basket/JNK: Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

Gene name - basket

Synonyms - Jun amino terminal kinase (DJNK)

Cytological map position - 31B-C

Function - MAP kinase - Jun N-terminal kinase

Keywords - dorsal closure, immune response, JNK pathway

Symbol - bsk

FlyBase ID:FBgn0000229

Genetic map position - 2-32

Classification - MAP kinase homolog

Cellular location - cytoplasmic and nuclear

NCBI link: Entrez Gene
bsk orthologs: Biolitmine

Recent literature
Huang, J. and Xue, L. (2015). Loss of flfl triggers JNK-dependent cell death in Drosophila. Biomed Res Int 2015: 623573. PubMed ID: 26583122
falafel (flfl) encodes a Drosophila homolog of human SMEK whose in vivo functions remain elusive. Gain-of-function and loss-of-function analysis was performed in in Drosophila and flfl was identified as a negative regulator of JNK pathway-mediated cell death. While ectopic expression of flfl suppresses TNF-triggered JNK-dependent cell death, loss of flfl promotes JNK activation and cell death in the developing eye and wing. These data report for the first time an essential physiological function of flfl in maintaining tissue homeostasis and organ development. As the JNK signaling pathway has been evolutionary conserved from fly to human, a similar role of PP4R3 in JNK-mediated physiological process is speculated.

Bornstein, B., et al. (2015). Developmental axon pruning requires destabilization of cell adhesion by JNK signaling. Neuron 88: 926-940. PubMed ID: 26586184
Developmental axon pruning is essential for normal brain wiring in vertebrates and invertebrates. How axon pruning occurs in vivo is not well understood. In a mosaic loss-of-function screen, this study found that Bsk, the Drosophila JNK, is required for axon pruning of mushroom body γ neurons, but not their dendrites. By combining in vivo genetics, biochemistry, and high-resolution microscopy, this study demonstrated that the mechanism by which Bsk is required for pruning is through reducing the membrane levels of the adhesion molecule Fasciclin II (FasII), the NCAM ortholog. Conversely, overexpression of FasII is sufficient to inhibit axon pruning. Finally, this study showed that overexpressing other cell adhesion molecules, together with weak attenuation of JNK signaling, strongly inhibits pruning. Taken together, this study uncovered a novel and unexpected interaction between the JNK pathway and cell adhesion and found that destabilization of cell adhesion is necessary for efficient pruning.

Khoshnood, B., Dacklin, I. and Grabbe, C. (2015). Urm1: an essential regulator of JNK signaling and oxidative stress in Drosophila melanogaster. Cell Mol Life Sci [Epub ahead of print]. PubMed ID: 26715182
Ubiquitin-related modifier 1 (Urm1) is a ubiquitin-like molecule (UBL) with the dual capacity to act both as a sulphur carrier and posttranslational protein modifier. This study characterizes the Drosophila homologues of Urm1 (CG33276) and its E1 activating enzyme Uba4 (CG13090) and shows that they function together to induce protein urmylation in vivo. Urm1 conjugation to target proteins in general, and to the evolutionary conserved substrate Peroxiredoxin 5 (Prx5) specifically, is dependent on Uba4. A complete loss of Urm1 is lethal in flies, although a small number of adult zygotic Urm1 n123 mutant escapers can be recovered. These escapers display a decreased general fitness and shortened lifespan, but in contrast to their S. cerevisiae counterparts, they are resistant to oxidative stress. Providing a molecular explanation, this study demonstrates that cytoprotective JNK signaling is increased in Urm1 deficient animals. In agreement, molecular and genetic evidence suggest that elevated activity of the JNK downstream target genes Jafrac1 (thioredoxin peroxidase 1) and gstD1 (Glutathione S transferase D1) strongly contributes to the tolerance against oxidative stress displayed by Urm1 n123 null mutants. In conclusion, Urm1 is a UBL that is involved in the regulation of JNK signaling and the response against oxidative stress in the fruit fly.

