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Abstract Cdk8 in Drosophila is the orthologue of vertebrate CDK8 and CDK19. These proteins have been shown to modulate transcriptional control by RNA polymerase II. We found that neuronal loss of Cdk8 severely reduces fly lifespan and causes bang sensitivity. Remarkably, these defects can be rescued by expression of human CDK19, found in the cytoplasm of neurons, suggesting a non-nuclear function of CDK19/Cdk8. Here we show that Cdk8 plays a critical role in the cytoplasm, with its loss causing elongated mitochondria in both muscles and neurons. We find that endogenous GFP-tagged Cdk8 can be found in both the cytoplasm and nucleus. We show that Cdk8 promotes the phosphorylation of Drp1 at S616, a protein required for mitochondrial fission. Interestingly, Pink1, a mitochondrial kinase implicated in Parkinson’s disease, also phosphorylates Drp1 at the same residue. Indeed, overexpression of Cdk8 significantly suppresses the phenotypes observed in flies with low levels of Pink1, including elevated levels of ROS, mitochondrial dysmorphology, and behavioral defects. In summary, we propose that Pink1 and Cdk8 perform similar functions to promote Drp1-mediated fission.

Neurodegenerative Diseases (NDDs) are a group of disorders that cause progressive deficits of neuronal function. Recent evidence argues that sphingolipid metabolism is affected in a surprisingly broad set of NDDs. These include some lysosomal storage diseases (LSDs), hereditary sensory and autonomous neuropathy (HSAN), hereditary spastic paraplegia (HSP), infantile neuroaxonal dystrophy (INAD), Friedreich’s ataxia (FRDA), as well as some forms of amyotrophic lateral sclerosis (ALS) and Parkinson’s disease (PD). Many of these diseases have been modeled in Drosophila melanogaster and are associated with elevated levels of ceramides. Similar changes have also been reported in vertebrate cells and mouse models. Here, we summarize studies using fly models and/or patient samples which demonstrate the nature of the defects in sphingolipid metabolism, the organelles that are implicated, the cell types that are initially affected, and potential therapeutics for these diseases.

Previously, we described a large collection of Drosophila strains that each carry an artificial exon containing a T2AGAL4 cassette inserted in an intron of a target gene based on CRISPR-mediated homologous recombination. These alleles permit numerous applications and have proven to be very useful. Initially, the homologous recombination-based donor constructs had long homology arms (>500 bps) to promote precise integration of large constructs (>5 kb). Recently, we showed that in vivo linearization of the donor constructs enables insertion of large artificial exons in introns using short homology arms (100–200 bps). Shorter homology arms make it feasible to commercially synthesize homology donors and minimize the cloning steps for donor construct generation. Unfortunately, about 58% of Drosophila genes lack a suitable coding intron for integration of artificial exons in all of the annotated isoforms. Here, we report the development of new set of constructs that allow the replacement of the coding region of genes that lack suitable introns with a KozakGAL4 cassette, generating a knock-out/knock-in allele that expresses GAL4 similarly as the targeted gene. We also developed custom vector backbones to further facilitate and improve transgenesis. Synthesis of homology donor constructs in custom plasmid backbones that contain the target gene sgRNA obviates the need to inject a separate sgRNA plasmid and significantly increases the transgenesis efficiency. These upgrades will enable the targeting of nearly every fly gene, regardless of exon–intron structure, with a 70–80% success rate.

Abstract Objectives Oncogenic potential of Notch signaling and its cooperation with other factors to affect proliferation are widely established. Notch exhibits a cooperative effect with loss of a cell polarity gene, scribble to induce neoplastic overgrowth. Oncogenic Ras also show cooperative effect with loss of cell polarity genes such as scribble (scrib), lethal giant larvae (lgl) and discs large to induce neoplastic overgrowth and invasion. Our study aims at assessing the cooperation of activated Notch with loss of function of lgl in tumor overgrowth, and the mode of JNK signaling activation in this context. Results In the present study, we use Drosophila as an in vivo model to show the synergy between activated Notch (N act ) and loss of function of lgl (lgl-IR) in tumor progression. Coexpression of N act and lgl-IR results in massive tumor overgrowth and displays hallmarks of cancer, such as MMP1 upregulation and loss of epithelial integrity. We further show activation of JNK signaling and upregulation of its receptor, Grindelwald in N act /lgl-IR tumor. In contrast to previously described Notch act /scrib −/− tumor, our experiments in N act /lgl-IR tumor showed the presence of dying cells along with tumorous overgrowth.

Chromatin-remodeling proteins have a profound role in the transcriptional regulation of gene expression during development. Here, we have shown that the chromodomain-containing protein Hat-trick is predominantly expressed within the oocyte nucleus, specifically within the heterochromatinized karyosome, and that a mild expression is observed in follicle cells. Colocalization of Hat-trick with Heterochromatin Protein 1 and synaptonemal complex component C(3)G along with the diffused karyosome after hat-trick downregulation shows the role of this protein in heterochromatin clustering and karyosome maintenance. Germline mosaic analysis reveals that hat-trick is required for maintaining the dorso-ventral patterning of eggs by regulating the expression of Gurken. The increased incidence of double-strand breaks (DSBs), delayed DSB repair, defects in karyosome formation, altered Vasa mobility, and, consequently, misexpression and altered localization of Gurken in hat-trick mutant egg chambers clearly suggest a putative involvement of Hat-trick in the early stages of oogenesis. In addition, based on phenotypic observations in hat-trick mutant egg chambers, we speculate a substantial role of hat-trick in cystoblast proliferation, oocyte determination, nurse cell endoreplication, germ cell positioning, cyst encapsulation, and nurse cell migration. Our results demonstrate that hat-trick has profound pleiotropic functions during oogenesis in Drosophila melanogaster.