Molecular and cellular mechanisms of teneurin signaling in synaptic partner matching.

In developing brains, axons exhibit remarkable precision in selecting synaptic partners among many non-partner cells. Evolutionarily conserved teneurins are transmembrane proteins that instruct synaptic partner matching. However, how intracellular signaling pathways execute teneurins' functions is unclear. Here, we use in situ proximity labeling to obtain the intracellular interactome of a teneurin (Ten-m) in the Drosophila brain. Genetic interaction studies using quantitative partner matching assays in both olfactory receptor neurons (ORNs) and projection neurons (PNs) reveal a common pathway: Ten-m binds to and negatively regulates a RhoGAP, thus activating the Rac1 small GTPases to promote synaptic partner matching. Developmental analyses with single-axon resolution identify the cellular mechanism of synaptic partner matching: Ten-m signaling promotes local F-actin levels and stabilizes ORN axon branches that contact partner PN dendrites. Combining spatial proteomics and high-resolution phenotypic analyses, this study advanced our understanding of both cellular and molecular mechanisms of synaptic partner matching.

Cellular bases of olfactory circuit assembly revealed by systematic time-lapse imaging.

Neural circuit assembly features simultaneous targeting of numerous neuronal processes from constituent neuron types, yet the dynamics is poorly understood. Here, we use the Drosophila olfactory circuit to investigate dynamic cellular processes by which olfactory receptor neurons (ORNs) target axons precisely to specific glomeruli in the ipsi- and contralateral antennal lobes. Time-lapse imaging of individual axons from 30 ORN types revealed a rich diversity in extension speed, innervation timing, and ipsilateral branch locations and identified that ipsilateral targeting occurs via stabilization of transient interstitial branches. Fast imaging using adaptive optics-corrected lattice light-sheet microscopy showed that upon approaching target, many ORN types exhibiting "exploring branches" consisted of parallel microtubule-based terminal branches emanating from an F-actin-rich hub. Antennal nerve ablations uncovered essential roles for bilateral axons in contralateral target selection and for ORN axons to facilitate dendritic refinement of postsynaptic partner neurons. Altogether, these observations provide cellular bases for wiring specificity establishment.

The nutrient sensor OGT regulates Hipk stability and tumorigenic-like activities in Drosophila.

Environmental cues such as nutrients alter cellular behaviors by acting on a wide array of molecular sensors inside cells. Of emerging interest is the link observed between effects of dietary sugars on cancer proliferation. Here, we identify the requirements of hexosamine biosynthetic pathway (HBP) and O-GlcNAc transferase (OGT) for Drosophila homeodomain-interacting protein kinase (Hipk)-induced growth abnormalities in response to a high sugar diet. On a normal diet, OGT is both necessary and sufficient for inducing Hipk-mediated tumor-like growth. We further show that OGT maintains Hipk protein stability by blocking its proteasomal degradation and that Hipk is O-GlcNAcylated by OGT. In mammalian cells, human HIPK2 proteins accumulate posttranscriptionally upon OGT overexpression. Mass spectrometry analyses reveal that HIPK2 is at least O-GlcNAc modified at S852, T1009, and S1147 residues. Mutations of these residues reduce HIPK2 O-GlcNAcylation and stability. Together, our data demonstrate a conserved role of OGT in positively regulating the protein stability of HIPKs (fly Hipk and human HIPK2), which likely permits the nutritional responsiveness of HIPKs.

Hyperpolarized mitochondria accumulate in Drosophila Hipk-overexpressing cells to drive tumor-like growth.

Both functional and dysfunctional mitochondria are known to underlie tumor progression. Here, we establish use of the proto-oncogene Drosophila Homeodomain-interacting protein kinase (Hipk) as a new tool to address this paradox. We find that, in Hipk-overexpressing tumor-like cells, mitochondria accumulate and switch from fragmented to highly fused interconnected morphologies. Moreover, elevated Hipk promotes mitochondrial membrane hyperpolarization. These mitochondrial changes are at least in part driven by the upregulation of Myc. Furthermore, we show that the altered mitochondrial energetics, but not morphology, is required for Hipk-induced tumor-like growth, because knockdown of pdsw (also known as nd-pdsw; NDUFB10 in mammals; a Complex I subunit) abrogates the growth. Knockdown of ATPsynß (a Complex V subunit), which produces higher levels of reactive oxygen species (ROS) than pdsw knockdown, instead synergizes with Hipk to potentiate JNK activation and the downstream induction of matrix metalloproteinases. Accordingly, ATPsynß knockdown suppresses Hipk-induced tumor-like growth only when ROS scavengers are co-expressed. Together, our work presents an in vivo tumor model featuring the accumulation of hyperfused and hyperpolarized mitochondria, and reveals respiratory complex subunit-dependent opposing effects on tumorigenic outcomes.This article has an associated First Person interview with the first author of the paper.