Clemente-Ruiz, M., Murillo-Maldonado, J.M., Benhra, N., Barrio, L., Pérez, L., Quiroga, G., Nebreda, A.R. and Milán, M. (2016). Gene dosage imbalance contributes to chromosomal instability-induced tumorigenesis. Dev Cell 36: 290-302. PubMed ID: 26859353
Chromosomal instability (CIN) is thought to be a source of mutability in cancer. However, CIN often results in aneuploidy, which compromises cell fitness. This study used the dosage compensation mechanism (DCM) of Drosophila to demonstrate that chromosome-wide gene dosage imbalance contributes to the deleterious effects of CIN-induced aneuploidy and its pro-tumorigenic action. Resetting of the DCM counterbalances the damaging effects caused by CIN-induced changes in X chromosome number. Importantly, interfering with the DCM suffices to mimic the cellular effects of aneuploidy in terms of reactive oxygen species (ROS) production, JNK-dependent cell death, and tumorigenesis upon apoptosis inhibition. A role of ROS was found in JNK activation and a variety of cellular and tissue-wide mechanisms that buffer the deleterious effects of CIN, including DNA-damage repair, activation of the p38 pathway, and cytokine induction were found to promote compensatory proliferation. These data reveal the existence of robust compensatory mechanisms that counteract CIN-induced cell death and tumorigenesis.
Lim, B., Dsilva, C. J., Kevrekidis, I. G. and Shvartsman, S. Y. (2017). Reconstructing ERK signaling in the Drosophila embryo from fixed images. Methods Mol Biol 1487: 337-351. PubMed ID: 27924579
The early Drosophila embryo provides unique opportunities for quantitative studies of ERK signaling. This system is characterized by simple anatomy, the ease of obtaining large numbers of staged embryos, and the availability of powerful tools for genetic manipulation of the ERK pathway. This paper describes how these experimental advantages can be combined with recently developed microfluidic devices for high throughput imaging of ERK activation dynamics. Focus was placed on the stage during the third hour of development, when ERK activation is essential for patterning of the future nerve cord. This approach starts with an ensemble of fixed embryos stained with an antibody that recognizes the active, dually phosphorylated form of ERK. Each embryo in this ensemble provides a snapshot of the spatial and temporal pattern of ERK activation during development. Then the ages of fixed embryos are quantitatively estimated using a model that links their morphology and developmental time. This model is learned based on live imaging of cellularization and gastrulation, two highly stereotyped morphogenetic processes at this stage of embryogenesis. Applying this approach, ERK signaling can be characterized at high spatial and temporal resolution. This methodology can be readily extended to studies of ERK regulation and function in multiple mutant backgrounds, providing a versatile assay for quantitative studies of developmental ERK signaling.
Nitta, Y. and Sugie, A. (2017). DISCO interacting protein 2 determines direction of axon projection under the regulation of c-Jun N-terminal kinase in the Drosophila mushroom body. Biochem Biophys Res Commun. PubMed ID: 28396149
Precisely controlled axon guidance for complex neuronal wiring is essential for appropriate neuronal function. c-Jun N-terminal kinase (JNK) was found to play a role in axon guidance recently as well as in cell proliferation, protection and apoptosis. In spite of many genetic and molecular studies on these biological processes regulated by JNK, how JNK regulates axon guidance accurately has not been fully explained thus far. To address this question, this study used the Drosophila mushroom body (MB) as a model since the α/β axons project in two distinct directions. This study showns that DISCO interacting protein 2 (DIP2) is required for the accurate direction of axonal guidance. DIP2 expression is under the regulation of Basket (Bsk), the Drosophila homologue of JNK. The Bsk/DIP2 pathway is independent from the AP-1 transcriptional factor complex pathway, which is directly activated by Bsk. In conclusion, these findings revealed DIP2 as a novel effector downstream of Bsk modulating the direction of axon projection.
Ma, X., Lu, J. Y., Dong, Y., Li, D., Malagon, J. N. and Xu, T. (2017). PP6 disruption synergizes with oncogenic Ras to promote JNK-dependent tumor growth and invasion. Cell Rep 19(13): 2657-2664. PubMed ID: 28658615
RAS genes are frequently mutated in cancers, yet an effective treatment has not been developed, partly because of an incomplete understanding of signaling within Ras-related tumors. To address this, a genetic screen was performed in Drosophila, aiming to find mutations that cooperate with oncogenic Ras (RasV12) to induce tumor overgrowth and invasion. fiery mountain (fmt; CG10289), a regulatory subunit of the protein phosphatase 6 (PP6) complex, was identified as a tumor suppressor that synergizes with RasV12 to drive c-Jun N-terminal kinase (JNK)-dependent tumor growth and invasiveness. Fmt was shown to negatively regulate JNK upstream of dTAK1. It was further demonstrated that disruption of PpV, the catalytic subunit of PP6, mimics fmt loss-of-function-induced tumorigenesis. Finally, Fmt synergizes with PpV to inhibit JNK-dependent tumor progression. These data here further highlight the power of Drosophila as a model system to unravel molecular mechanisms that may be relevant to human cancer biology.
Bushnell, H. L., Feiler, C. E., Ketosugbo, K. F., Hellerman, M. B., Nazzaro, V. L. and Johnson, R. I. (2017). JNK is antagonized to ensure the correct number of interommatidial cells pattern the Drosophila retina. Dev Biol [Epub ahead of print]. PubMed ID: 29133184
Apoptosis is crucial during the morphogenesis of most organs and tissues, and is utilized for tissues to achieve their proper size, shape and patterning. Many signaling pathways contribute to the precise regulation of apoptosis. This study shows that Jun N-terminal Kinase (JNK) activity contributes to the coordinated removal of interommatidial cells via apoptosis in the Drosophila pupal retina. This is consistent with previous findings that JNK activity promotes apoptosis in other epithelia. However, JNK activity was found to be repressed by Cindr (the CIN85 and CD2AP ortholog) in order to promote cell survival. Reducing the amount of Cindr resulted in ectopic cell death. Increased expression of the Drosophila JNK basket in the setting of reduced cindr expression was found to result in even more severe apoptosis, whilst ectopic death was found to be reduced if retinas were heterozygous for basket. Hence Cindr is required to properly restrict JNK-mediated apoptosis in the pupal eye, resulting in the correct number of interommatidial cells. A lack of precise control over developmental apoptosis can lead to improper tissue morphogenesis.
Ogura, Y., Wen, F. L., Sami, M. M., Shibata, T. and Hayashi, S. (2018). A switch-like activation relay of EGFR-ERK signaling regulates a wave of cellular contractility for epithelial invagination. Dev Cell 46(2): 162-172.e165. PubMed ID: 29983336
The dynamics of extracellular signal-regulated kinase (ERK) signaling underlies its versatile functions in cell differentiation, cell proliferation, and cell motility. Classical studies in Drosophila established that a gradient of epidermal growth factor receptor (EGFR)-ERK signaling is essential for these cellular responses. However, this study challenges this view by the real-time monitoring of ERK activation; a switch-like ERK activation is shown to be essential for the invagination movement of the Drosophila tracheal placode. This switch-like ERK activation stems from the positive feedback regulation of the EGFR-ERK signaling and a resultant relay of EGFR-ERK signaling among tracheal cells. A key transcription factor Trachealess (Trh) permissively regulates the iteration of the relay, and the ERK activation becomes graded in trh mutant. A mathematical model based on these observations and a molecular link between ERK activation dynamics and myosin shows that the relay mechanism efficiently promotes epithelial invagination while the gradient mechanism does not.
Chi, C., Wang, L., Lan, W., Zhao, L. and Su, Y. (2018). PpV, acting via the JNK pathway, represses apoptosis during normal development of Drosophila wing. Apoptosis. PubMed ID: 30159848
Apoptosis is one of the main fundamental biological processes required for development of multicellular organisms. Inappropriate regulation of apoptosis can lead to severe developmental abnormalities and diseases. Therefore, the control of apoptosis, not only for its activation but also for its inhibition, is critically important during development. In contrast to the extensive studies of apoptosis induction, its inhibitory mechanisms that are even more vital in certain populations of cells actually are very far from being well understood. This study reports an inhibitory role of protein phosphatase V (PpV), a serine/threonine protein phosphatase, in controlling the apoptosis during Drosophila wing development. Inhibition of ppv by RNAi in wing imaginal discs induced ectopic cell death and caspase activation, thus, resulted in a defective adult wing. Moreover, knocking-down ppv triggered the activation of c-Jun N-terminal kinase (JNK) signal, an evolutionarily conserved intracellular signaling that has been implicated to modulate the apoptotic machinery in many biological and experimental systems. Disrupting the JNK signal transduction was adequate to suppress the ppv effects for wing development. Together, this study provides the evidence to demonstrate that ppv is required for normal wing development in maintaining the silence of apoptotic signal possibly through JNK pathway.
Wang, X., Huang, X., Wu, C. and Xue, L. (2018). Pontin/Tip49 negatively regulates JNK-mediated cell death in Drosophila. Cell Death Discov 5: 8. PubMed ID: 30062057
Pontin (Pont), also known as Tip49, encodes a member of the AAA+ (ATPases Associated with Diverse Cellular Activities) superfamily and plays pivotal roles in cell proliferation and growth, yet its function in cell death has remained poorly understood. This study performed a genetic screen for dominant modifiers of Eiger-induced JNK-dependent cell death in Drosophila, and identified Pont as a negative regulator of JNK-mediated cell death. In addition, loss of function of Pont is sufficient to induce cell death and activate the transcription of JNK target gene puc. Furthermore, the epistasis analysis indicates that Pont acts downstream of Hep. Finally, this study found that Pont is also required for JNK-mediated thorax development and acts as a negative regulator of JNK phosphorylation. Together, these data suggest that pont encodes a negative component of Egr/JNK signaling pathway in Drosophila through negatively regulating JNK phosphorylation, which provides a novel role of ATPase in Egr-JNK signaling.
Moreno, E., Valon, L., Levillayer, F. and Levayer, R. (2019). Competition for space induces cell elimination through compaction-driven ERK downregulation. Curr Biol 29(1): 23-34. PubMed ID: 30554899
The plasticity of developing tissues relies on the adjustment of cell survival and growth rate to environmental cues. This includes the effect of mechanical cues on cell survival. Accordingly, compaction of an epithelium can lead to cell extrusion and cell death. This process was proposed to contribute to tissue homeostasis but also to facilitate the expansion of pretumoral cells through the compaction and elimination of the neighboring healthy cells. However, very little is known about the pathways that can trigger apoptosis upon tissue deformation, and the contribution of compaction-driven death to clone expansion has never been assessed in vivo. Using the Drosophila pupal notum and a new live sensor of ERK, it was shown first that tissue compaction induces cell elimination through the downregulation of epidermal growth factor receptor/extracellular signal regulated kinase (EGFR/ERK) pathway and the upregulation of the pro-apoptotic protein Hid. Those results suggest that the sensitivity of EGFR/ERK pathway to mechanics could play a more general role in the fine tuning of cell elimination during morphogenesis and tissue homeostasis. Second, the contribution of compaction-driven death to pretumoral cell expansion was assessed in vivo. The activation of the oncogene Ras in clones was found to downregulate ERK and activate apoptosis in the neighboring cells through their compaction, which eventually contributes to Ras clone expansion. The mechanical modulation of EGFR/ERK during growth-mediated competition for space may contribute to tumor progression.
Wang, L., Davis, S. S., Borch Jensen, M., Rodriguez-Fernandez, I. A., Apaydin, C., Juhasz, G., Gibson, B. W., Schilling, B., Ramanathan, A., Ghaemmaghami, S. and Jasper, H. (2019). JNK modifies neuronal metabolism to promote proteostasis and longevity. Aging Cell: e12849. PubMed ID: 30810280
Aging is associated with a progressive loss of tissue and metabolic homeostasis. This loss can be delayed by single-gene perturbations, increasing lifespan. How such perturbations affect metabolic and proteostatic networks to extend lifespan remains unclear. This question was addressed by comprehensively characterizing age-related changes in protein turnover rates in the Drosophila brain, as well as changes in the neuronal metabolome, transcriptome, and carbon flux in long-lived animals with elevated Jun-N-terminal Kinase signaling. These animals exhibit a delayed age-related decline in protein turnover rates, as well as decreased steady-state neuronal glucose-6-phosphate levels and elevated carbon flux into the pentose phosphate pathway due to the induction of glucose-6-phosphate dehydrogenase (G6PD). Over-expressing G6PD in neurons is sufficient to phenocopy these metabolic and proteostatic changes, as well as extend lifespan. This study identifies a link between metabolic changes and improved proteostasis in neurons that contributes to the lifespan extension in long-lived mutants.
Li, P., Ma, Z., Yu, Y., Hu, X., Zhou, Y. and Song, H. (2019). FER promotes cell migration via regulating JNK activity. Cell Prolif: e12656. PubMed ID: 31264309
Cell migration has a key role in cancer metastasis, which contributes to drug resistance and tumour recurrence. Better understanding of the mechanisms involved in this process will potentially reveal new drug targets for cancer therapy. Fer is a non-receptor protein tyrosine kinase aberrantly expressed in various human cancers, whereas its role in tumour progression remains elusive. Transgenic flies and epigenetic analysis were employed to investigate the role of Drosophila Fer (FER) in cell migration and underlying mechanisms. Co-immunoprecipitation assay was used to monitor the interaction between FER and Drosophila JNK (Bsk). The conservation of Fer in regulating JNK signalling was explored in mammalian cancer and non-cancer cells. Overexpression of FER triggered cell migration and activated JNK signalling in the Drosophila wing disc. Upregulation and downregulation in the basal activity of Bsk exacerbated and eliminated FER-mediated migration, respectively. In addition, loss of FER blocked signal transduction of the JNK pathway. Specifically, FER interacted with and promoted the activity of Bsk, which required both the kinase domain and the C-terminal of Bsk. Lastly, Fer regulated JNK activities in mammalian cells. This study reveals FER as a positive regulator of JNK-mediated cell migration and suggests its potential role as a therapeutic target for cancer metastasis.
Weavers, H., Wood, W. and Martin, P. (2019). Injury activates a dynamic cytoprotective network to confer stress resilience and drive repair. Curr Biol 29(22): 3851-3862 PubMed ID: 31668626
In healthy individuals, injured tissues rapidly repair themselves following damage. Within a healing skin wound, recruited inflammatory cells release a multitude of bacteriocidal factors, including reactive oxygen species (ROS), to eliminate invading pathogens. Paradoxically, while these highly reactive ROS confer resistance to infection, they are also toxic to host tissues and may ultimately delay repair. Repairing tissues have therefore evolved powerful cytoprotective "resilience" machinery to protect against and tolerate this collateral damage. This study used in vivo time-lapse imaging and genetic manipulation in Drosophila to dissect the molecular and cellular mechanisms that drive tissue resilience to wound-induced stress. A dynamic, cross-regulatory network of stress-activated cytoprotective pathways was identified, linking calcium, JNK, Nrf2, and Gadd45, that act to both "shield" tissues from oxidative damage and promote efficient damage repair. Ectopic activation of these pathways confers stress protection to naive tissue, while their inhibition leads to marked delays in wound closure. Strikingly, the induction of cytoprotection is tightly linked to the pathways that initiate the inflammatory response, suggesting evolution of a fail-safe mechanism for tissue protection each time inflammation is triggered. A better understanding of these resilience mechanisms-their identities and precise spatiotemporal regulation-is of major clinical importance for development of therapeutic interventions for all pathologies linked to oxidative stress, including debilitating chronic non-healing wounds.
Zhou, J. and Boutros, M. (2020). JNK-dependent intestinal barrier failure disrupts host-microbe homeostasis during tumorigenesis. Proc Natl Acad Sci U S A 117(17): 9401-9412. PubMed ID: 32277031
In all animals, the intestinal epithelium forms a tight barrier to the environment. The epithelium regulates the absorption of nutrients, mounts immune responses, and prevents systemic infections. This study investigate the consequences of tumorigenesis on the microbiome using a Drosophila intestinal tumor model. Upon loss of BMP signaling, tumors lead to aberrant activation of JNK/Mmp2 signaling, followed by intestinal barrier dysfunction and commensal imbalance. In turn, the dysbiotic microbiome triggers a regenerative response and stimulates tumor growth. Inhibiting JNK signaling or depletion of the microbiome restores barrier function of the intestinal epithelium, leading to a reestablishment of host-microbe homeostasis, and organismic lifespan extension. These experiments identify a JNK-dependent feedback amplification loop between intestinal tumors and the microbiome. They also highlight the importance of controlling the activity level of JNK signaling to maintain epithelial barrier function and host-microbe homeostasis.
von Frieling, J., Faisal, M. N., Sporn, F., Pfefferkorn, R., Nolte, S. S., Sommer, F., Rosenstiel, P. and Roeder, T. (2020). A high-fat diet induces a microbiota-dependent increase in stem cell activity in the Drosophila intestine. PLoS Genet 16(5): e1008789. PubMed ID: 32453733
Over-consumption of high-fat diets (HFDs) is associated with several pathologies. Although the intestine is the organ that comes into direct contact with all diet components, the impact of HFD has mostly been studied in organs that are linked to obesity and obesity related disorders. Drosophila was used as a simple model to disentangle the effects of a HFD on the intestinal structure and physiology from the plethora of other effects caused by this nutritional intervention. A HFD, composed of triglycerides with saturated fatty acids, was shown to trigger activation of intestinal stem cells in the Drosophila midgut. This stem cell activation was transient and dependent on the presence of an intestinal microbiota, as it was completely absent in germ free animals. Moreover, major components of the signal transduction pathway have been elucidated. In this study, JNK (basket) in enterocytes was necessary to trigger synthesis of the cytokine upd3 in these cells. This ligand in turn activated the JAK/STAT pathway in intestinal stem cells. Chronic subjection to a HFD markedly altered both the microbiota composition and the bacterial load. Although HFD-induced stem cell activity was transient, long-lasting changes to the cellular composition, including a substantial increase in the number of enteroendocrine cells, were observed. Taken together, a HFD enhances stem cell activity in the Drosophila gut and this effect is completely reliant on the indigenous microbiota and also dependent on JNK signaling within intestinal enterocytes.
Ugbode, C., Garnham, N., Fort-Aznar, L., Evans, G. J. O., Chawla, S. and Sweeney, S. T. (2020). JNK signalling regulates antioxidant responses in neurons. Redox Biol 37: 101712. PubMed ID: 32949970
Reactive oxygen species (ROS) are generated during physiological bouts of synaptic activity and as a consequence of pathological conditions in the central nervous system. How neurons respond to and distinguish between ROS in these different contexts is currently unknown. In Drosophila mutants with enhanced JNK activity, lower levels of ROS are observed and these animals are resistant to both changes in ROS and changes in synapse morphology induced by oxidative stress. In wild type flies, disrupting JNK-AP-1 signalling perturbs redox homeostasis suggesting JNK activity positively regulates neuronal antioxidant defense. This hypothesis was validated in mammalian neurons, finding that JNK activity regulates the expression of the antioxidant gene Srxn-1, in a c-Jun dependent manner. This study describes a conserved 'adaptive' role for neuronal JNK in the maintenance of redox homeostasis that is relevant to several neurodegenerative diseases.
Esteban-Collado, J., Corominas, M. and Serras, F. (2021). Nutrition and PI3K/Akt signaling are required for p38-dependent regeneration. Development 148(8). PubMed ID: 33913483
Regeneration after damage requires early signals to trigger the tissue repair machinery. Reactive oxygen species (ROS) act as early signals that are sensed by the MAP3 kinase Ask1, which in turn activates by phosphorylation the MAP kinases p38 and JNK. The sustained or high activation of these kinases can result in apoptosis, whereas short or low activation can promote regeneration. Using the Ask1-dependent regeneration program, this study demonstrated in Drosophila wing that PI3K/Akt signaling is necessary for Ask1 to activate p38, but not JNK. In addition, nutrient restriction or mutations that target Ser83 of the Drosophila Ask1 protein, a PI3K/Akt-sensitive residue, block regeneration. However, these effects can be reversed by the ectopic activation of p38, but not of JNK. These results demonstrate that Ask1 controls the activation of p38 through Ser83, and that the phosphorylation of p38 during regeneration is nutrient sensitive. This mechanism is important for discriminating between p38 and JNK in the cells involved in tissue repair and regenerative growth.
Joy, J., Barrio, L., Santos-Tapia, C., Romao, D., Giakoumakis, N. N., Clemente-Ruiz, M. and Milan, M. (2021). Proteostasis failure and mitochondrial dysfunction leads to aneuploidy-induced senescence. Dev Cell. PubMed ID: 34216545
Aneuploidy, an unbalanced number of chromosomes, is highly deleterious at the cellular level and leads to senescence, a stress-induced response characterized by permanent cell-cycle arrest and a well-defined associated secretory phenotype. This study used a Drosophila epithelial model to delineate the pathway that leads to the induction of senescence as a consequence of the acquisition of an aneuploid karyotype. Whereas aneuploidy induces, as a result of gene dosage imbalance, proteotoxic stress and activation of the major protein quality control mechanisms, near-saturation functioning of autophagy leads to compromised mitophagy, accumulation of dysfunctional mitochondria, and the production of radical oxygen species (ROS). This study uncovered a role of c-Jun N-terminal kinase (JNK) in driving senescence as a consequence of dysfunctional mitochondria and ROS. Activation of the major protein quality control mechanisms and mitophagy dampens the deleterious effects of aneuploidy, and this study has identified a role of senescence in proteostasis and compensatory proliferation for tissue repair.
Wagner, C., Uliczka, K., Bossen, J., Niu, X., Fink, C., Thiedmann, M., Knop, M., Vock, C., Abdelsadik, A., Zissler, U. M., Isermann, K., Garn, H., Pieper, M., Wegmann, M., Koczulla, A. R., Vogelmeier, C. F., Schmidt-Weber, C. B., Fehrenbach, H., Konig, P., Silverman, N., Renz, H., Pfefferle, P., Heine, H. and Roeder, T. (2021). Constitutive immune activity promotes JNK- and FoxO-dependent remodeling of Drosophila airways. Cell Rep 35(1): 108956. PubMed ID: 33826881
Extensive remodeling of the airways is a major characteristic of chronic inflammatory lung diseases such as asthma or chronic obstructive pulmonary disease (COPD). To elucidate the importance of a deregulated immune response in the airways for remodeling processes, a matching Drosophila model was established. Here, triggering the Imd (immune deficiency) pathway in tracheal cells induced organ-wide remodeling. This structural remodeling comprises disorganization of epithelial structures and comprehensive epithelial thickening. These structural changes do not depend on the Imd pathway's canonical branch terminating on nuclear factor κB (NF-κB) activation. Instead, activation of a different segment of the Imd pathway that branches off downstream of Tak1 and comprises activation of c-Jun N-terminal kinase (JNK) and forkhead transcription factor of the O subgroup (FoxO) signaling is necessary and sufficient to mediate the observed structural changes of the airways. These findings imply that targeting JNK and FoxO signaling in the airways could be a promising strategy to interfere with disease-associated airway remodeling processes.
Cong, B., Nakamura, M., Sando, Y., Kondo, T., Ohsawa, S. and Igaki, T. (2021). JNK and Yorkie drive tumor malignancy by inducing L-amino acid transporter 1 in Drosophila. PLoS Genet 17(11): e1009893. PubMed ID: 34780467.
Identifying a common oncogenesis pathway among tumors with different oncogenic mutations is critical for developing anti-cancer strategies. This study performed transcriptome analyses on two different models of Drosophila malignant tumors caused by Ras activation with cell polarity defects (RasV12/scrib-/-) or by microRNA bantam overexpression with endocytic defects (bantam/rab5-/-), followed by an RNAi screen for genes commonly essential for tumor growth and malignancy. Juvenile hormone Inducible-21 (JhI-21), a Drosophila homolog of the L-amino acid transporter 1 (LAT1), was identified is upregulated in these malignant tumors with different oncogenic mutations and knocking down of JhI-21 strongly blocked their growth and invasion. JhI-21 expression was induced by simultaneous activation of c-Jun N-terminal kinase (JNK) and Yorkie (Yki) in these tumors and thereby contributed to tumor growth and progression by activating the mTOR-S6 pathway. Pharmacological inhibition of LAT1 activity in Drosophila larvae significantly suppressed growth of RasV12/scrib-/- tumors. Intriguingly, LAT1 inhibitory drugs did not suppress growth of bantam/rab5-/- tumors and overexpression of bantam rendered RasV12/scrib-/- tumors unresponsive to LAT1 inhibitors. Further analyses with RNA sequencing of bantam-expressing clones followed by an RNAi screen suggested that bantam induces drug resistance against LAT1 inhibitors via downregulation of the TMEM135-like gene CG31157. These observations unveil an evolutionarily conserved role of LAT1 induction in driving Drosophila tumor malignancy and provide a powerful genetic model for studying cancer progression and drug resistance.
Gera, J., Budakoti, P., Suhag, M., Mandal, L. and Mandal, S. (2022). Physiological ROS controls Upd3-dependent modeling of ECM to support cardiac function in Drosophila. Sci Adv 8(7): eabj4991. PubMed ID: 35179958
Despite their highly reactive nature, reactive oxygen species (ROS) at the physiological level serve as signaling molecules regulating diverse biological processes. While ROS usually act autonomously, they also function as local paracrine signals by diffusing out of the cells producing them. Using in vivo molecular genetic analyses in Drosophila, this study provides evidence for ROS-dependent paracrine signaling that does not entail ROS release. Elevated levels of physiological ROS within the pericardial cells activate a signaling cascade transduced by Ask1, c-Jun N-terminal kinase, and p38 to regulate the expression of the cytokine Unpaired 3 (Upd3). Upd3 released by the pericardial cells controls fat body-specific expression of the extracellular matrix (ECM) protein Pericardin, essential for cardiac function and healthy life span. Therefore, this work reveals an unexpected inter-organ communication circuitry wherein high physiological levels of ROS regulate cytokine-dependent modulation of cardiac ECM with implications in normal and pathophysiological conditions.

