Abstract Here we report novel tumour suppressor activity for the Drosophila Argonaute family RNA binding protein AGO1, a component of the miRNA-dependent RNA-induced silencing complex (RISC). The mechanism for growth inhibition does not, however, involve canonical roles as part of the RISC; rather AGO1 controls cell and tissue growth by functioning as a direct transcriptional repressor of the master regulator of growth, Myc. AGO1 depletion in wing imaginal discs drives a significant increase in ribosome biogenesis, nucleolar expansion, and cell growth in a manner dependent on Myc abundance. Moreover, increased Myc promoter activity and elevated Myc mRNA in AGO1 depleted animals requires RNA Pol II transcription. Further support for transcriptional AGO1 functions is provided by physical interaction with the RNA Pol II transcriptional machinery (chromatin remodelling factors and Mediator Complex), punctate nuclear localisation in euchromatic regions and overlap with Polycomb Group transcriptional silencing loci. Moreover, significant AGO1 enrichment is observed on the Myc promoter and AGO1 interacts with the Myc transcriptional activator Psi. Together our data show AGO1 functions outside of the RISC to repress Myc transcription and inhibit developmental cell and tissue growth.
ABSTRACT The tumour suppressor, Lethal (2) giant larvae [Lgl; also known as L(2)gl], is an evolutionarily conserved protein that was discovered in the vinegar fly Drosophila, where its depletion results in tissue overgrowth and loss of cell polarity. Lgl links cell polarity and tissue growth through regulation of the Notch and the Hippo signalling pathways. Lgl regulates the Notch pathway by inhibiting V-ATPase activity via Vap33. How Lgl regulates the Hippo pathway was unclear. In this current study, we show that V-ATPase activity inhibits the Hippo pathway, whereas Vap33 acts to activate Hippo signalling. Vap33 physically and genetically interacts with the actin cytoskeletal regulators RtGEF (Pix) and Git, which also bind to the Hippo protein (Hpo) and are involved in the activation of the Hippo pathway. Additionally, we show that the ADP ribosylation factor Arf79F (Arf1), which is a Hpo interactor, is involved in the inhibition of the Hippo pathway. Altogether, our data suggest that Lgl acts via Vap33 to activate the Hippo pathway by a dual mechanism: (1) through interaction with RtGEF, Git and Arf79F, and (2) through interaction and inhibition of the V-ATPase, thereby controlling epithelial tissue growth.
Abstract The tumour suppressor, Lethal (2) giant larvae (Lgl), is an evolutionarily conserved protein that was discovered in the vinegar fly, Drosophila , where its depletion results in tissue overgrowth and loss of cell polarity and tissue architecture. Our previous studies have revealed a new role for Lgl in linking cell polarity and tissue growth through regulation of the Notch (proliferation and differentiation) and the Hippo (negative tissue growth control) signalling pathways. Moreover, Lgl regulates vesicle acidification, via the Vacuolar ATPase (V-ATPase), and we showed that Lgl inhibits V-ATPase activity through Vap33 (a Vamp (v-SNARE)-associated protein, involved in endo-lysosomal trafficking) to regulate the Notch pathway. However, how Lgl acts to regulate the Hippo pathway was unclear. In this current study, we show that V-ATPase activity inhibits the Hippo pathway, whereas Vap33 acts to activate Hippo signalling. Using an in vivo affinity-purification approach we found that Vap33 binds to the actin cytoskeletal regulators RtGEF (Pix, a Rho-type guanine nucleotide exchange factor) and Git (G protein-coupled receptor kinase interacting ArfGAP), which also bind to the Hpo protein kinase, and are involved in the activation of the Hippo pathway. Vap33 genetically interacts with RtGEF and Git in Hippo pathway regulation. Additionally, we show that the ADP ribosylation factor Arf79F (Arf1), which is a Hpo interactor, is involved in the inhibition of the Hippo pathway. Altogether our data suggests that Lgl acts via Vap33 to activate the Hippo pathway by a dual mechanism, 1) through interaction with RtGEF/Git/Arf79F, and 2) through interaction and inhibition of the V-ATPase, thereby controlling epithelial tissue growth.
