small gene in a favorable genomic location for CRISPR-Cas9 mediated deletion. After deletion, I reinserted the gene at the endogenous locus with individual allelic variants chosen from those segregating in a wild-derived inbred population to produce five lines varying at single nucleotides in a common genetic background. Different alleles, both within and near the gene (potentially regulatory) and both common and rare, have different, large effects on organismal fitness traits as well as on genome-wide coregulated ensembles of transcripts. These effects are at the level of mean and microenvironmental variance in both fitness traits and the transcriptome. However, these alleles have only small effects on fitness traits in the wild-derived inbred population indicating that the effects of individual alleles can be context-specific and are perhaps suppressed in natural populations via epistatic interactions. Next, I studied how acute cocaine consumption and developmental alcohol exposure affect the transcriptome at single-cell resolution. The Drosophila brain is small, allowing for comprehensive whole-brain studies. Further, previous studies have characterized effects of acute cocaine consumption and developmental alcohol exposure on flies, which resemble those in humans. Single-cell RNA sequencing revealed that the transcriptomes of cells in the fly brain are affected in a cell-type and sex-dependent manner after the flies consumed fixed amounts of cocaine or are exposed to developmental alcohol exposure. These effects are sexually dimorphic, with males showing a greater degree of differential expression and are particularly prominent in glial and mushroom body cells. Developmental alcohol exposure leads to a similar, but different, sexually dimorphic and cell-type dependent pattern of differential expression as cocaine consumption. Some mechanisms are shared between the experimental paradigms indicating common processes. The strategies used in the studies described in this dissertation can be generally applied to explore genotype-phenotype relationships at high resolution

Bioinformatic Characterization of P-Type ATPases Encoded Within the Fully Sequenced Genomes of 26 Eukaryotes

P-type ATPases play essential roles in numerous processes, which in humans include nerve impulse propagation, relaxation of muscle fibers, secretion and absorption in the kidney, acidification of the stomach and nutrient absorption in the intestine. Published evidence suggests that uncharacterized families of P-type ATPases with novel specificities exist. In this study, the fully sequenced genomes of 26 eukaryotes, including animals, plants, fungi and unicellular eukaryotes, were analyzed for P-type ATPases. We report the organismal distributions, phylogenetic relationships, probable topologies and conserved motifs of nine functionally characterized families and 13 uncharacterized families of these enzyme transporters. We have classified these proteins according to the conventions of the functional and phylogenetic IUBMB-approved transporter classification system (www.tcdb.org, Saier et al. in Nucleic Acids Res 34:181–186, 2006; Nucleic Acids Res 37:274–278, 2009)

Axon Death Pathways Converge on Axed to Promote Axon Disassembly

Axons use a conserved program to actively drive their own destruction after injury. Axon degeneration is present in many neurological disorders and an axon death program could be a major pharmaceutical target to preserve neuronal function. This intrinsic signaling cascade activates pro-degenerative dSarm/Sarm1, rapidly depletes axonal stores of NAD+, and terminates in cytoskeletal breakdown. Conversely, loss of dSarm/Sarm1, maintenance of NAD+ levels or its biosynthetic enzyme Nmnat, result in long-term morphological perseveration of severed axons. Exactly how dSarm/Sarm1 and loss of NAD+ execute axon death remains poorly defined. We sought to uncover novel regulators of axon death and maintenance by performing a deficiency screen and a forward genetic mutagenesis screen in axotomized Drosophila wing sensory neurons. We identified a BTB domain protein enriched in neurons, we named Axundead (Axed), which is specifically required for axon death. Severed axons harboring loss of function mutations in axed, similar to dSarm mutants, remain preserved for 50 days post axotomy. Spontaneous neurodegeneration induced by activated dSarm or dNmnat depletion are both suppressed in axed mutants, but not in dSarm mutant alleles. Additionally, severed axed mutant axons also expressing activated dSarm or lacking Nmnat are preserved. These results indicate that dSarm acts upstream of dNmnat loss, and both events precede essential Axed function and axon destruction. Thus, the axon death pathway converges on Axed function