For many years immunologists have held the view that the primary goal of the immune system is discrimination between self and non-self. Because Drosophila does not have an immunoglobulin-based immune response, the fly affords a unique opportunity to examine the nature of an immune system that evolved prior to the evolution of immunoglobulin as a primary driving force in an organism's response to pathogens. An examination of Drosophila suggests that an inducible system exists and it is based on the need to detect and protect against danger. The immunologist Matzinger (1994) has argued that such a system also remains functional as a controlling influence in the vertebrate immune response, in addition to the immunoglobulin-based system. The protein JNK is one of the pivotal actors in the Drosophila immune response, and appears to be functionally conserved in the vertebrate immune response as well. What does JNK do in both Drosophila and vertebrates, and what does this teach about the nature of the immune response?

Before taking a closer look at basket/JNK, a word on the pathway in which it functions is in order. Cascades of mitogen activated protein kinases (MAPKs) transduce signals from multiple extracellular stimuli, mediating responses such as cell proliferation, differentiation, and the regulation of metabolic pathways. There are multiple MAPKs in eukaryotes. In Drosophila, two such pathways have been identified. The first is a MAPK pathway involving Ras and Rolled, two components of the Ras pathway. It is required at least three times during development: as the terminal system which mediates responses to the Torso receptor, as neurogenic and wing vein pathways mediate responses to the EGF-receptor, and for the differentiation of photoreceptors, which mediate responses to the Sevenless receptor.

The second MAPK pathway in Drosophila involves the MAPK called Jun-N-terminal kinase (DJNK), also known as Basket. This pathway involves an additional kinase, Hemipterous, which serves to phosphorylate DJNK. Both proteins are required for dorsal closure, the sealing of the dorsal region of the embryo late in embryonic development. Dorsal closure involves coordinated changes in the shape of ectodermal cells, driven by cytoskeletal changes. (For a discussion of dorsal closure see Hemipterous). The phosphorylation cascade involving HEP and DJUN terminates in the phosphorylation of DJUN. Both DJNK and DJUN are activated in the the immune response of the fly. DJNK is closely associated on chromosome 2 with dror, a Drosophila receptor tyrosine kinase homolog of vertebrate TRK kinases, which mediate responses to neurotropins. Close chromosomal linkage often, but not always, occurs as a consequence of shared developmental function.

Bacterial infection causes insect immune tissues to synthesize a spectrum of antibacterial peptides, including Cecropin and Diptericin, which lyse the invading microorganisms (Hultmark, 1993 and Peterson, 1995). Exposure of Drosophila to environmental stress (such as a pathogenic onslaught) activates two groups of transcription factors: AP-1 (coded for by Drosophila jun and fos) and two related proteins; Dorsal and Dorsal related factor (Dif).

Dorsal and Dif are homologs of NFkappaB (also known as Rel), an essential transcription factor involved in the vertebrate immune response (Ip, 1993). Both Dif and Dorsal act in the insect immune defense against bacterial infection (see Dorsal for more information).

Drosophila JNK is activated by endotoxic lipopolysaccharide (LPS). LPS is a component of bacterial cell walls, and is known to be a stimulant for the immune response in both insects and mammals. The response of Drosophila to bacterial infection can be studied by examining the effect of LPS on cultured cell lines. Addition of LPS to cultured cell lines, including mbn-2 hemocytes and Schneider S2 embryonic cells, causes marked induction of cecropin and diptercin genes. basket is activated within 5 minutes of LPS addition. However, basket activation is transient and returns to basal levels after 1 hour. The activation of DJun by DJNK in LPS-treated cells may lead to increased AP-1 (a heterodimer of DFos and DJun) transcriptional activity. Targets of Drosophila AP-1 may include the DJun promoter (Sluss, 1996).

Mammalian JNK has been implicated in a variety of immune-related signaling pathways, including the response to pro-inflammatory cytokines, the respone to LPS, and T-cell activation (Su, 1994, Westwick, 1994 and 1995 and Raingeaud, 1995). The role of JNK and of Rel proteins in the vertebrate immune response harkens back to the roles of these proteins in Drosophila: both proteins are involved in systems that have developed to detect and protect against danger. When viewed from the perspective of signal transduction cascades and transcription factors it is clear that at its core, the vertebrate immune response system is one which detects and protects against danger. This conclusion is made clearer by looking at the roles of JNK and Rel in vertebrate responses to danger and in the related immune responses (see the basket/JNK Evolutionary homologs section).

Regulation of JNK by Src during Drosophila development

Basket (Bsk) is regulated by Src during dorsal closure. Mutants for Src42A, a Drosophila c-src protooncogene homolog, are described. Src42A functions in epidermal closure during both embryogenesis and metamorphosis. The severity of the epidermal closure defect in the Src42A mutant depends on the amount of Bsk activity, and the amount of Bsk activity depends on the amount of Src42A. Thus, activation of the Bsk pathway is required downstream of Src42A in epidermal closure. This work confirms mammalian studies that demonstrate a physiological link between Src and JNK (Tateno, 2000).

Genes that regulate cell shape changes in Drosophila are required for dorsal closure of the embryonic epidermis and thorax closure of the pupal epidermis. Mutations in genes such as hemipterous (hep) and basket (bsk, also known as DJNK) result in abnormal embryos with a dorsal hole or abnormal adults with a dorsal midline cleft. Hep and Bsk are homologous to the mammalian MKK7 (MAPK kinase 7) and JNK, and they are components of a MAPK (mitogen-activated protein kinase) cascade. Although the role of the Hep-Bsk cascade during dorsal closure has been extensively studied, the upstream trigger of this cascade is poorly understood. To identify the trigger, a screen was carried out for mutants showing the dorsal midline cleft phenotype, like a mild hep mutant. The mutant for Src42A shows this phenotype and Src42A regulates Bsk during Drosophila development (Tateno, 2000).

One line, Jp45, from the mutant collection of the P-element-inserted semilethal lines has been identified that survives to adulthood but shows various degrees of the dorsal midline cleft phenotype. Excision of the P-element eliminates the semilethality and restores the cleft phenotype. The P-element is inserted in the 5' untranslated region (UTR) of the Src42A gene, which encodes a Src-family nonreceptor tyrosine kinase. Ethyl methanesulfonate (EMS) mutant screening was used to isolate two strong alleles of Src42A, Src42AE1 and Src42Amyristylation (myri). In Src42AE1, a stop codon at codon 483 eliminates the COOH-terminal part of the kinase domain of Src42A. Src42Amyri has a point mutation in codon 2, which causes an amino acid substitution from Gly2 to Asp. Gly2 is conserved in all members of the Src family and must be myristylated for localization of Src to the cellular membrane in mammals. About 50% of the Src42Amyri homozygotes die before they hatch, and most of the remainder die during the first-instar larval stage. Therefore, Gly2 is required for development (Tateno, 2000).

Because the adult Src42AJp45 phenotype resembles that of hep, it was suspected that Src42A is involved in Hep and Bsk function. A mutation in hep or bsk dominantly enhances the lethality and the phenotypic severity of Src42AJp45 homozygotes. Conversely, reducing the gene dosage of puckered (puc), a gene encoding a phosphatase that inactivates Bsk, suppresses the lethality and the severity of the cleft phenotype of Src42AJp45. Thus, Src42A may function in the Bsk pathway during metamorphosis (Tateno, 2000).

Dorsal closure is the process by which a pair of epidermal layers elongate dorsally and fuse at the dorsal midline of the embryo. This process is not completed in hep and bsk mutants, yielding a dorsal open phenotype. Strong Src42A mutants do not show the dorsal open phenotype but display malformed mouth parts. This defect is similar to the defect in the embryo of the Tec29 mutant. Tec29 is a Src-related nonreceptor tyrosine kinase and is regulated by SRC64, another Drosophila Src homolog, during oogenesis. Thus, Tec29 may be involved in the function of Src42A. A mutation in Tec29 dominantly enhances the lethality of Src42AJp45 (Tateno, 2000).

Furthermore, the Tec29 Src42A double mutant shows complete embryonic lethality, and a certain fraction of the dead embryos show the dorsal open phenotype. Activated DJun, a transcription factor downstream of Bsk, partially rescues the dorsal open phenotype in the Tec29 Src42A double mutant. Thus, Src42A appears to regulate Bsk in the fusion of epithelial sheets during embryogenesis and metamorphosis, and Tec29 is involved in this regulation. The double mutant for Src64 and Src42A manifests a mild but clear dorsal open phenotype, which suggests a functional redundancy between Src64 and Src42A (Tateno, 2000).

Expression of puc is known to be induced by the Bsk signal. In the wing disc of the wild-type third-instar larva, puc is expressed in the dorsal midline of the adult notum. In the wing disc of the Src42AJp45 mutant, puc expression is reduced. In contrast, larvae with a constitutively activated form of Src42A (Src42ACA) shows ectopic expression of puc. Further, introduction of a hep null mutation reduces the amount of ectopic puc expression. It is known that Bsk induces expression of puc and decapentaplegic (dpp) during embryonic dorsal closure. The embryos of the Tec29 Src42A double mutant do not show any puc or dpp expression in the leading edge cells. These results indicate that Src42A, Tec29, Hep, and Bsk regulate dpp and puc expression during embryonic dorsal closure (Tateno, 2000).

To investigate the ability of Src42ACA to activate Bsk, the amount of phosphorylated Bsk was directly assessed by immunoblot analysis. Forced expression of Src42ACA does not affect the quantity of total Bsk protein but induces more phosphorylated Bsk than the controls. Thus, Src42A appears to regulate the phosphorylation level of Bsk (Tateno, 2000).

During embryonic dorsal closure, the Hep-Bsk signal is required for elongation of the leading edge cells. In the absence of the Bsk signal, these cells do not fully elongate. The accumulation of F-actin and phosphotyrosine (P-Tyr) in leading edge cells is associated with the elongation of these cells. Accumulation of these substances is disturbed in the DJun and the puc mutants. In the double mutant for Tec29 and Src42A, the leading edge cells contain reduced quantities of F-actin and P-Tyr, and these cells are only partially elongated. Thus, the defect in embryonic dorsal closure in the Tec29 Src42A double mutant is caused by this failure in cell shape change, as is the case in the DJun mutant (Tateno, 2000).

A model is proposed in which Src42A, upon receiving an unidentified signal, activates the Hep-Bsk pathway to regulate cell shape change and epidermal layer movement. This is consistent with the observation in mammals that c-Src regulates the cell morphogenetic and migratory processes and is known to activate JNK. As in Drosophila, c-Src definitely affects F-actin organization and P-Tyr localization during cell morphogenesis. Therefore, Src regulation of JNK activity toward a change in cell shape may be conserved (Tateno, 2000).

It can be also interpreted that Src42A acts upstream of DFos, a dimerization partner of DJun. Although the Src42A, Tec29, and Src64 single mutants do not show a dorsal open phenotype, the DFos mutant clearly exhibits it. This relationship is also analogous to that in mammals. Both c-src and c-fos knockout mice have a similar defect, osteopetrosis caused by reduced osteoclast function. But the phenotypic severity is milder in c-src than in c-fos knockouts; this can be explained by the functional overlap in multiple Src-family tyrosine kinases. Accordingly, in both Drosophila and mammals, multiple nonreceptor tyrosine kinases may cooperate to regulate the function of the Jun/Fos complex (Tateno, 2000).

Cell competition modifies adult stem cell and tissue population dynamics in a JAK-STAT-dependent manner

Throughout their lifetime, cells may suffer insults that reduce their fitness and disrupt their function, and it is unclear how these potentially harmful cells are managed in adult tissues. This question was addressed using the adult Drosophila posterior midgut as a model of homeostatic tissue and ribosomal Minute mutations to reduce fitness in groups of cells. A quantitative approach was taken, combining lineage tracing and biophysical modeling, and how cell competition affects stem cell and tissue population dynamics was addressed. Healthy cells were shown to induce clonal extinction in weak tissues, targeting both stem and differentiated cells for elimination. It was also found that competition induces stem cell proliferation and self-renewal in healthy tissue, promoting selective advantage and tissue colonization. Finally, winner cell proliferation was shown to be fueled by the JAK-STAT ligand Unpaired-3, produced by Minute-/+ cells in response to chronic JNK stress signaling (Kolahgar, 2015).

Recent studies have shown that cell competition can also take place in adult tissues. This work has taken this notion forward and delineated quantitatively how adult stem cells and tissue population dynamics are affected by cell competition. In the subfit population, differentiated cells are killed by apoptosis followed by cell delamination; stem cells are also eliminated, possibly via induction of differentiation, as dying stem cells have not been detected. In parallel, as this study has shown, the healthy tissue expands due to an increase in stem cell proliferation and self-renewal. Indeed, biophysical modeling shows that changes in these parameters of a magnitude comparable to what observed experimentally is sufficient to recapitulate the stem cell dynamics of wild-type tissue undergoing Minute cell competition. Interestingly, accelerated proliferation of fitter stem cells has been seen in mouse embryonic stem cells using in vitro models of cell competition. However, in those studies, increased stem cell self-renewal has not been observed, probably because stemness in vitro is artificially maintained by exogenous factors in the culture medium (Kolahgar, 2015).