A nerve growth factor (NGF) receptor interactive monoclonal antibody (192-IgG) which enhances B-NGF binding to PC12 cells has been produced.The hybridoma clone was obtained by fusing Sp2/0-Ag14 myeloma cells with splenocytes from Balb/C mice which had been immunized with n-octyl glucoside solubilized proteins from isolated PC12 cell plasma membranes.The antibody is an IgG, which does not bind P-NGF.It binds to the same number of sites on PC12 cells at low temperature as does 8-NGF.The 192-IgG increases the apparent affinity of B-NGF binding to fast receptors on PC12 cells at low temperature by a factor of 2.5-to 4-fold and enhances the photoactivatable cross-linking of B-NGF to the same receptor while decreasing the cross-linking of B-NGF to the slow NGF receptor.At 37 "C 192-IgG partially inhibits the regeneration of neurites from primed PC12 cells.The 192-IgG also reduces the rate of appearance of binding to slow NGF receptors and increases the proportion of 8-NGF bound to fast receptors at 37 "C.These results implicate the slow receptor as the mediator of the biological response.This antibody provides a tool for examining steps in the mechanism of action of ,B-NGF after binding to the receptor.NGF' is a polypeptide which is required for the development and maintenance of sympathetic and some sensory neurons (1, 2) and is one of the factors involved in the regeneration of sympathetic and sensory axons after injury (3).A specific retrograde flow of NGF occurs from the peripheral target to the neuronal cell body (4).The flow is initiated by the binding
In both Drosophila melanogaster and mammalian systems, epithelial structure and underlying cell polarity are essential for proper tissue morphogenesis and organ growth. Cell polarity interfaces with multiple cellular processes that are regulated by the phosphorylation status of large protein networks. To gain insight into the molecular mechanisms that coordinate cell polarity with tissue growth, we screened a boutique collection of RNAi stocks targeting the kinome for their capacity to modify Drosophila "cell polarity" eye and wing phenotypes. Initially, we identified kinase or phosphatase genes whose depletion modified adult eye phenotypes associated with the manipulation of cell polarity complexes (via overexpression of Crb or aPKC). We next conducted a secondary screen to test whether these cell polarity modifiers altered tissue overgrowth associated with depletion of Lgl in the wing. These screens identified Hippo, Jun kinase (JNK), and Notch signaling pathways, previously linked to cell polarity regulation of tissue growth. Furthermore, novel pathways not previously connected to cell polarity regulation of tissue growth were identified, including Wingless (Wg/Wnt), Ras, and lipid/Phospho-inositol-3-kinase (PI3K) signaling pathways. Additionally, we demonstrated that the "nutrient sensing" kinases Salt Inducible Kinase 2 and 3 (SIK2 and 3) are potent modifiers of cell polarity phenotypes and regulators of tissue growth. Overall, our screen has revealed novel cell polarity-interacting kinases and phosphatases that affect tissue growth, providing a platform for investigating molecular mechanisms coordinating cell polarity and tissue growth during development.
Despite two decades of research, the major function of FBP-family KH domain proteins during animal development remains controversial. The literature is divided between RNA processing and transcriptional functions for these single stranded nucleic acid binding proteins. Using Drosophila, where the three mammalian FBP proteins (FBP1-3) are represented by one ortholog, Psi, we demonstrate the primary developmental role is control of cell and tissue growth. Co-IP-mass spectrometry positioned Psi in an interactome predominantly comprised of RNA Polymerase II (RNA Pol II) transcriptional machinery and we demonstrate Psi is a potent transcriptional activator. The most striking interaction was between Psi and the transcriptional mediator (MED) complex, a known sensor of signaling inputs. Moreover, genetic manipulation of MED activity modified Psi-dependent growth, which suggests Psi interacts with MED to integrate developmental growth signals. Our data suggest the key target of the Psi/MED network in controlling developmentally regulated tissue growth is the transcription factor MYC. As FBP1 has been implicated in controlling expression of the MYC oncogene, we predict interaction between MED and FBP1 might also have implications for cancer initiation and progression.
We investigated the time course for development of functional tolerance to the ataxic effects of ethanol in four genetically distinct mouse populations. Two inbred-strains (C57BL/6J and DBA/2J) and two lines of mice (long-sleep and short-sleep) selectively bred for differences in acute ethanol sensitivity were used. Mice were injected i.p. with ethanol in doses that produced ataxia and were tested repeatedly for their ability to balance on a wooden rod. When they regained their balance at threshold, brain ethanol levels were measured in some mice and booster injections of ethanol were administered to the remaining animals. This sequence was repeated for five injections, delivering a total of 6 g/kg of ethanol to the final group of animals. Functional tolerance developed in all four populations of mice as evidence by threshold brain ethanol levels that were significantly higher after two or three successive injections than after one injection. The magnitude of tolerance was not increased by practice on the dowel. To investigate whether alterations in membrane lipid composition accompanied this rapid development of tolerance, we used erythrocytes as a model system and measured the cholesterol and phospholipid content of their membranes. The erythrocyte membranes from ethanol-tolerant mice of each population contained more cholesterol than those from controls. The erythrocyte membrane phospholipid content of ethanol-tolerant animals changed only slightly in two populations.
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