Clueless forms dynamic, insulin-responsive bliss particles sensitive to stress.

Drosophila Clueless (Clu) is a ribonucleoprotein that directly affects mitochondrial function. Loss of clu causes mitochondrial damage, and Clu associates with proteins on the mitochondrial outer membrane. Clu's subcellular pattern is diffuse throughout the cytoplasm, but Clu also forms large mitochondria-associated particles. Clu particles are reminiscent of ribonucleoprotein particles such as stress granules and processing bodies. Ribonucleoprotein particles play critical roles in the cell by regulating mRNAs spatially and temporally. Here, we show that Clu particles are unique, highly dynamic and rapidly disperse in response to stress in contrast to processing bodies and autophagosomes. In addition, Clu particle formation is dependent on diet as ovaries from starved females no longer contain Clu particles, and insulin signaling is necessary and sufficient for Clu particle formation. Oxidative stress also disperses particles. Since Clu particles are only present under optimal conditions, we have termed them "bliss particles". We also demonstrate that many aspects of Clu function are conserved in the yeast homolog Clu1p. These observations identify Clu particles as stress-sensitive cytoplasmic particles whose absence corresponds with altered cell stress and mitochondrial localization.

Fly Models of Human Diseases: Drosophila as a Model for Understanding Human Mitochondrial Mutations and Disease.

Mitochondrial diseases are a prevalent, heterogeneous class of diseases caused by defects in oxidative phosphorylation, whose severity depends upon particular genetic mutations. These diseases can be difficult to diagnose, and current therapeutics have limited efficacy, primarily treating only symptoms. Because mitochondria play a pivotal role in numerous cellular functions, especially ATP production, their diminished activity has dramatic physiological consequences. While this in and of itself makes treating mitochondrial disease complex, these organelles contain their own DNA, mtDNA, whose products are required for ATP production, in addition to the hundreds of nucleus-encoded proteins. Drosophila offers a tractable whole-animal model to understand the mechanisms underlying loss of mitochondrial function, the subsequent cellular and tissue damage that results, and how these organelles are inherited. Human and Drosophila mtDNAs encode the same set of products, and the homologous nucleus-encoded genes required for mitochondrial function are conserved. In addition, Drosophila contain sufficiently complex organ systems to effectively recapitulate many basic symptoms of mitochondrial diseases, yet are relatively easy and fast to genetically manipulate. There are several Drosophila models for specific mitochondrial diseases, which have been recently reviewed (Foriel, Willems, Smeitink, Schenck, & Beyrath, 2015). In this review, we highlight the conservation between human and Drosophila mtDNA, the present and future techniques for creating mtDNA mutations for further study, and how Drosophila has contributed to our current understanding of mitochondrial inheritance.

miR-16 rescues F508del-CFTR function in native cystic fibrosis epithelial cells.

Cystic fibrosis (CF) is due to mutations in the CFTR gene, which prevents correct folding, trafficking and function of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) protein. The dysfunctional effect of CFTR mutations, principally the F508del-CFTR mutant, is further manifested by hypersecretion of the pro-inflammatory chemokine interleukin-8 into the airway lumen, which further contributes to morbidity and mortality. We have hypothesized that microRNA (miR)-based therapeutics could rescue the dysfunctional consequences of mutant CFTR. Here we report that a miR-16 mimic can effectively rescue F508del-CFTR protein function in airway cell lines and primary cultures, of differentiated human bronchial epithelia from F508del homozygotes, which express mutant CFTR endogenously. We also identify two other miRs, miR-1 and miR-302a, which are also active. Although miR-16 is expressed at basal comparable levels in CF and control cells, miR-1 and miR-302a are undetectable. When miR mimics are expressed in CF lung or pancreatic cells, the expression of the F508del-CFTR protein is significantly increased. Importantly, miR-16 promotes functional rescue of the cyclic AMP-activated apical F508del-CFTR chloride channel in primary lung epithelial cells from CF patients. We interpret these findings to suggest that these miRs may constitute novel targets for CF therapy.

Motional enhancement of exciton magnetic moments in zinc-blende semiconductors.