In many adult homeostatic tissues, stem cells stochastically differentiate or self-renew, and this leads to clonal extinction balanced by clonal expansion. This is known as neutral drift competition, because through this process, stem cell compartments stochastically tend toward monoclonality. It has also been shown that stem cell competition can be nonneutral (i.e., biased) when stem cells acquire a cell-autonomous advantage. In these cases, the bias derives from intrinsic differences (e.g., faster proliferation) and does not rely on cell interactions. This study shows instead that in adult homeostatically maintained tissues, competitive cell interactions can act as extrinsic cues that actively modify stem cell behavior, and that this confers on winners an advantage (e.g., as this study observed, increased proliferation rate and self-renewal) and on losers a disadvantage (e.g., as observed induced cell death), influencing tissue colonization. It is important to note that clones of wild-type cells that have lost proliferative capability because they are devoid of ISCs are equally able to induce death in neighboring M/+cells. This rules out the possibility that physical displacement due to a faster clonal expansion is the cause of cell competition in this case. This process instead, like the recent reports of cell competition in the mouse heart and fly nervous system, likely corresponds to the adult equivalent of the cellular competition observed in developing tissues (Kolahgar, 2015).

This work shows that M/+ midguts suffer from a chronic inflammatory response, which through JNK signaling activation and the ensuing production of the JAK-STAT ligand Upd-3 promotes wild-type tissue overgrowth. Thus, in this tissue, the overproliferation of winner cells stems from the increased availability of proliferative signals in the M/+ environment. The results suggest that wild-type cells respond more efficiently than M/+ cells to this proliferation stimulus, and that this difference results in their preferential overgrowth, contributing to cell competition. It has long been suggested that cell competition may result from the limiting availability of growth factors, which would compromise the viability of loser cells. Here it was instead found that excess production of a growth factor (Upd-3) can boost cell competition by promoting preferential proliferation of fitter cells. Given that JNK and JAK-STAT are frequently activated in response to stress or deleterious mutations, it would be interesting to test whether this is a general mechanism used by loser cells to promote the overgrowth of fitter neighbors. Notably, differences in JAK-STAT signaling are sufficient to trigger cell competition and, consistent with this, reducing JAK-STAT signaling in wild-type cells compromises their ability to eliminate scribble/losers. Thus, increased JAK-STAT signaling may in addition provide wild-type cells with a heightened fitness state and help promote the elimination of M/losers (Kolahgar, 2015).

Ribosomal mutations are linked with many adult disorders, not just in Drosophila but more importantly in humans, where they are associated with a number of severe pathologies, collectively known as ribosomopathies. Given that 79 proteins make up the eukaryotic ribosome (and several more are involved in ribosomal production) and that many Minute mutations are dominant, the sporadic insurgence of M/+ in adult tissues is likely to be one of the most common spontaneous generations of somatic mutant cells in our bodies. The elimination of these cells via cell competition is likely to play an unappreciated role in maintaining healthy adult tissues (Kolahgar, 2015).

A striking feature emerging from the results is that, in response to cell competition, normal cells can efficiently repopulate adult tissues, thus effectively replacing potentially diseased cells. This bears striking resemblance to the phenomenon of mosaic revertants, observed in a number of human skin and blood diseases. Spontaneous sporadic reversion of genetically inherited, disease-bearing mutations leads to the generation of revertant cells, which effectively repopulate tissues, at times ameliorating the condition. In some instances, the revertants' expansion is so efficient that selective advantage has been. Intriguingly, ichthyosis with confetti, a skin disease characterized by confetti-like appearance of revertant skin spots, is associated with a mutation in Keratin 10, which, due to its nucleolar mislocalization, could affect ribosome production similar to M-/+mutants. Thus, based on these findings, it is tentative to speculate that selective advantage in mosaic revertants could in some cases be driven by cell competition (Kolahgar, 2015).

The work shows that M/+ midguts suffer from a chronic inflammatory response, which through JNK signaling activation and the ensuing production of the JAK-STAT ligand Upd-3 promotes wild-type tissue overgrowth. Thus, in this tissue, the overproliferation of winner cells stems from the increased availability of proliferative signals in the M/+ environment. The results suggest that wild-type cells respond more efficiently than M/+ cells to this proliferation stimulus, and that this difference results in their preferential overgrowth, contributing to cell competition. It has long been suggested that cell competition may result from the limiting availability of growth factors, which would compromise the viability of loser cells. This study found instead that excess production of a growth factor (Upd-3) can boost cell competition by promoting preferential proliferation of fitter cells. Given that JNK and JAK-STAT are frequently activated in response to stress or deleterious mutations, it would be interesting to test whether this is a general mechanism used by 'loser' cells to promote the overgrowth of fitter neighbors. Notably, differences in JAK-STAT signaling are sufficient to trigger cell competition and, consistent with this, reducing JAK-STAT signaling in wild-type cells compromises their ability to eliminate scribble/ losers. Thus, increased JAK-STAT signaling may in addition provide wild-type cells with a heightened fitness state and help promote the elimination of M/+ losers (Kolahgar, 2015).

Ribosomal mutations are linked with many adult disorders, not just in but more importantly in humans, where they are associated with a number of severe pathologies, collectively known as ribosomopathies. Given that 79 proteins make up the eukaryotic ribosome (and several more are involved in ribosomal production) and that many Minute mutations are dominant, the sporadic insurgence of M/+ cells in adult tissues is likely to be one of the most common spontaneous generations of somatic mutant cells in our bodies. The elimination of these cells via cell competition is likely to play an unappreciated role in maintaining healthy adult tissues (Kolahgar, 2015).

A striking feature emerging from the current results is that, in response to cell competition, normal cells can efficiently repopulate adult tissues, thus effectively replacing potentially diseased cells. This bears striking resemblance to the phenomenon of mosaic revertants, observed in a number of human skin and blood diseases. Spontaneous sporadic reversion of genetically inherited, disease-bearing mutations leads to the generation of revertant cells, which effectively repopulate tissues, at times ameliorating the condition. In some instances, the revertants' expansion is so efficient that selective advantage has been proposed. Intriguingly, ichthyosis with confetti, a skin disease characterized by confetti-like appearance of revertant skin spots, is associated with a mutation in Keratin 10, which, due to its nucleolar mislocalization, could affect ribosome production similar to M/+ mutants. Thus, based on the current findings, it is tentative to speculate that selective advantage in mosaic revertants could in some cases be driven by cell competition (Kolahgar, 2015).

Tankyrase mediates K63-linked ubiquitination of JNK to confer stress tolerance and influence lifespan in Drosophila
Tankyrase (Tnks) transfers poly(ADP-ribose) on substrates. Whereas studies have highlighted the pivotal roles of Tnks in cancer, cherubism, systemic sclerosis, and viral infection, the requirement for Tnks under physiological contexts remains unclear. This study report that the loss of Tnks or its muscle-specific knockdown impairs lifespan, stress tolerance, and energy homeostasis in adult Drosophila. Tnks is a positive regulator in the JNK signaling pathway, and modest alterations in the activity of JNK signaling can strengthen or suppress the Tnks mutant phenotypes. JNK was identified as a direct substrate of Tnks. Although Tnks-dependent poly-ADP-ribosylation is tightly coupled to proteolysis in the proteasome, it was demonstrated that Tnks initiates degradation-independent ubiquitination on two lysine residues of JNK to promote its kinase activity and in vivo functions. This study uncovers a type of posttranslational modification of Tnks substrates and provides insights into Tnks-mediated physiological roles (Li, 2018).

Tankyrase (Tnks) belongs to the poly(ADP-ribose) polymerase (PARP) superfamily, which consists of 17 members in human. PARP-1 is the founding member of the family and has a critical role in the repair of DNA damage. The PARPs are characterized by a structurally similar PARP catalytic domain that successively transfers ADP-ribose from NAD+ onto substrate proteins. This post-translational modification is referred to as poly-ADP-ribosylation, or PARsylation. It has been shown that some PARPs actually catalyze mono-ADP-ribosylation rather than the polymerization of poly(ADP-ribose) chains. Because of this reason, the PARP family members have been renamed as ADP-ribosyltransferases diphtheria toxin-like (ARTDs). Tnks was identified as a telomere-associated protein that binds to the telomere-specific DNA binding protein TRF1. In addition to the PARP domain, Tnks contains two unique domains that distinguish it from other PARPs, including an Ankyrin repeat domain that is involved in the recruitment of substrate and a sterile alpha motif (SAM) that mediates oligomerization. Tnks is evolutionarily conserved in human, mouse, rat, chicken, C. elegans, and Drosophila. The human and mouse genome encodes two Tnks proteins (Tnk1/PARP5A/ARTD5 and Tnk2/PARP5B/ARTD6), whereas there is only one Tnks homolog in Drosophila (Li, 2018).

Tnks catalyzes PARsylation on its substrates and is involved in a variety of cellular processes, such as telomere homeostasis, cell-cycle progression, Wnt/β-catenin signaling, PI3K signaling, Hippo signaling, glucose metabolism, stress granule formation, and proteasome regulation. Various amino acid residues including lysine, arginine, aspartate, glutamate, asparagine, cysteine, phospho-serine, and diphthamide may serve as acceptors for PARsylation. In many cases, proteins modified by Tnks are subsequently poly-ubiquitinated and targeted for proteasomal degradation. For example, Tnks promotes telomere elongation by mediating the degradation of TRF1, a negative regulator of telomere length maintenance. Tnks PARsylates the β-catenin destruction complex component Axin, triggers its degradation, and thereby activates Wnt signaling. Tnks also regulates the stability of centrosomal P4.1-associated protein CPAP to limit centriole elongation during mitosis. On the other hand, the effects of PARsylation on some Tnks substrates, such as the nuclear mitotic apparatus protein NUMA, have not been elucidated, implying that alternative regulation following PARsylation may exist (Li, 2018).

Aberrant Tnks expression or activity has been implicated in a diversity of diseases including cancer, systemic sclerosis, cherubism, Herpes simplex, and Epstein-Barr viral infections. Particularly, the pro-oncogenic role of Tnks in many types of cancer strongly suggests a therapeutic benefit of Tnks inhibition. Whereas a substantial amount of studies have highlighted the important functions of Tnks under pathological conditions, the requirement for Tnks under physiological contexts is largely unexplored (Li, 2018).

This study investigate the physiological functions of Tnks during the adult stage in Drosophila with its mutant alleles and RNAi strains. The loss of Tnks was shown to impair lifespan, stress tolerance, and energy storage in adult flies. Ubiquitous or muscle-specific knockdown of Tnks causes defects similar to those observed in the mutant alleles. It was further shown that Tnks is specifically required for the activity of the c-Jun N-terminal kinase (JNK) and positively regulates the outputs of JNK signaling during organ development. In addition, mild reduction and slight increase in the activity of JNK signaling via genetic manipulations can strengthen and suppress the phenotypes of the Tnks mutants, respectively. Last, the results reveal for the first time that Tnks mediates degradation-independent ubiquitination on its substrate. Drosophila JNK was identified as a direct substrate protein of Tnks. The PARsylation by Tnks triggers K63-linked poly-ubiquitination on JNK, enhancing its kinase activity and maintaining its in vivo functions. Together, this study uncovers that Tnks is a positive regulator of JNK activity, mediates a novel type of post-translational modification, and functions through JNK signaling to affect lifespan, stress resistance, and energy homeostasis in Drosophila (Li, 2018).

PARsylation by Tnks appears to be tightly coupled to poly-ubiquitination and subsequent proteasome-dependent degradation, as observed for known Tnks substrates including Axin, TRF1, PTEN, 3BP2, CPAP, and AMOT family proteins. This study found that Tnks mediated PARsylation and poly-ubiquitination of Drosophila JNK (Bsk), however, without affecting its protein levels. This observation prompted investigation og the form of poly-ubiquitin chain assembled on Bsk in the presence of Tnks. Although all the seven lysine residues (K6, K11, K27, K29, K33, K48, and K63) and the N-terminal methionine residue in the ubiquitin can serve as the attachment site to generate poly-ubiquitin chain, K48-linked and K63-linked poly-ubiquitination are two predominant forms in cells. It is well-established that K48-linked poly-ubiquitination targets substrates to the 26S proteasome and promotes protein degradation, while K63-linked poly-ubiquitin chain performs non-proteolytic functions. Consistently, this study observed that Tnks promoted the assembly of K63-linked poly-ubiquitin chain on Bsk and increased its kinase activity. In contrast, Tnks did not affect K48-linked poly-ubiquitination of Bsk. Although the possibility cannot be completely that these modifications occur on a Bsk partner that tightly binds to Bsk even after stringent washing, this study reveals for the first time that Tnks mediates degradation-independent ubiquitination of its substrate, which may represent a novel type of modification induced by PARsylation. Indeed, it has been reported that PARsylation of some Tnks substrates such as the proteasome regulator PI31 and the mitotic spindle-pole protein NuMA may affect the activities of these proteins. It will be interesting to investigate whether similar modification occurs during these processes (Li, 2018).

JNK is an evolutionarily conserved stress-activated protein kinase (SAPK) and is one of the most versatile stress sensors in metazoans. The JNK signaling pathway adapts growth and metabolism to environmental conditions, and mediates stress tolerance, damage repair, and apoptosis. Therefore, JNK signaling has pivotal roles in regulating homeostasis, longevity, and organ development. This study reports an essential role of Tnks in regulating JNK signaling. As an initial hint, adult Tnks mutants are more sensitive to oxidative stress and to starvation, and live much shorter than the wild-type controls. Although several signaling pathways are known to coordinate these functions in Drosophila, this study observed that the loss of Tnks specifically decreased the activity of Bsk without affecting that of ERK, p38, Akt, and Nrf2 signaling. It was further found that Tnks was required for the outputs of JNK signaling during the development of eye, wing, and thorax. The knockdown of Tnks in the thorax strengthened the thoracic cleft phenotype caused by bsk knockdown. The loss of Tnks, or reducing its gene dosage, suppressed ectopic JNK signaling-induced phenotypes during the formation of compound eye and wing vein. In addition, Tnks overexpression in the wing disc activated puc expression in a bsk-dependent manner. Last, it was observed that bsk with mutations in Tnks-induced ubiquitination sites lacked the ability to rescue the lethality of the bsk1/2 mutant and displayed impaired activity in regulating stress tolerance, climbing, energy storage, and organ development compared to wild-type bsk. This work thus reveals Tnks as a positive regulator of the JNK signaling pathway (Li, 2018).