LRP6 lets Merlin go in times of nutrient scarcity.

Cells take advantage of cross-talk in signaling pathways to integrate diverse signals and produce coordinated responses. In this issue of EMBO Reports, Jeong et al discover that the Wnt co-receptor, low-density lipoprotein (LDL) receptor-related protein LRP6, negatively regulates Hippo signaling by serving as a binding sink to sequester and inhibit Merlin, an activator of Hippo signaling (Jeong et al, 2020). This regulation is nutrient-responsive, likely using LRP6 O-GlcNAcylation as a molecular switch.

ß-Spectrin regulates the hippo signaling pathway and modulates the basal actin network.

Emerging evidence suggests functional regulation of the Hippo pathway by the actin cytoskeleton, although the detailed molecular mechanism remains incomplete. In a genetic screen, we identified a requirement for ß-Spectrin in the posterior follicle cells for the oocyte repolarization process during Drosophila mid-oogenesis. ß-spectrin mutations lead to loss of Hippo signaling activity in the follicle cells. A similar reduction of Hippo signaling activity was observed after ß-Spectrin knockdown in mammalian cells. We further demonstrated that ß-spectrin mutations disrupt the basal actin network in follicle cells. The abnormal stress fiber-like actin structure on the basal side of follicle cells provides a likely link between the ß-spectrin mutations and the loss of the Hippo signaling activity phenotype.

Cdk8/CDK19 promotes mitochondrial fission through Drp1 phosphorylation and can phenotypically suppress pink1 deficiency in Drosophila.

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.

The AMPK-like protein kinases Sik2 and Sik3 interact with Hipk and induce synergistic tumorigenesis in a Drosophila cancer model.

Homeodomain-interacting protein kinases (Hipks) regulate cell proliferation, apoptosis, and tissue development. Overexpression of Hipk in Drosophila causes tumorigenic phenotypes in larval imaginal discs. We find that depletion of Salt-inducible kinases Sik2 or Sik3 can suppress Hipk-induced overgrowth. Furthermore, co-expression of constitutively active forms of Sik2 or Sik3 with Hipk caused significant tissue hyperplasia and tissue distortion, indicating that both Sik2 and Sik3 can synergize with Hipk to promote tumorous phenotypes, accompanied by elevated dMyc, Armadillo/ß-catenin, and the Yorkie target gene expanded. Larvae expressing these hyperplastic growths also display an extended larval phase, characteristic of other Drosophila tumour models. Examination of total protein levels from fly tissues showed that Hipk proteins were reduced when Siks were depleted through RNAi, suggesting that Siks may regulate Hipk protein stability and/or activity. Conversely, expression of constitutively active Siks with Hipk leads to increased Hipk protein levels. Furthermore, Hipk can interact with Sik2 and Sik3 by co-immunoprecipitation. Co-expression of both proteins leads to a mobility shift of Hipk protein, suggesting it is post-translationally modified. In summary, our research demonstrates a novel function of Siks in synergizing with Hipk to promote tumour growth.

Origin of wiring specificity in an olfactory map revealed by neuron type-specific, time-lapse imaging of dendrite targeting.

How does wiring specificity of neural maps emerge during development? Formation of the adult Drosophila olfactory glomerular map begins with the patterning of projection neuron (PN) dendrites at the early pupal stage. To better understand the origin of wiring specificity of this map, we created genetic tools to systematically characterize dendrite patterning across development at PN type-specific resolution. We find that PNs use lineage and birth order combinatorially to build the initial dendritic map. Specifically, birth order directs dendrite targeting in rotating and binary manners for PNs of the anterodorsal and lateral lineages, respectively. Two-photon- and adaptive optical lattice light-sheet microscope-based time-lapse imaging reveals that PN dendrites initiate active targeting with direction-dependent branch stabilization on the timescale of seconds. Moreover, PNs that are used in both the larval and adult olfactory circuits prune their larval-specific dendrites and re-extend new dendrites simultaneously to facilitate timely olfactory map organization. Our work highlights the power and necessity of type-specific neuronal access and time-lapse imaging in identifying wiring mechanisms that underlie complex patterns of functional neural maps.