We report a remarkable enhancement of the magnetic moments of excitons as a result of their motion. This surprising result, which we have observed in magneto-optical studies of three distinct zinc-blende semiconductors, GaAs, CdTe, and ZnSe, becomes significant as the kinetic energy of the exciton becomes comparable with its Rydberg energy and is attributed to motionally induced changes in the internal structure of the exciton. The enhancement of the magnetic moment as a function of the exciton translational wave vector can be represented by a universal equation.

A screen for mutations that suppress the phenotype of Drosophila armadillo, the beta-catenin homolog.

During development signaling pathways coordinate cell fates and regulate the choice between cell survival or programmed cell death. The well-conserved Wingless/Wnt pathway is required for many developmental decisions in all animals. One transducer of the Wingless/Wnt signal is Armadillo/beta-catenin. Drosophila Armadillo not only transduces Wingless signal, but also acts in cell-cell adhesion via its role in the epithelial adherens junction. While many components of both the Wingless/Wnt signaling pathway and adherens junctions are known, both processes are complex, suggesting that unknown components influence signaling and junctions. We carried out a genetic modifier screen to identify some of these components by screening for mutations that can suppress the armadillo mutant phenotype. We identified 12 regions of the genome that have this property. From these regions and from additional candidate genes tested we identified four genes that suppress arm: dTCF, puckered, head involution defective (hid), and Dpresenilin. We further investigated the interaction with hid, a known regulator of programmed cell death. Our data suggest that Wg signaling modulates Hid activity and that Hid regulates programmed cell death in a dose-sensitive fashion.

The canonical Wg and JNK signaling cascades collaborate to promote both dorsal closure and ventral patterning.

Elaboration of the Drosophila body plan depends on a series of cell-identity decisions and morphogenetic movements regulated by intercellular signals. For example, Jun N-terminal kinase signaling regulates cell fate decisions and morphogenesis during dorsal closure, while Wingless signaling regulates segmental patterning of the larval cuticle via Armadillo. wingless or armadillo mutant embryos secrete a lawn of ventral denticles; armadillo mutants also exhibit dorsal closure defects. We found that mutations in puckered, a phosphatase that antagonizes Jun N-terminal kinase, suppress in a dose-sensitive manner both the dorsal and ventral armadillo cuticle defects. Furthermore, we found that activation of the Jun N-terminal kinase signaling pathway suppresses armadillo-associated defects. Jun N-terminal kinase signaling promotes dorsal closure, in part, by regulating decapentaplegic expression in the dorsal epidermis. We demonstrate that Wingless signaling is also required to activate decapentaplegic expression and to coordinate cell shape changes during dorsal closure. Together, these results demonstrate that MAP-Kinase and Wingless signaling cooperate in both the dorsal and ventral epidermis, and suggest that Wingless may activate both the Wingless and the Jun N-terminal kinase signaling cascades.

Roles of the C terminus of Armadillo in Wingless signaling in Drosophila.

Drosophila melanogaster Armadillo and its vertebrate homolog beta-catenin play multiple roles during development. Both are components of cell-cell adherens junctions and both transduce Wingless (Wg)/Wnt intercellular signals. The current model for Wingless signaling proposes that Armadillo binds the DNA-binding protein dTCF, forming a bipartite transcription factor that activates Wingless-responsive genes. In this model, Armadillo's C-terminal domain is proposed to serve an essential role as a transcriptional activation domain. In Xenopus, however, overexpression of C-terminally truncated beta-catenin activates Wnt signaling, suggesting that the C-terminal domain might not be essential. We reexamined the function of Armadillo's C terminus in Wingless signaling. We found that C-terminally truncated mutant Armadillo has a deficit in Wg-signaling activity, even when corrected for reduced protein levels. However, we also found that Armadillo proteins lacking all or part of the C terminus retain some signaling ability if overexpressed, and that mutants lacking different portions of the C-terminal domain differ in their level of signaling ability. Finally, we found that the C terminus plays a role in Armadillo protein stability in response to Wingless signal and that the C-terminal domain can physically interact with the Arm repeat region. These data suggest that the C-terminal domain plays a complex role in Wingless signaling and that Armadillo recruits the transcriptional machinery via multiple contact sites, which act in an additive fashion.

Membrane-tethered Drosophila Armadillo cannot transduce Wingless signal on its own.