Aberrant Tnks expression or activity has been implicated in a variety of disease states including cancer, cherubism, systemic sclerosis, and viral infection. The proposed roles of Tnks in telomere homeostasis, mitosis, and Wnt signaling have made it an attractive drug target in many types of cancer. Tnks is implied in a diversity of additional cellular processes, such as glucose metabolism, stress granule assembly, and proteasome regulation. However, the requirement for Tnks under physiological contexts remains poorly understood. While a double knockout of Tnks1 and Tnk2 is embryonically lethal in mice, Tnks mutant flies do not display any noticeable developmental defects. Although a previous study reported that Tnks-deficient mice exhibited substantially reduced adiposity, the underlying molecular mechanism is unclear. This work has focused on the physiological changes in Tnks mutant flies during the adult stage. It is reported that the loss of Tnks or its knockdown shortens lifespan, decreases climbing ability, reduces resistance to oxidative stress and starvation, and impairs energy storage in adult flies. Through tissue-specific knockdown, it is concluded that the above functions of Tnks are mainly mediated via its activity in the muscle. These physiological functions of Tnks are attributed to its regulation of JNK signaling activity, which is supported by several lines of evidence. First, the loss of Tnks specifically weakens Bsk activity and JNK signaling. Second, mild reduction in JNK signaling activity by removing one copy of bsk gene aggravates the phenotypes in Tnks mutants, whereas slightly elevated JNK signaling largely rescues these phenotypes. Third, this study shows that Tnks triggers PARsylation and K63-linked poly-ubiquitination of Bsk and enhances the kinase activity and in vivo functions of Bsk (Li, 2018).

JNK and Yorkie drive tumor progression by generating polyploid giant cells in Drosophila

Epithelial cancer tissues often possess polyploid giant cells, which are thought to be highly oncogenic. However, the mechanisms by which polyploid giant cells are generated in tumor tissues and how such cells contribute to tumor progression remain elusive. Cells mutant for the endocytic gene rab5 in Drosophila imaginal epithelium exhibit enlarged nuclei. This study finds that mutations in endocytic 'neoplastic tumor-suppressor' genes, such as rab5, vps25, erupted, or avalanche result in generation of polyploid giant cells. Genetic analyses on rab5-defective cells reveal that cooperative activation of JNK and Yorkie generates polyploid giant cells via endoreplication. Mechanistically, Yorkie-mediated upregulation of Diap1 cooperates with JNK to downregulate the G2/M cyclin CycB, thereby inducing endoreplication. Interestingly, malignant tumors induced by Ras activation and cell polarity defect also consist of polyploid giant cells, which are generated by JNK and Yorkie-mediated downregulation of CycB. Strikingly, elimination of polyploid giant cells from such malignant tumors by blocking endoreplication strongly suppressed tumor growth and metastatic behavior. These observations suggest that JNK and Yorkie, two oncogenic proteins activated in many types of human cancers, cooperatively drive tumor progression by generating oncogenic polyploid giant cells (Cong, 2018).

Polyploid giant cells, which contain multiples of the diploid genome equivalents, are often observed in human cancer tissues. Such polyploidy can be generated by endoreplication, a cell cycle variation that gives rise to genomic contents by replicating DNA in the absence of cell division. Polyploid giant cancer cells were shown to be more tumorigenic than normal diploid cancer cells. However, the mechanisms by which polyploid giant cells are generated in tumors and how they contribute to tumor progression remain elusive (Cong, 2018).

In Drosophila imaginal epithelia, loss-of-function mutations in the endocytic genes, such as rab5, vps25, erupted (ept), or avalanche (avl) cause neoplastic tissue overgrowth and therefore these genes are called 'neoplastic tumor-suppressors'. Previously found that cells deficient for Rab5, a small GTPase essential for generating early endosomes, induce non-autonomous overgrowth of surrounding tissue when induced as mosaic clones in the imaginal disc (Takino, 2014). Mechanistically, loss-of-Rab5 causes activation of Eiger (a tumor necrosis factor (TNF) homolog)-JNK signaling and EGFR-Ras signaling, which cooperate together to activate the Hippo pathway effector Yorkie (Yki, a YAP homolog), leading to upregulation of a secreted growth factor Unpaired (Upd, an IL-6 homolog) (Takino, 2014). Intriguingly, this study also noticed that such Rab5-deficient cells exhibited enlarged nuclei, which was suppressed by inhibition of JNK signaling, Ras signaling, or Yki activity, although the underlying mechanisms and its function have been unknown (Cong, 2018).

This study also analyzed Rab5-defective cells in detail and found that rab5 mutation generates polyploid giant cells through endoreplication. Genetic analyses reveal that JNK and a Yki-target Diap1 (Drosophila inhibitor-of-apoptosis protein 1) cooperate to induce endoreplication in Rab5-defective cells via downregulation of the G2/M cyclin CyclinB (CycB). Furthermore, this study also showed that generation of such polyploid giant cells is essential for tumor growth and metastasis in a Drosophila model of malignant tumors bearing Ras activation and cell polarity defect (Cong, 2018).

Serpin facilitates tumor-suppressive cell competition by blocking Toll-mediated Yki activation in Drosophila

Normal epithelial tissue exerts an intrinsic tumor-suppressive effect against oncogenically transformed cells. In Drosophila imaginal epithelium, clones of oncogenic polarity-deficient cells mutant for scribble (scrib) or discs large (dlg) are eliminated by cell competition when surrounded by wild-type cells. In this study, a genetic screen in Drosophila identified Serpin5 (Spn5), a secreted negative regulator of Toll signaling, as a crucial factor for epithelial cells to eliminate scrib mutant clones from epithelium. Downregulation of Spn5 in wild-type cells leads to elevation of Toll signaling in neighboring scrib cells. Strikingly, forced activation of Toll signaling or Toll-related receptor (TRR) signaling in scrib clones transforms scrib cells from losers to supercompetitors, resulting in tumorous overgrowth of mutant clones. Mechanistically, Toll activation in scrib clones leads to c-Jun N-terminal kinase (JNK) activation and F-actin accumulation, which cause strong activation of the Hippo pathway effector Yorkie that blocks cell death and promotes cell proliferation. These data suggest that Spn5 secreted from normal epithelial cells acts as a component of the extracellular surveillance system that facilitates elimination of pre-malignant cells from epithelium (Katsukawa, 2018).

Clones of oncogenic polarity-deficient cells are actively eliminated from Drosophila imaginal epithelium when surrounded by normal tissue, indicating the existence of intrinsic tumor-suppression mechanism by cell competition. The present study shows that normal epithelial cells secrete Spn5 to facilitate the tumor-suppressive cell competition by antagonizing Toll signaling activation in polarity-deficient cells. Elevation of Toll signaling in polarity-deficient cells transforms them from losers to supercompetitors, which leads to tumorous overgrowth of mutant tissue. Thus, Spn5 acts as a component of the extracellular surveillance system that eliminates oncogenic cells by cell competition. It is not known at this stage why scrib cells are more sensitive to loss of spn5 to upregulate Toll signaling compared to surrounding wild-type cells. One possible mechanism that drives Toll activation in scrib cells would be JNK activation, which was shown to be sufficient to activate Toll signaling (Katsukawa, 2018).

Interestingly, it has been shown that activation of TRR signaling in losers of Myc- or Minute-induced cell competition causes losers' death through nuclear factor κB (NF-κB)-mediated induction of cell death gene hid or rpr, respectively. Consistent with this report, it has been shown in Drosophila larval fat bodies that activation of Toll signaling leads to inactivation of Yki, which may cause hid- or rpr-mediated cell death because one of the important Yki targets is a caspase inhibitor diap1. These observations intriguingly indicate that Toll signaling has opposite roles in different types of cell competition; while Toll activation promotes elimination of losers in Myc- or Minute-induced cell competition, it suppresses elimination of polarity-deficient losers in tumor-suppressive cell competition. Importantly, however, in both cases, Toll or TRR signaling seems to act as an oncogenic signaling that promotes expansion of pre-malignant winner clones within the tissue. Consistent with these findings in Drosophila, it has been reported that upregulation of Toll-like receptors (TLRs) is associated with tumor growth and progression in some human cancers. In addition, one of the human orthologs of Drosophila Spn5, SpnA5, has been shown to inhibit breast cancer growth and metastasis, and its expression level is decreased in renal cell carcinoma and sarcoma. These observations, together with the data from Drosophila genetics, suggest that Toll signaling drives tumorigenesis by promoting supercompetition of oncogenic cell clones (Katsukawa, 2018).

The mechanism by which Toll activation in polarity-deficient cells leads to Yki activation is an important open question for future studies. One possible mechanism is co-activation of JNK and Ras signaling in Toll-activated scrib cells, as these two pathways have shown to cooperate to induce Yki activation through F-actin accumulation and Wts inactivation. Interestingly, it has been shown in mammalian systems that the TLR signaling activates JNK signaling and that several TLRs activate EGFR-Ras signaling upon immune response. Given that signaling molecules identified in Drosophila are all conserved, similar Toll-mediated regulation of tumorigenesis could be involved in human cancer (Katsukawa, 2018).

Long-term memory engram cells are established by c-Fos/CREB transcriptional cycling

Training-dependent increases in c-fos have been used to identify engram cells encoding long-term memories (LTMs). However, the interaction between transcription factors required for LTM, including CREB and c-Fos, and activating kinases such as phosphorylated ERK (pERK) in the establishment of memory engrams has been unclear. Formation of LTM of an aversive olfactory association in flies requires repeated training trials with rest intervals between trainings. This study finds that prolonged rest interval-dependent increases in pERK induce transcriptional cycling between c-Fos and CREB in a subset of KCs in the mushroom bodies, where olfactory associations are made and stored. Preexisting CREB is required for initial c-fos induction, while c-Fos is required later to increase CREB expression. Blocking or activating c-fos-positive engram neurons inhibits memory recall or induces memory-associated behaviors. These results suggest that c-Fos/CREB cycling defines LTM engram cells required for LTM (Miyashita, 2018).

This study has found that activation of CREB is only part of a c-Fos/CREB cycling program that occurs in specific cells to generate memory engrams. Previous studies have shown that LTM is encoded in a subset of neurons that are coincidently activated during training. The data suggest that these coincidently activated neurons differ from other neurons because they activate c-Fos/CREB cycling, which then likely induces expression of downstream factors required for memory maintenance. Thus, memory engram cells can be identified by the colocalization of c-Fos, CREB, and pERK activities. Inhibiting synaptic outputs from these neurons suppresses memory-associated behaviors, while artificial activation of these neurons induces memory-based behaviors in the absence of the conditioned stimulus (Miyashita, 2018).

The importance of rest intervals during training for formation of LTM is well known. 10x spaced training produces LTM in flies, while 48x massed trainings, which replace rest intervals with further training, does not. It has been shown that pERK is induced in brief waves after each spaced training trial, and it has been proposed that the number of waves of pERK activity gates LTM formation. While the current results are generally consistent with previous studies, this study found that LTM is formed after 48x massed training in CaNB2/+ and PP1/+ flies, which show sustained pERK activity instead of wave-like activity. Thus, it is suggest that either sustained pERK activity or several bursts of pERK activity are required, first to activate endogenous CREB, then to activate induced c-Fos, and later to activate induced CREB (Miyashita, 2018).

In this study, 10x massed training of CaNB2/+ flies produces an intermediate form of protein synthesis-dependent LTM that declines to baseline within 7 days. This result is consistent with results from a previous study, which identified two components of LTM: an early form that decays within 7 days and a late form that lasts more than 7 days. 10x massed training takes the same amount of time as 3x spaced training, which is insufficient to produce 7-day LTM and instead produces only the early form of LTM from preexisting dCREB2. It is proposeed that long-lasting LTM requires increased dCREB2 expression generated from c-Fos/CREB cycling. This increased dCREB2 expression allows engram cells to sustain expression of LTM genes for more than 7 days (Miyashita, 2018).

Although it is proposed that c-Fos/CREB cycling forms a positive feedback loop, this cycling does not result in uncontrolled increases in c-Fos and dCREB2. Instead, spaced training induces an early dCREB2-dependent increase in c-fos and other LTM-related genes, and subsequent c-Fos/CREB cycling maintains this increase and sustains LTM. It is believed that c-Fos/CREB cycling does not cause uncontrolled activation, because dCREB2 activity depends on an increase in the ratio of activator to repressor isoforms. The data indicate that splicing to dCREB2 repressor isoforms is delayed relative to expression of activator isoforms, leading to a transient increase in the activator-to-repressor ratio during the latter half of spaced training. However, the ratio returns to basal by the 10th training cycle, suggesting that the splicing machinery catches up to the increase in transcription. The transience of this increase prevents uncontrolled activation during c-Fos/CREB cycling and may explain the ceiling effect observed in which training in excess of 10 trials does not further increase LTM scores or duration (Miyashita, 2018).

Why does ERK activity increase during rest intervals, but not during training? ERK is phosphorylated by MEK, which is activated by Raf. Amino acid homology with mammalian B-Raf suggests that Drosophila Raf (DRaf) is activated by cAMP-dependent protein kinase (PKA) and deactivated by CaN. The current results indicate that ERK activation requires D1-type dopamine receptors and rut-AC, while a previous study demonstrates that ERK activation also requires Ca2+ influx through glutamate NMDA receptors. Thus, training-dependent increases in glutamate and dopamine signaling may activate rut-AC, which produces cAMP and activates PKA. PKA activates the MAPK pathway, resulting in ERK phosphorylation. At the same time, training-dependent increases in Ca2+/CaM activate CaN and PP1 to deactivate MEK signating in increased ERK activation during the rest interval after training (Miyashita, 2018).

This study examined the role of ERK phosphorylation and activation in LTM and did not observe significant effects of ERK inhibition in short forms of memory. However, a previous study reported that ERK suppresses forgetting of 1-hr memory, suggesting that ERK may have separate functions in regulating STM and LTM. c-Fos/CREB cycling distinguishes engram cells from non-engram cells, and it is suggested that this cycling functions to establish and maintain engrams. However, studies in mammals indicate that transcription and translation after fear conditioning is required for establishing effective memory retrieval pathways instead of memory storage. Thus, c-Fos/CREB cycling may be required for establishment and maintenance of engrams or for retrieval of information from engrams (Miyashita, 2018).

The engram cells identified in this study consist of α/β KCs, a result consistent with previous studies demonstrating the importance of these cells in LTM. Although some α/β neurons are seen expressing high amounts of dCREB2 in naive and massed trained animals (6.5% ± 0.5% of pERK-positive cells in massed trained animals), few c-fos-positive cells are seen and no overlap between c-fos expression and dCREB2 in these animals. After spaced training, the percentage of cells that express both c-fos and dCREB2 jumps to 18.9% ± 1.2%, and these cells fulfill the criteria for engram cells, because they are reactivated upon recall and influence memory-associated behaviors. The phosphatase pathway may predominate during training, inhibiting ERK phosphorylation. However, phosphatase activity may deactivate faster at the end of training compared to the Rut/PKA activity, While some mammalian studies suggest that neurons that express high amounts of CREB are preferentially recruited to memory engrams, this study found that the percentage of neurons that express high dCREB2 and low c-fos remains relatively unchanged between massed trained and spaced trained flies. Furthermore, this study finds that the increase in neurons expressing high amounts of dCREB2 after spaced training corresponds to the increase in c-Fos/CREB cycling engram cells. Thus, in flies, LTM-encoding engram cells might not be recruited from cells that previously expressed high amounts of dCREB2 but instead may correspond to cells in which c-Fos/CREB cycling is activated by coincident odor and shock sensory inputs (Miyashita, 2018).