The brain's ability to sense, act and remember relies on the intricate network of connections between neurons. Organization of these connections into neural maps is critical for processing sensory information. For instance, different odors are represented by specific neurons in a part of the brain known as the olfactory bulb, allowing animals to distinguish between smells. Projection neurons in the olfactory bulb have extensions known as dendrites that receive signals from sensory neurons. Scientists have extensively used the olfactory map in adult fruit flies to study brain wiring because of the specific connections between their sensory and projection neurons. This has led to the discovery of similar wiring strategies in mammals. But how the olfactory map is formed during development is not fully understood. To investigate, Wong et al. built genetic tools to label specific types of olfactory projection neurons during the pupal stage of fruit fly development. This showed that a group of projection neurons directed their dendrites in a clockwise rotation pattern depending on the order in which they were born: the first-born neuron sent dendrites towards the top right of the antennal lobe (the fruit fly equivalent of the olfactory bulb), while the last-born sent dendrites towards the top left. Wong et al. also carried out high-resolution time-lapse imaging of live brains grown in the laboratory to determine how dendrites make wiring decisions. This revealed that projection neurons send dendrites in all directions, but preferentially stabilize those that extend in the direction which the neurons eventually target. Also, live imaging showed neurons could remove old dendrites (used in the larvae) and build new ones (to be used in the adult) simultaneously, allowing them to quickly create new circuits. These experiments demonstrate the value of imaging specific types of neurons to understand the mechanisms that assemble neural maps in the developing brain. Further work could use the genetic tools created by Wong et al. to study how wiring decisions are determined in this and other neural maps by specific genes, potentially yielding insights into neurological disorders associated with wiring defects.

Metabolic reprogramming in cancer: mechanistic insights from Drosophila.

Cancer cells constantly reprogram their metabolism as the disease progresses. However, our understanding of the metabolic complexity of cancer remains incomplete. Extensive research in the fruit fly Drosophila has established numerous tumor models ranging from hyperplasia to neoplasia. These fly tumor models exhibit a broad range of metabolic profiles and varying nutrient sensitivity. Genetic studies show that fly tumors can use various alternative strategies, such as feedback circuits and nutrient-sensing machinery, to acquire and consolidate distinct metabolic profiles. These studies not only provide fresh insights into the causes and functional relevance of metabolic reprogramming but also identify metabolic vulnerabilities as potential targets for cancer therapy. Here, we review the conceptual advances in cancer metabolism derived from comparing and contrasting the metabolic profiles of fly tumor models, with a particular focus on the Warburg effect, mitochondrial metabolism, and the links between diet and cancer.

A positive feedback loop between Myc and aerobic glycolysis sustains tumor growth in a Drosophila tumor model.

Cancer cells usually exhibit aberrant cell signaling and metabolic reprogramming. However, mechanisms of crosstalk between these processes remain elusive. Here, we show that in an in vivo tumor model expressing oncogenic Drosophila Homeodomain-interacting protein kinase (Hipk), tumor cells display elevated aerobic glycolysis. Mechanistically, elevated Hipk drives transcriptional upregulation of Drosophila Myc (dMyc; MYC in vertebrates) likely through convergence of multiple perturbed signaling cascades. dMyc induces robust expression of pfk2 (encoding 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase; PFKFB in vertebrates) among other glycolytic genes. Pfk2 catalyzes the synthesis of fructose-2,6-bisphosphate, which acts as a potent allosteric activator of Phosphofructokinase (Pfk) and thus stimulates glycolysis. Pfk2 and Pfk in turn are required to sustain dMyc protein accumulation post-transcriptionally, establishing a positive feedback loop. Disruption of the loop abrogates tumorous growth. Together, our study demonstrates a reciprocal stimulation of Myc and aerobic glycolysis and identifies the Pfk2-Pfk governed committed step of glycolysis as a metabolic vulnerability during tumorigenesis.

Homeodomain-interacting protein kinase promotes tumorigenesis and metastatic cell behavior.

Aberrations in signaling pathways that regulate tissue growth often lead to tumorigenesis. Homeodomain-interacting protein kinase (Hipk) family members are reported to have distinct and contradictory effects on cell proliferation and tissue growth. From these studies, it is clear that much remains to be learned about the roles of Hipk family protein kinases in proliferation and cell behavior. Previous work has shown that Drosophila Hipk is a potent growth regulator, thus we predicted that it could have a role in tumorigenesis. In our study of Hipk-induced phenotypes, we observed the formation of tumor-like structures in multiple cell types in larvae and adults. Furthermore, elevated Hipk in epithelial cells induces cell spreading, invasion and epithelial-to-mesenchymal transition (EMT) in the imaginal disc. Further evidence comes from cell culture studies, in which we expressed Drosophila Hipk in human breast cancer cells and showed that it enhances proliferation and migration. Past studies have shown that Hipk can promote the action of conserved pathways implicated in cancer and EMT, such as Wnt/Wingless, Hippo, Notch and JNK. We show that Hipk phenotypes are not likely to arise from activation of a single target, but rather through a cumulative effect on numerous target pathways. Most Drosophila tumor models involve mutations in multiple genes, such as the well-known RasV12 model, in which EMT and invasiveness occur after the additional loss of the tumor suppressor gene scribble. Our study reveals that elevated levels of Hipk on their own can promote both hyperproliferation and invasive cell behavior, suggesting that Hipk family members could be potent oncogenes and drivers of EMT.