Drosophila Armadillo and its vertebrate homolog beta-catenin are key effectors of Wingless/Wnt signaling. In the current model, Wingless/Wnt signal stabilizes Armadillo/beta-catenin, which then accumulates in nuclei and binds TCF/LEF family proteins, forming bipartite transcription factors which activate transcription of Wingless/Wnt responsive genes. This model was recently challenged. Overexpression in Xenopus of membrane-tethered beta-catenin or its paralog plakoglobin activates Wnt signaling, suggesting that nuclear localization of Armadillo/beta-catenin is not essential for signaling. Tethered plakoglobin or beta-catenin might signal on their own or might act indirectly by elevating levels of endogenous beta-catenin. We tested these hypotheses in Drosophila by removing endogenous Armadillo. We generated a series of mutant Armadillo proteins with altered intracellular localizations, and expressed these in wild-type and armadillo mutant backgrounds. We found that membrane-tethered Armadillo cannot signal on its own; however it can function in adherens junctions. We also created mutant forms of Armadillo carrying heterologous nuclear localization or nuclear export signals. Although these signals alter the subcellular localization of Arm when overexpressed in Xenopus, in Drosophila they have little effect on localization and only subtle effects on signaling. This supports a model in which Armadillo's nuclear localization is key for signaling, but in which Armadillo intracellular localization is controlled by the availability and affinity of its binding partners.

Drosophila Tcf and Groucho interact to repress Wingless signalling activity.

Wingless/Wnt signalling directs cell-fate choices during embryonic development. Inappropriate reactivation of the pathway causes cancer. In Drosophila, signal transduction from Wingless stabilizes cytosolic Armadillo, which then forms a bipartite transcription factor with the HMG-box protein Drosophila Tcf (dTcf) and activates expression of Wingless-responsive genes. Here we report that in the absence of Armadillo, dTcf acts as a transcriptional repressor of Wingless-responsive genes, and we show that Groucho acts as a corepressor in this process. Reduction of dTcf activity partially suppresses wingless and armadillo mutant phenotypes, leading to derepression of Wingless-responsive genes. Furthermore, overexpression of wild-type dTcf enhances the phenotype of a weak wingless allele. Finally, mutations in the Drosophila groucho gene also suppress wingless and armadillo mutant phenotypes as Groucho physically interacts with dTcf and is required for its full repressor activity.

Armadillo is required for adherens junction assembly, cell polarity, and morphogenesis during Drosophila embryogenesis.

Morphological and biochemical analyses have identified a set of proteins which together form a structure known as the adherens junction. Elegant experiments in tissue culture support the idea that adherens junctions play a key role in cell-cell adhesion and in organizing cells into epithelia. During normal embryonic development, cells quickly organize epithelia; these epithelial cells participate in many of the key morphogenetic movements of gastrulation. This prompted the hypothesis that adherens junctions ought to be critical for normal embryonic development. Drosophila Armadillo, the homologue of vertebrate beta-catenin, is a core component of the adherens junction protein complex and has been hypothesized to be essential for adherens junction function in vivo. We have used an intermediate mutant allele of armadillo, armadilloXP33, to test these hypotheses in Drosophila embryos. Adherens junctions cannot assemble in the absence of Armadillo, leading to dramatic defects in cell-cell adhesion. The epithelial cells of the embryo lose adhesion to each other, round up, and apparently become mesenchymal. Mutant cells also lose their normal cell polarity. These disruptions in the integrity of epithelia block the appropriate morphogenetic movements of gastrulation. These results provide the first demonstration of the effect of loss of adherens junctions on Drosophila embryonic development.

An analysis of the inhibitory responses of dopamine and octopamine on Helix central neurons.

1. Electrophysiological recordings were made from identified neurons in the isolated suboesophageal ganglionic mass of Helix aspersa. Cells were voltage clamped at testing membrane potential. 2. Bath addition of 1 microM dibutyryl cAMP caused a time dependent enhancement of an evoked IPSP and the dopamine (DA) and octopamine (OA) induced outward currents obtained in these neurons. Forskolin, 0.1 microM, which enhances and MDL 12,330A, 0.12 microM, which depresses adenylate cyclase activity also modified these responses. 3. The DA and OA inhibitory responses were both shown to be potassium mediated events. They were preferentially antagonised by low micromolar concentrations of 4-aminopyridine. Two other potassium channel antagonists, tetraethylammonium and apamin had little effect on the DA and OA responses. 4. Cell sensitivity to DA and OA was greatly enhanced in calcium free/2 mM cobalt Ringer. The reversal potential of the DA response was shifted to a more negative value in calcium free Ringer. Sodium free Ringer was also found to alter the responses to DA or OA but those results were not consistent.