Ets21c governs tissue renewal, stress tolerance, and aging in the Drosophila intestine

Homeostatic renewal and stress-related tissue regeneration rely on stem cell activity, which drives the replacement of damaged cells to maintain tissue integrity and function. The Jun N-terminal kinase (JNK) signaling pathway has been established as a critical regulator of tissue homeostasis both in intestinal stem cells (ISCs) and mature enterocytes (ECs), while its chronic activation has been linked to tissue degeneration and aging. This study shows that JNK signaling requires the stress-inducible transcription factor Ets21c to promote tissue renewal in Drosophila. Ets21c controls ISC proliferation as well as EC apoptosis through distinct sets of target genes that orchestrate cellular behaviors via intrinsic and non-autonomous signaling mechanisms. While its loss appears dispensable for development and prevents epithelial aging, ISCs and ECs demand Ets21c function to mount cellular responses to oxidative stress. Ets21c thus emerges as a vital regulator of proliferative homeostasis in the midgut and a determinant of the adult healthspan (Mundorf, 2019).

The intestinal epithelium undergoes continuous homeostatic and acute, stress-induced cellular turnover to ensure tissue integrity and function throughout an organism's lifetime. The replacement of damaged and aberrant cells is fueled by somatic stem cells, whose proliferation is tightly controlled and coordinated with differentiation to satisfy tissue needs while preventing organ degeneration or tumor formation. In the Drosophila adult midgut, which is a functional equivalent of the mammalian small intestine, the intestinal stem cells (ISCs) divide asymmetrically to self-renew and generate two different cell types: the transient enteroblasts (EBs) and the enteroendocrine (EE) lineage-determined cells. Following several rounds of endoreplication, the EBs mature into the large, polyploid, and polarized enterocytes (ECs), which represent the major building blocks of the midgut epithelium. While primarily involved in nutrient resorption, the ECs also serve as a physical and chemical barrier protecting the organism against toxins, pathogens, oxidative stress, and mechanical damage. The runaway stem cell activity and loss of intestinal integrity due to chronic inflammation and increased stress load have been recognized as the prime underlying causes of aging-associated tissue decline and lifespan shortening. How stress signals are transduced and integrated with the homeostatic maintenance mechanisms at the cellular level and the organ level is only partially understood (Mundorf, 2019).

The evolutionarily conserved Jun N-terminal kinase (JNK) signaling is among the key pathways that govern regenerative responses to stress, infection, and damage in the intestine. Its chronic activation has been linked to the breakdown of epithelial integrity and accelerated aging. JNK signaling affects both ISCs and differentiated ECs. In the ECs, JNK confers stress tolerance and promotes epithelial turnover by triggering the apoptosis of damaged ECs and compensatory ISC proliferation. At the same time, cell-autonomous JNK activation in ISCs accelerates ISC mitosis in cooperation with the epidermal growth factor receptor (EGFR/Ras/ERK) signaling pathway, which provides the permissive signal for division. In contrast, JNK suppression prevents age-associated ISC hyperproliferation, accumulation of mis-differentiated cells, and epithelial dysplasia, resulting in lifespan extension. The canonical response to JNK signaling culminates in the activation of transcription factors that orchestrate gene expression. The basic leucine zipper (bZIP) transcription factors Fos (kayak) and Jun (jun-related antigen) are the best-characterized JNK pathway transcriptional effectors during development. In the adult intestine, however, the relation between JNK, Jun, and Fos is less clear. Deficiency for either Fos or Jun interferes with ISC survival, a response that is not observed upon JNK inhibition. In addition, the transcription factor Foxo has been shown to orchestrate adaptive metabolic responses downstream of JNK in ECs. However, Foxo overexpression does not drive epithelial renewal as JNK activation does. These data strongly suggest that other transcription factors may play a role in mediating the pleiotropic, adaptive JNK responses in the different cell types of the intestine (Mundorf, 2019).

The transcription factors of the E-twenty six (ETS) family are functionally conserved in all metazoans and are implicated in a plethora of processes, including cell-cycle control, differentiation, proliferation, apoptosis, and tissue remodeling. Several genome-wide sequencing approaches have determined that Drosophila ets21c, the ortholog of human proto-oncogenes FLI1 and ERG, is transcriptionally induced by infections, wounding, tumorigenesis, and aging. In the case of epithelial tumors and lipopolysaccharide treatment, ets21c upregulation required JNK activity, thus making Ets21c a plausible candidate to act as a JNK effector in the adult intestine (Mundorf, 2019).

This study shows that Ets21c acts as a critical and specific regulator of ISC and EC functions in the adult fly intestine downstream of JNK signaling and in response to oxidative stress and aging. Ets21c is necessary and sufficient to coordinate epithelial turnover by controlling ISC proliferation and the removal of ECs. By regulating specific sets of target genes, Ets21c orchestrates distinct cellular behaviors of midgut cells via intrinsic and non-autonomous signaling mechanisms. While dispensable for normal development, Ets21c functions as a vital determinant of stress tolerance and lifespan (Mundorf, 2019).

By targeted manipulation of Ets21c in progenitor cells, this study shows that Ets21c levels affect the rate of ISC proliferation intrinsically while their maintenance and survival remain unaltered. The reduced ERK activation in ISCs as a consequence of ets21c deficiency in the stress-free context could provide a mechanism for the observed decline in ISC mitotic capacity. This would be consistent with a described dependency of the JNK-induced ISC hyperproliferation on the EGFR/Ras/ERK signaling pathway. The precise mechanism by which Ets21c regulates ERK activity and ISC proliferation remains to be determined. However, the identification of pvf1 as a direct transcriptional target of Ets21c implies that Ets21c could modulate mitotic Pvr/Ras signaling in progenitors through the ISC-derived autocrine and EC-specific paracrine production of Pvf1 (Mundorf, 2019).

Differentiated ECs also require intrinsic Ets21c activity for proper function. EC-specific Ets21c activation drives epithelial turnover, which involves the apoptotic removal of mature ECs and ISC proliferation to renew the pool of differentiated cells. The Ets21c-mediated EC removal and compensatory ISC proliferation response could be suppressed by both co-expression of the pan-caspase inhibitor p35 or knockdown of the Ets21c target eip93F that controls Dcp1 activity. Neither upd3 nor pvf1 silencing in ECs interfered with Dcp1 activation, although both were indispensable for the non-autonomous induction of ISC proliferation by ets21c-expressing ECs. Based on these data, it is concluded that Ets21c orchestrates epithelial turnover by promoting EC apoptosis and stimulating compensatory ISC proliferation by apoptosis-dependent and -independent mechanisms exploiting intercellular signaling molecules such as Pvf1 growth factor and the chief stress-inducible cytokine Upd3. Apoptosis-dependent and -independent induction of ISC proliferation has also been demonstrated for JNK signaling in ECs. The cell death-independent mechanism would also explain why the ISC mitotic rate remains high in paraquat-exposed flies despite Dcp1 inactivation due to EC-specific ets21c silencing. In this respect, it is important to note that other transcription factors besides Ets21c have been shown to regulate Upd3 expression under stress conditions -- for example, in infected ECs or upon oncogene activation in imaginal discs (Mundorf, 2019).

Of note, increased JNK activity has been associated with age- and oxidative stress-related changes in the posterior midgut. Consistent with its role as a JNK-dependent transcriptional effector, Ets21c levels build up in response to paraquat and during aging. ISC/EB- and EC-specific TaDa profiling revealed that only a small fraction of the Ets21c-associated genes was actively transcribed, indicating that in the unstressed state, Ets21c contributes to the fine-tuning of gene expression that supports the steady-state epithelial replenishment. Its seemingly 'unproductive' binding to DNA, however, likely primes a genetic program that can be rapidly executed in response to JNK activation upon challenge. This notion is supported by the significant enrichment for functional GO terms associated with stress-related JNK signaling. Furthermore, Ets21c was shown to regulate genes that have been functionally linked to JNK signaling, including the autophagy-related gene 1 (atg1) and the insulin signaling intersecting-target (Jafrac1), or identified as JNK- and paraquat-responsive genes, such as the eukaryotic translation initiation factor 2α kinase (PEK), a thioredoxin-like protein (fax), or a glutathione S-transferase (gstD10). It is suggested that failure to accelerate intestinal regeneration and mount a robust cytoprotective response underlie the increased sensitivity of ets21c-deficient flies to oxidative stress. The capacity of Ets21c to confer cytoprotection but also trigger apoptosis is in line with the described roles of JNK signaling. It is proposed that the repertoire of Ets21c-regulated target genes and the biological outcome of Ets21c activation depends on the strength and duration of cellular stress and the signaling landscape in which Ets21c operates. Such a model would be in accordance with a showing that low stress levels can accelerate epithelial renewal in the absence of EC death due to moderate JNK induction that stimulates ISC division, while additional input from Hippo signaling accelerates apoptosis to prevent EC overcrowding (Mundorf, 2019).

In addition to its role in coordinating cellular behaviors within the intestine, Ets21c emerges as an important determinant of the adult intestinal healthspan and overall lifespan. Optimizing proliferative homeostasis in high-turnover tissues by, for example, moderate inhibition of insulin/IGF or JNK signaling activities has proven effective in prolonging the lifespan. As Ets21c represents a key effector of JNK in the adult gut and its knockdown reduced ISC proliferation, it is plausible that balanced intestinal function may contribute to the lifespan extension of unchallenged ets21c mutant flies. However, the use of a full-body ets21c mutant prevents drawing of a causal relation between the gut-specific role of Ets21c and longevity. The tissue- and cell-type-specific contribution of Ets21c to adult lifespan remains a question for future studies to address (Mundorf, 2019).

Finally, ets21c has been repeatedly picked up by gene expression profiling studies to be markedly increased in response to immune challenge, injury, oncogene activation, or aging. While functionally linked to JNK signaling in epithelial tumor models, Ets21c has also been classified as an effector of EGFR/ERK signaling in the intestine based on the binding of Capicua, the EGFR/Ras/ERK-regulated transcriptional repressor, to the ets21c locus and upregulation of ets21c expression following Capicua loss. However, the functional evidence placing Ets21c downstream of EGFR/Ras/ERK signaling has been missing. The current data show that while knockdown of ets21c completely rescues the phenotypic consequences of JNK activation in the ISCs or ECs, it fails to mitigate the effects of activated EGFR/Ras/ERK signaling. Therefore, it is proposed that the regulation of ets21c levels results from an integration of positive and negative signaling inputs. This regulatory network includes Capicua, which acts as a gatekeeper of ets21c transcription. Such regulatory mechanisms ensure that JNK-Ets21c-mediated responses are fast but transient in supporting efficient tissue renewal while preventing chronic or excessive Ets21c activation, which drives tissue dysplasia and epithelial degeneration (Mundorf, 2019).

Electron transport chain biogenesis activated by a JNK-insulin-Myc relay primes mitochondrial inheritance in Drosophil

Oogenesis features an enormous increase in mitochondrial mass and mtDNA copy number, which are required to furnish mature eggs with an adequate supply of mitochondria and to curb the transmission of deleterious mtDNA variants. Quiescent in dividing germ cells, mtDNA replication initiates upon oocyte determination in the Drosophila ovary, which necessitates active mitochondrial respiration. However, the underlying mechanism for this dynamic regulation remains unclear. This study shows that an feedforward insulin-Myc loop promotes mitochondrial respiration and biogenesis by boosting the expression of electron transport chain subunits and of factors essential for mtDNA replication and expression, and for the import of mitochondrial proteins. Transient activation of JNK enhances the expression of the insulin receptor and initiates the insulin-Myc signaling loop. This signaling relay promotes mitochondrial biogenesis in the ovary, and thereby plays a role in limiting the transmission of deleterious mtDNA mutations. This study demonstrates cellular mechanisms that couple mitochondrial biogenesis and inheritance with oocyte development (Wang, 2019).

Mitochondria host a number of biosynthetic pathways and produce most of the cell's ATP through oxidative phosphorylation, which is carried out by the electron transport chain (ETC) complexes located on the mitochondrial inner membrane. While the majority of mitochondrial proteins are encoded on the nuclear genome, synthesized in the cytoplasm, and imported into the mitochondria, a subset of core ETC components are encoded on the mitochondrial genome (mtDNA) and synthesized inside the mitochondrial matrix. Thus, mitochondria biogenesis and ETC activity in particular, rely on the coordinated expression of both nuclear- and mtDNA-encoded mitochondrial genes. Mitochondria vary in number and activity to meet the different energy and metabolic demands of different tissues and developmental processes. Mitochondria are transmitted exclusively through the maternal lineage in most metazoans, which demands a complex regulation of mitochondrial biogenesis and ETC activity during oogenesis. Animal oocytes are hundreds of times larger than their progenitors. During this tremendous oocyte growth, mitochondria undergo prodigious biogenesis and increase mtDNA copy number over a thousand folds. The massive amount of mitochondria in the mature oocyte is necessary to power early embryonic development, as inadequate mitochondrial contents often lead to embryonic lethality. However, the mechanism by which the germline couples mitochondrial biogenesis to oocyte development remains elusive (Wang, 2019).

While furnishing mature oocytes with sufficient number of mitochondria, oogenesis also limits the transmission of harmful mtDNA mutations. The mitochondrial genome is prone to accumulating mutations because of its close vicinity to the highly mutagenic free radicals present in the mitochondrial matrix and of a lack of effective repair mechanisms. Yet, harmful mtDNA mutations are rare in populations, underscoring the presence of efficient mechanisms to limit their transmission through the female germline. It has been reported that mtDNA replication depends on active respiration in the Drosophila ovary. Healthy mitochondria with wild-type genomes propagate more vigorously than defective ones carrying harmful mutations, thereby curbing the transmission of deleterious mtDNA mutations to the next generation. Therefore, an active ETC appears to be a stress test for the functionality of mtDNA, and is essential for mtDNA selective inheritance. Nonetheless, how the activity of the ETC is regulated during oogenesis is not well understood (Wang, 2019).

Insulin signaling (IIS), an evolutionary conserved pathway that controls cell growth and proliferation, has also been shown to regulate ETC biogenesis and ATP production in human skeletal muscles. In the Drosophila ovary, IIS promotes the growth of follicles from the early to the middle stages of oogenesis. IIS activity decreases before the nurse cells dump their content into the oocyte. This decrease relieves the inhibition of GSK3, thereby shutting down mitochondrial respiration. However, oogenesis begins with germline stem cells (GSCs) that are thought not to rely on oxidative phosphorylation to ATP production. It is predicted there had to be developmental cues to activate mitochondrial respiration in the late germarium stage when mtDNA replication commences. IIS represents a logical candidate to modulate this metabolic transition in early oogenesis. Nonetheless, it remains to be explored how IIS is dynamically regulated during oogenesis and whether it is indeed involved in the aforementioned metabolic transition. Furthermore, little is known regarding how IIS modulates ETC activity and mtDNA biogenesis in general (Wang, 2019).

This study found that mitochondrial respiration is quiescent in GSCs and dividing cysts, but markedly upregulated in the late germarium, the same spatial-temporal pattern as mtDNA replication. A feedforward loop was found between IIS and Myc protein which orchestrates the transcriptional activation of respiration and mtDNA replication. Furthermore, transient JNK activity boosts insulin receptor (InR) transcription to enhance the IIS-Myc loop. This work uncovers how developmental programs couple mitochondrial biogenesis with cell growth and mitochondrial inheritance (Wang, 2019).

mtDNA replication in the Drosophila ovary relies on active respiration, suggesting that ETC activity and mtDNA replication might be subject to the same spatio-temporal regulation. This study has addressed this question and has further elucidated the developmental mechanisms regulating ETC activity and mtDNA biogenesis in the ovary. Utilizing the COX/SDH dual activity staining, it was revealed that ETC complexes are inactive in the germline stem cells (GSCs) and dividing cysts from germarium region 1 to 2A, but sharply activated in region 2B and active through stage-10 follicles. This spatial pattern mirrors that of mtDNA replication in the Drosophila ovary, supporting an essential role of mitochondrial respiration in mtDNA inheritance, both quantitively and qualitatively. It was also demonstrated that ETC activation is accompanied with an upregulation of the expression of ETC genes of both nuclear and mitochondrial origin. Interestingly, MDI, which drives the local translation of nuclear encoded mitochondrial proteins on the mitochondrial outer membrane and TFAM, which governs mtDNA replication and transcription, exhibit the same developmental pattern as mitochondrial respiration in the germarium. Collectively, these proteins would boost the biogenesis of ETC in region 2B of the germarium and in growing egg chambers. In an ovariole, different stages of developing germ cells reside in the same microenvironment and experience the same oxygen tension. Thus, the mitochondrial respiratory activity is likely to be determined by the abundance of ETC components, which itself is controlled by transcriptional activationx (Wang, 2019).

To understand how mitochondrial respiration is regulated, an RNAi screen was conducted for genes that boost COX/SDH activity in the ovary. The myc gene emerged as one of the strongest hits, and a hypomorphic allele, mycP0, largely abolished ETC activity and mtDNA replication in the germarium. Moreover, the spatial pattern of Myc protein mirrors mtDNA replication and ETC activity, further supporting its essential role in transcriptional activation of ETC biogenesis. RNA sequencing data demonstrate that Myc broadly stimulates gene expression in the Drosophila ovary, including many nuclear-encoded ETC genes and factors required for mtDNA replication and expression. These observations are consistent with previous studies in mammals showing that MYC can promote mitochondrial biogenesis by directly elevating the expression of nuclear-encoded mitochondrial genes. Among 198 annotated human mitochondrial genes that are up-regulated by Myc overexpression, 185 have homologs in the Drosophila genome. Of note, 44.9% (101 out of 225) of the fly homologs are down-regulated in mycP0 mutant ovaries, suggesting an evolutionarily conserved function of Myc in regulating mitochondrial biogenesis through gene expression. The finding that Myc induces ETC biogenesis and respiration is also in line with the studies in mammals demonstrating the multi-faceted roles of Myc in the regulation of mitochondria, including boosting mitochondrial biogenesis, stimulating oxidative metabolism , and regulating mitochondrial structure and dynamics (Wang, 2019).

Myc overexpression sometimes gives rise to different transcriptional output in different cell types. This observation reflects the fact that Myc-family proteins often associate with other cofactors and exert a broad and complex transcriptional role in a cell- or tissue-specific manner. This study also found that 130 transcription regulators, including Spargel Srl (fly homolog of human PGC-1) and CG32343 (fly homolog of GABPB2), were affected by the mycP0 mutation. PGC-1 proteins belong to an evolutionarily conserved family that integrates mitochondrial biogenesis and energy metabolism with a variety of cellular processes. In Drosophila, Srl regulates the expression of a subset of nuclear encoded mitochondrial genes. Mammalian GABPB2 is a regulatory subunit of the Nuclear Respiratory Factor complex 2 that regulates the expression of a small set of nuclear encoded mitochondrial proteins. Therefore, additional tiers of transcriptional regulations downstream of Myc are likely involved in boosting ETC biogenesis (Wang, 2019).

While myc transcription is uniform in the germarium, Myc protein is elevated at region 2B and remains high until the stage-10 egg chamber, indicating that Myc abundance is mainly regulated via post transcriptional mechanisms. IIS and JNK also emerged from the RNAi screen, and both were further confirmed to be required for triggering ETC biogenesis and mtDNA replication. IIS activity, marked by both p-AKT and p-GSK3 staining, displayed a pattern similar to that of Myc. Additionally, elevated IIS activity was required to establish a high level of Myc and to activate ETC in the late germarium stage. GSK3 directly phosphorylates Myc and promotes its ubiquitination and degradation in both mammalian and fly cultured cells. Thus, IIS likely stabilizes Myc protein by inhibiting GSK activity. This result is also in line with a previous study showing that decreased IIS activity relieves the inhibition on GSK3, which leads to mitochondrial quiescence at later stages of oogenesis. Importantly, this work uncovers Myc as the downstream effector of IIS in the regulation of respiration and mtDNA biogenesis in the ovary (Wang, 2019).

It was noticed that InR transcription was down-regulated in the myc mutant ovary, suggesting a positive feedback regulation between IIS and Myc. This regulatory loop maintains high levels of both Myc protein and IIS activity in the mid-stage follicles, where massive mitochondrial biogenesis and massive cell growth take place. However, it does not explain how this loop is activated in the first place at the late germarium stages. It was found that JNK was transiently activated in germ cells in the germarium region 2B, but decreased in budding egg chambers and sharply diminished thereafter. High level and sustained JNK activity often lead to apoptosis. However, cell death is rarely observed in the germaria of flies cultured under normal conditions. Thus, JNK activation in the late germarium must be triggered by cellular processes unrelated to apoptosis. Transiently elevated JNK activity was sufficient to increase InR mRNA level, which in-turn boosted IIS activity and stabilized Myc protein. Currently, the link between JNK and IIS is not well-understood. In the metastatic Drosophila epithelium, cell survival and proliferation entail upregulation of InR expression by JNK through wingless signaling. However, no genes in the wingless signaling pathway emerged from the RNAi screen in germ cells. The molecular mechanisms that links JNK activation to InR expression in ovary remain to be explored (Wang, 2019).

The JNK-dependent transcriptional program can be activated by various cellular stresses and cell-cell signaling events. In region 2B of the germarium, the follicle cells extend and migrate laterally across the germarium to wrap around the 16 cells cyst. Thus, JNK activation in germ cells may reflect paracrine signaling from the follicle cells, for instance via TNF-α. Alternatively, the process of follicle cells enveloping and compressing the 16-cell cyst may generate mechanical stress that subsequently activates JNK. Regardless, this work uncovers a novel function of JNK in energy metabolism and mitochondrial biogenesis besides its well-established roles in controlling cell apoptosis, growth, and proliferation (Wang, 2019).

Studies in a variety of animal models have shown that reproductive aging in females is tightly associated with decreased IIS activity. Interestingly, oocytes of aged females often have higher incidence of mtDNA lesions and lower mtDNA copy number. Thus, developmental control of mitochondrial biogenesis and mtDNA replication via IIS may be a conserved mechanism in metazoans. Previous studies demonstrated that prodigious mitochondrial biogenesis during oogenesis underlies the selective inheritance of functional mtDNA by allowing proliferation competition between healthy mitochondria and mitochondria carrying deleterious mtDNA mutations. This study has shown that the JNK/IIS/Myc signaling relay governs mitochondrial biogenesis in the ovary, and thereby influences mitochondrial inheritance both quantitively and quantitively. These studies could provide a molecular framework to further understand the control of mitochondrial biogenesis and mtDNA inheritance in animals (Wang, 2019).

Control of intestinal cell fate by dynamic mitotic spindle repositioning influences epithelial homeostasis and longevity

Tissue homeostasis depends on precise yet plastic regulation of stem cell daughter fates. During growth, Drosophila intestinal stem cells (ISCs) adjust fates by switching from asymmetric to symmetric lineages to scale the size of the ISC population. Using a combination of long-term live imaging, lineage tracing, and genetic perturbations, this study demonstrates that this switch is executed through the control of mitotic spindle orientation by Jun-N-terminal kinase (JNK) signaling. JNK interacts with the WD40-repeat protein Wdr62 at the spindle and transcriptionally represses the kinesin Kif1a to promote planar spindle orientation. In stress conditions, this function becomes deleterious, resulting in overabundance of symmetric fates and contributing to the loss of tissue homeostasis in the aging animal. Restoring normal ISC spindle orientation by perturbing the JNK/Wdr62/Kif1a axis is sufficient to improve intestinal physiology and extend lifespan. These findings reveal a critical role for the dynamic control of SC spindle orientation in epithelial maintenance (Hu, 2019).

This study directly demonstrates that cell fate and spindle orientation are tightly linked and identifies a function for JNK signaling in promoting symmetric lineages through the realignment of the mitotic spindle. The data support a model in which the mutual recruitment of phosphorylated JNK (pJNK) and Wdr62 to the spindle, as well as the JNK-dependent transcriptional repression of Kif1a, is required for spindle positioning toward a planar orientation. Because the activation of JNK also prevents cortical localization of Mud, it is proposed that JNK activity disrupts engagement of the spindle with cortical determinants of spindle orientation and limits the force exerted on astral microtubules by repressing Kif1a expression (Hu, 2019).

Live long-term lineage tracing results reveal that planar spindles result in symmetric division outcomes, whereas oblique spindles precede asymmetric outcomes. As such, changes in spindle orientation (after paraquat, short-term refeeding, and age) reflect changes in division modes. Although live imaging is a powerful tool to directly visualize spindle orientation and fate outcomes, the lower resolution compared with fixed imaging could potentially cause a wider error range in spindle angle measurements. Nonetheless, the ability to clearly visualize the vector bisecting the segregation of the two cell bodies during telophase and the vector lining the basal region of neighboring stem cells helps alleviate this issue. Another potential caveat in this analysis is that fates of ISC daughter cells may have been mis-scored because of a delay in Su(H) activation. However, an asymmetric outcome was never observed to derive from planar spindles, and division outcomes were scored as symmetric only if Su(H) activity was not observed at the end of the time-lapse recording, which was ~4 h after Su(H) activation was first observed in divisions with outcomes scored as asymmetric. In paraquat-exposed animals that overexpressed Kif1a in ISCs, Su(H) expression was detected in outcomes scored as asymmetric at roughly the same time frame as in control conditions, suggesting that stress conditions like paraquat exposure do not grossly perturb regulation of Su(H) expression (Hu, 2019).

The spindle angle that separates symmetric and asymmetric divisions is ~15°, and it is unclear whether cell fate specification during divisions with spindle orientation around that angle is deterministic or stochastic. A small subset of spindle orientations above 20° (2 of 22 examples) resulted in 2 YFP+ cells rather than 1 YFP+ cell and 1 YFP+/mCherry+ cell. It is possible that these divisions still result in an asymmetric outcome but may have generated an mCherry- EE cell rather than an mCherry+ EB cell. The rare occurrence of these events is consistent with the smaller population of EEs compared with EB/ECs in the intestine, and spindle orientation during EE fate specification may be important to segregate prospero (Hu, 2019).

Although the results are thus compatible with a deterministic model for cell fate specification, they do not rule out a role for neutral drift. In a neutral drift model, the stem cell pool is maintained by a balance of ISC loss (by generating two differentiated cells) and duplication. It is unknown how regulation of spindle orientation affects neutral drift and whether spindle orientation differs between divisions leading to two ISCs or two EBs. Addressing these issues will be important for comprehensive understanding of cell fate determination in this system (Hu, 2019).

The disparity between spindle behaviors after paraquat treatment and those after Ecc15 infection shows that the nature of the environmental trigger is critical. Although both stresses induce strong proliferative responses, their effects on spindle orientation and the corresponding cell fate outcome are different. Based on the data in this study, this disparity is likely caused by differing levels of JNK activity. JNK is activated by oxidative stress and is thus strongly induced by paraquat exposure. Ecc15 infection, in turn, promotes ISC proliferation by predominantly stimulating JAK/signal transducer and activator of transcription (STAT) activation in ISCs and only transiently activating JNK. JNK was shown to be activated immediately after Ecc15 infection (30 min post-infection), but the genes encoding components of the JNK pathway were no longer upregulated as early as 4 h post-infection. These observations are consistent with analysis 16-20 h post-infection, particularly the absence of phosphorylated JNK at the mitotic spindle in Ecc15-infected animals. However, a possible role of JNK on spindle orientation at earlier time points after infection cannot be ruled out (Hu, 2019).

Previous studies have reported that similar to the current observations with Ecc15, infection of another strain of bacteria, Pseudomonas entomophila, largely promoted asymmetric fate outcomes. However, JNK activity was still detected in the entire gut 2 days post-infection, although the specific cell type (stem cells versus differentiated cells) in which JNK was activated was not examined. The possibility that JNK is activated in ISCs after P. entomophila infection was not ruled out. The difference in pathology of P. entomophila-which is lethal, unlike Ecc15-may contribute to a different response in JNK activation. One hypothesis is that although JNK may be activated after P. entomophila infection in ISCs, it is not recruited to the mitotic spindle and therefore would not affect spindle orientation. Future studies are needed to test this hypothesis and explore possible mechanisms of a pathogen-specific difference (Hu, 2019).