An analysis of the adenosine receptors responsible for modulation of an excitatory acetylcholine response on an identified Helix neuron.

1. Electrophysiological recordings were made from an identified neuron, F1, in the isolated suboesophageal ganglionic mass of Helix aspersa. 2. Bath applied adenosine (AD) (60-600 nM) depressed the depolarisation induced in the cell F1 by bath or iontophoretically applied acetylcholine (ACh). L-Phenylisopropyladenosine (L-PIA) also depressed the ACh response but NECA had no depressive effect. This effect of AD or L-PIA is inhibited by 12 microM 8-phenyltheophylline and is believed to be mediated by an A1 receptor. 3. If the proportion of the excitatory ACh response that was carried by calcium ions was increased, the percentage depression of this modified response by AD was significantly greater. 4. There was a residual current evoked by ACh in high calcium/sodium free Ringer. This ACh induced current was antagonized by 3 mM cobalt or 50 microM verapamil suggesting that it was calcium mediated. This residual current was also completely abolished by 0.6 microM AD. 5. Lower bath concentrations of AD (0.6-6 nM) and L-PIA than caused the depression of the ACh response and also adenosine triphosphate (ATP) (0.7 microM) and alpha,beta-methylene ATP (0.6 microM), enhanced the ACh D response. The relative potencies of AD and its two analogues 5'-N-ethylcarboxamideadenosine (NECA) and L-PIA in causing this enhancement of the ACh response were: NECA greater than AD greater than L-PIA. This is the potency ranking described for an A2 receptor.

Effect of silver nitrate on pregnancy termination in cynomolgus monkeys.

The effect of intrauterine injection of 1% silver nitrate on pregnancy was studied in 13 cynomolgus monkeys. The monkeys received, in a random manner, intrauterine injections of 1 ml of either 1% silver nitrate (n = 7) or 0.9% NaCl solution (n = 6) between days 27 and 43 of pregnancy. Silver nitrate injection resulted in vaginal bleeding, beginning 1 or 2 days after treatment, that lasted for an average of 5.3 days. In all of these cases, pregnancy was terminated. Injection of normal saline had variable sequelae, but four of six monkeys were delivered of healthy offspring. All of the silver nitrate-treated animals recycled and, after remating, two of the seven subsequently became pregnant again and were delivered of normal healthy infants. This study demonstrates the efficacy of intrauterine injection of 1% silver nitrate in terminating early pregnancy.

Intrauterine ethanol-induced termination of pregnancy in cynomolgus monkeys (Macaca fascicularis).

The effectiveness of locally applied ethanol in terminating early pregnancy in cynomolgus monkeys was studied. In a randomized trial ethanol (70 per cent, 1 ml.) or saline (0.9 per cent, 1 ml.) was injected extra-amniotically through a sterile blunt-end needdle inserted through the cervix into the uterine cavity. Six of seven monkeys receiving ethanol showed vaginal bleeding beginning one or two days after treatment. With the one failure, alcohol leakage occurred from the Luer-lock needle joint at the time of injection and the amount entering the uterus was unknown. No animal receiving saline showed any vaginal bleeding. The six monkeys showing vaginal bleeding were found to have nonenlarged or subnormally enlarged uteri one month after treatment. All monkeys subsequently resumed menstrual cycles within one year. Three monkeys became pregnant and were delivered at term of healthy offspring. Histologic evaluation of uteri from ethanol-treated monkeys revealed necrosis of decidua and, to a lesser degree, of the placenta one day after injection. The high efficacy of 70 per cent ethanol in inducing endometrial sloughing and the documentation of normal subsequent pregnancies in 50 per cent of treated monkeys make this technique worthy of consideration as a menstrual induction agent in women.