In recruitment to the spindle, pJNK and Wdr62 depend mutually on each other. Although JNK clearly plays a critical role in this process, the data do not rule out a role for other kinases that have been reported to recruit Wdr62 to the centrosome, including Aurora A and Polo-like kinase. Unlike other reports in neural stem cells, this study did not find an obvious role for Wdr62 in maintaining bipolar spindles. Reports have also identified roles for Wdr62 in stabilizing microtubules and centrosomes in interphase neural stem cells, and although the effects of Wdr62 depletion during interphase was not tested in this study, the absence of gross mitotic defects suggests that in Drosophila ISCs, Wdr62 may function selectively in spindle orientation. However, somewhat smaller clone sizes were observed of ISC lineages deficient for Wdr62, and therefore a function for interphase Wdr62 cannot be ruled out. Disruption of Wdr62 activity during interphase may also contribute to the inconsistent effect on lifespan observed after Wdr62 depletion, despite the restoration of oblique spindles in ISCs of old flies (Hu, 2019).

The consequences of the loss of Pins and Mud seem to vary depending on the tissue: disrupting Pins and Mud in Drosophila neuroblasts randomizes the mitotic spindle, but in the mammalian skin, basal stem cells with depleted LGN favor planar spindles, similar to observations in Drosophila ISCs. A loss of cortical Mud was observed after JNK activation, supporting the notion that JNK regulates the interaction between the astral microtubules and the cell cortex to promote planar spindles. The extent to which JNK or Wdr62 interacts directly with Mud is an important question for further study (Hu, 2019).

The mechanism by which Kif1a promotes oblique spindle orientation in ISCs is unclear. Khc-73, a kinesin in the same Kinesin-3 family, is believed to interact with Pins or Disc Large in Drosophila S2 cells and neuroblasts to orient astral microtubules to the cell cortex, and Kif1a may play similar roles in ISCs. Although the data suggest that JNK regulates Kif1a levels transcriptionally, it is possible that JNK also regulates Kif1a at the protein level and may direct its possible interaction with the spindle recruitment machinery (Hu, 2019).

The data reveal how a physiological role for JNK signaling in regulating spindle positioning during periods of tissue resizing becomes hijacked under stress and age, limiting tissue homeostasis and shortening lifespan. It remains unclear how JNK is activated in ISCs during starvation-refeeding, but insulin signaling has been implicated in promoting symmetric outcomes during adaptive resizing of the Drosophila intestine. It will be interesting to test whether insulin signaling and JNK interact to regulate spindle positioning in ISCs, because elevated insulin signaling activity may also contribute to the age-related chronic activation of JNK. The age-related bias toward planar spindle orientations is reminiscent of the changes in spindle orientation of germline stem cells in old male flies, and restoring oblique spindle orientation in aged ISCs by increasing Kif1a expression is sufficient to improve intestinal physiology and extend lifespan. Understanding the exact mechanisms and consequences of ISC spindle positioning will be critical to identifying new intervention strategies to allay age-related dysfunction in barrier epithelia (Hu, 2019).

Such interventions are likely to have significant clinical relevance, because barrier epithelia in mammals regenerate and age through mechanisms that are similar to the Drosophila intestinal epithelium. However, although SC fate determination by changes in spindle orientation is observed in multiple mammalian tissues during development, the extent to which similar mechanisms determine cell fate in adult mammalian tissues is unclear. Mouse ISCs within the adult intestine use different mechanisms to establish cell fate, because spindle orientation is largely planar, and extrinsic cues preferentially differentiate one of the daughter cells. In the mouse trachea, however, it has been reported that spindle orientation fluctuates in basal stem cells in response to injury and may affect cell fate specification. Given the variation in lineage, cell composition, and organization in different adult tissues, it is likely that the importance of spindle orientation in cell specification differs among tissues. Determining the tissues in which spindle orientation is linked with cell fate, and testing whether the role of JNK in the regulation of spindle orientation in these SCs is conserved, will provide important insight into regenerative biology (Hu, 2019).

Microenvironmental innate immune signaling and cell mechanical responses promote tumor growth

Tissue homeostasis is achieved by balancing stem cell maintenance, cell proliferation and differentiation, as well as the purging of damaged cells. Elimination of unfit cells maintains tissue health: however, the underlying mechanisms driving competitive growth when homeostasis fails, for example, during tumorigenesis, remain largely unresolved. Using a Drosophila intestinal model, this study found that tumor cells outcompete nearby enterocytes (ECs) by influencing cell adhesion and contractility. This process relies on activating the immune-responsive Relish/NF-κB pathway to induce EC delamination and requires a JNK-dependent transcriptional upregulation of the peptidoglycan recognition protein PGRP-LA. Consequently, in organisms with impaired PGRP-LA function, tumor growth is delayed and lifespan extended. This study identifies a non-cell-autonomous role for a JNK/PGRP-LA/Relish signaling axis in mediating death of neighboring normal cells to facilitate tumor growth. It is proposed that intestinal tumors 'hijack' innate immune signaling to eliminate enterocytes in order to support their own growth (Zhou, 2021).

The intestinal epithelium separates the organism from the environment and plays essential roles in nutrient uptake and immune and regenerative processes. Intestinal renewal requires dynamic regulation of cell-cell contacts between enterocytes, and this is achieved by highly proliferative stem cells, proper differentiation, and cell loss by cell extrusion and apoptosis. Dysregulation of cell death in the intestinal epithelium can lead to pathologies such as intestinal bowel diseases and cancer (Zhou, 2021).

Studies in Drosophila have made important contributions toward an understanding of intestinal homeostasis, innate immunity, and aging. In adult flies, intestinal stem cells (ISCs) self-renew and produce progenitor cells called enteroblasts (EBs). These EBs can differentiate into either enteroendocrine cells (EEs) or enterocytes (ECs). The intestinal epithelium undergoes rapid stem cell division and differentiation to continuously replace damaged ECs and ensure tissue integrity and homeostasis, similar to mammalian intestines. Previous studies have shown that bacterial infection induces ISC proliferation and elimination of damaged ECs, thereby leading to remodeling of the intestinal epithelium. Enteric infection also triggers the evolutionarily conserved NF-ΚB pathway through the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs), leading to the production of antimicrobial peptides (AMPs) for host immune defense (Zhou, 2021).

In addition to the role of the NF-κB pathway in AMP production in different cell types, several studies have identified non-immune functions. For example, constitutive activation of NF-κB reduces animal lifespan, NF-κB activity has been implicated in age-related neurodegenerative diseases, and NF-κB regulates Mef2 to coordinate its immune functions with metabolism. Further evidence links NFκB to Ras/MAPK and JAK/STAT signaling pathways. This allows for the proper balance of immune responses with cell growth and proliferation. Moreover, it has been recently reported that the NF-κB pathway in Drosophila is involved in infection-induced EC shedding, which facilitates maintenance of barrier function during intestinal regeneration (Zhou, 2021).

The Drosophila BMP2/4 homolog Decapentaplegic (Dpp) is involved in multiple developmental processes. The Dpp signal is transduced by the type I receptor Thickveins (Tkv) and type II receptor Punt that phosphorylate Drosophila Smad transcriptional factors such as Mothers against Dpp (Mad), Medea (Med), and the coregulator Schnurri (Shn) to regulate gene expression. Inactivation of BMP signaling components in the Drosophila intestine leads to intestinal tumor formation resembling juvenile polyposis syndrome (JPS). In humans, loss of BMP signaling leads to JPS, which has been associated with increased risks of developing gastrointestinal cancer (Zhou, 2021).

Cell replenishment and rearrangement are common mechanisms to sustain tissue homeostasis, which is also essential for development. Tissue growth requires dynamic cell rearrangements including cell elimination by mechanical competition. For example, epithelial cells can be eliminated by cell extrusion to maintain tissue homeostasis. Recent studies also suggested that tumor cells outcompete and eliminate their neighboring cells to clear space for their expansion. However, the underlying mechanisms and how tumor cells eliminate normal cells in the tumor microenvironment remain largely unknown (Zhou, 2021).

This study demonstrates a critical role for mechanical competition in the tumor microenvironment to promote tumorigenesis. Mechanistically, it was shown that tumor induces DE-cadherin- and myosin-dysregulation-associated mechanical stresses to nearby ECs. These processes trigger the ROCK-associated JNK signaling and subsequent activation of PGRP-LA/NF-κrB/ Relish in surrounding ECs to regulate the expression of pro- apoptotic genes and thereby promote cell delamination and apoptosis. The dying ECs then induce paracrine JAK/STAT signaling to trigger regeneration and further promote tumorigenesis. Importantly, tumors with associated activation of JNK/ PGPR-LA/Relish cascades can be inhibited by preventing apoptosis or by administering ROCK inhibitors. These results thus establish a tumor-cell-driven inflammatory feedback mechanism for competitive growth (Zhou, 2021).

This study demonstrates a non-cell-autonomous feedback mechanism that facilitates tumor development. First, tumor growth outcompetes its microenvironment by inducing mechanical forces. This triggers stress related ROCK/JNK signaling and induces EC elimination through activation of PGRP-LA/Relish signaling and downstream pro-apoptotic genes. Subsequently, dying ECs produce cytokines that activate JAK/STAT signaling in tumor cells to further promote tumor growth, thereby establishing a positive amplification loop between the tumor and its microenvironment (Zhou, 2021).

Cells undergoing rapid proliferation will push on their neighbors, which leads to local increase in mechanical pressures and triggers cell delamination. Previous studies have shown that the expansion of tumor cells triggers cell competition, which involves mechanical interactions. For instance, ectopic expression of Ras oncogene drives mechanical competitive growth and induces delamination of nearby wild-type cells in Drosophila. Hence, these data are in line with the current interpretation that mechanical competition also drives tumor competitive growth in the Drosophila intestine. In the epithelium, the mechanical modulation of surface tension is regulated by the actomyosin complex, counterbalanced by the Cadherin-dependent cell-cell adhesions. This study shows that tumors interact with their microenvironment and trigger ROCK/ JNK related cell death. A similar mechanism was observed in mammalian Madin-Darbin canine kidney (MDCK) cells, which, when deficient in the polarity gene scribble, are eliminated by mechanical cell competition, a process that requires the activation of the ROCK-p38-p53 pathway (Zhou, 2021).

The intestinal epithelium requires homeostatic mechanisms to counterbalance stem cell division and elimination of damaged or unfit cells. Dysregulation of either of these homeostatic programs can lead to tumor development. This study found that tumor cells induce immune-responsive PGRP- LA/Relish signaling for cell delamination and apoptosis. The role of NF-κB in the regulation of apoptosis has been discussed before for Drosophila Imd and mammalian TNFR1 pathways, which share key components to regulate NF-κB-related immune response and caspase-dependent apoptosis. Several studies have shown that tumor cells displace the nearby ECs through activation of Hippo and JNK signaling for tumor progression. In addition, JNK acts in parallel with NF-κB to control EC shedding during intestinal regeneration. In mammals, intestinal TNFR1 signaling is also required for EC detachment and apoptosis (Zhou, 2021).

Previous studies revealed a role for NF-κB signaling in cell death of outcompeted cells during development. In Drosophila wing discs, Myc-induced cell competition triggers Imd/Relish-related activation of the pro-apoptotic gene Hid for cell death. The Toll-signaling transcription factors Dorsal and Dif have been suggested to be required for Minute-induced cell competition by inducing Reaper-dependent apoptosis of outcompeted cells. However, further evidence showed axenic conditions abolished Toll-inhibition-induced competitive growth in the outcompeted cells. This suggested that infection contributes to Toll pathway inhibition induced cell competition. The current experiments indicate that axenic conditions failed to abolish the Imd activation in the tumor-surrounding ECs, suggesting a tumor-associated role of Imd/Relish induced EC cell death. Furthermore, a recent study discovered that cells with growth advantages, such as high protein synthesis, induce NF-κB-dependent autophagy to eliminate neighboring unfit cells in developing tissues. NF-κB and its upstream activating receptor are also required for salivary gland degradation through autophagy. Eye disc tumors also trigger a cell-autonomous feedback loop to promote proliferation by activation of JNK, Yki, and JAK/STAT signaling. In a distinct organ with a high rate of turnover, the data suggest that the NF-κB/Rel-dependent EC cell death cooperates with compensatory stem cell proliferation through the non-cell-autonomous activation of JNK and JAK/ STAT signaling for tumor progression (Zhou, 2021).

PGRPs are known as immune modulators of NF-κB signaling through binding and recognizing bacterial peptidoglycans. Several PGRPs have been implicated in other important biological processes beyond immunity such as host-microbe homeostasis, systemic inflammatory response, tissue integrity, and aging. PGRPs contain a RIP RHIM domain which has been proposed to activate NF-κB signaling. However, unlike in mammals, the Drosophila RHIMs may not be required for cell death. Consistently, it was found that the PGRP-LAD containing the RHIM domain does not induce cell death. However, PGRP-LAF lacking RHIM drives EC delamination and apoptosis. Previous studies suggested a regulatory role of PGRP-LA in controlling NF-κB activity rather than binding to peptidoglycan. In this study, PGRP-LA depletion extends the lifespan of tumor-bearing flies. However, the expression of PGRP-LA is low in the intestine during normal homeostasis, suggesting PGRP-LA may not be involved in normal aging and intestinal homeostasis. Whether the programed cell death pathways impact metazoan lifespan remains unknown, and this requires further research (Zhou, 2021).

This study revealed that tumor induces cytoplasmic enrichment of DE-cadherin::GFP and activates p-Myosin signal in nearby ECs with elongated cell morphology. The DE-cadherin reporter and p-Myosin staining have been previously used as mechano-transduction sensors in Drosophila. The results therefore suggest that the tumor induces DE-cadherin- and myosin-dysregulation-associated mechanical stresses. However, changes in mechanical tension in cells adjacent to tumor cells is a rapid process and more evidence will be required to illustrate mechanical competition, e.g., by monitoring mechanosensors in live tissue. Unfortunately, this remains technically difficult in the Drosophila intestine because of constraints on live imaging and a lack of molecular markers. Moreover, how cells sense and respond to mechanical stress in the context of tumor growth requires further investigations (Zhou, 2021).


Genomic size - 4186 bp

cDNA clone length - 1831 bases

Bases in 5' UTR - 535

Exons - 6

Bases in 3' UTR - 141


Amino Acids - 372

Structural Domains

Drosophila Basket/JNK contains kinase subdomains I-XI. The dual phosphorylation motif Thr-Xaa-Tyr required for MAP kinase activation is located in kinase subdomain VIII (Thr-Pro-Tyr). Sequence comparison with other MAP kinases demonstrates that DJNK is a member of the JNK group of protein kinases. The amino acid sequence of DJNK is 65% identical to human JNK1. The amino acid sequence between kinase subdomains IX and X varies between the JNKs, and determines the efficiency of binding to c-Jun. In this region, DJNK is most homologous to JNK2. DJNK is related more distantly to the Drosophila rolled gene product ERKA (Sluss, 1996 and Riesgo-Escovar, 1996).

basket/JNK: | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References
date revised: 12 June 2022 
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