PubMed Health⌕ Search

Biomedical subjects

Ernst Hafen

Publications and source records attributed to Ernst Hafen.

At least 19 recordsLinked to original sources
PubMed2006

The Cdi/TESK1 kinase is required for Sevenless signaling and epithelial organization in the Drosophila eye.

How cellular behaviors such as cell-to-cell communication, epithelial organization and cell shape reorganization are coordinated during development is poorly understood. The developing Drosophila eye offers an ideal model system to study these processes. Localized actin polymerization is required to constrict the apical surface of epithelial cells of the eye imaginal disc to maintain the refined arrangement of the developing ommatidia. The identity of each photoreceptor cell within the epithelium is determined by cell-to-cell contacts involving signal transduction events. The R7 photoreceptor cell requires the activity of the Sevenless RTK to adopt a proper cell fate. We performed an EP screen for negative regulators of this inductive process, and we identified the serine/threonine kinase Center divider (cdi) as a suppressor of the phenotype caused by an activated Sevenless receptor. Cdi is homologous to the human testis-specific kinase 1 (TESK1), a member of the LIM kinases involved in cytoskeleton control through ADF/cofilin phosphorylation. We have analyzed the effects of gain- and loss-of-function of cdi and found alterations in actin organization and in the adherens junctions proteins DE-cadherin and beta-catenin, as well as in Sevenless apical localization. Interference with the function of the ADF/cofilin phosphatase Slingshot (ssh), which antagonizes Cdi, also results in a suppression of signaling triggered by the Sevenless RTK. Our results reveal a critical interplay between the localization of molecules involved in epithelial organization and signal transduction.

Actins↗
PubMed2006

Csk differentially regulates Src64 during distinct morphological events in Drosophila germ cells.

The Src family protein tyrosine kinases (SFKs) are crucial regulators of cellular morphology. In Drosophila, Src64 controls complex morphological events that occur during oogenesis. Recent studies have identified key Src64-dependent mechanisms that regulate actin cytoskeletal dynamics during the growth of actin-rich ring canals, which act as intercellular bridges between germ cells. By contrast, the molecular mechanisms that regulate Src64 activity levels and potential roles for Src64 in additional morphological events in the ovary have not been defined. In this report, we demonstrate that regulation of Src64 by Drosophila C-terminal-Src Kinase (Csk) contributes to the packaging of germline cysts by overlying somatic follicle cells during egg chamber formation. These results uncover novel roles for both Csk and Src64 in a dynamic event that involves adhesion, communication between cell types and control of cell motility. Strikingly, Src64 and Csk function in the germline to control packaging, not in migrating follicle cells, suggesting novel functions for this signaling cassette in regulating dynamic adhesion. In contrast to the role played by Csk in the regulation of Src64 activity during packaging, Csk is dispensable for ring canal growth control, indicating that distinct mechanisms control Src64 activity during different morphological events.

Animals↗
PubMed2006

Overgrowth caused by misexpression of a microRNA with dispensable wild-type function.

MicroRNAs (miRNAs) represent an abundant class of non-coding RNAs that negatively regulate gene expression, primarily at the post-transcriptional level. miRNA genes are frequently located in proximity to fragile chromosomal sites associated with cancers and amplification of a miRNA cluster has been correlated with the etiology of lymphomas and solid tumors. The oncogenic potential of a miRNA polycistron has recently been demonstrated in vivo. Here, we show that misexpression of the Drosophila miRNA mirvana/mir-278 in the developing eye causes massive overgrowth, in part due to inhibition of apoptosis. A single base substitution affecting the mature miRNA blocks the gain-of-function phenotype but is not associated with a detectable reduction-of-function phenotype when homozygous. This result demonstrates that misexpressed miRNAs may acquire novel functions that cause unscheduled proliferation in vivo and thus exemplifies the potential of miRNAs to promote tumor formation.

Animals↗
PubMed2005

A novel, evolutionarily conserved protein phosphatase complex involved in cisplatin sensitivity.

Using a combination of tandem affinity purification tagging and mass spectrometry, we characterized a novel, evolutionarily conserved protein phosphatase 4 (PP4)-containing complex (PP4cs, protein phosphatase 4, cisplatin-sensitive complex) that plays a critical role in the eukaryotic DNA damage response. PP4cs is comprised of the catalytic subunit PP4C; a known regulatory subunit, PP4R2; and a novel protein that we termed PP4R3. The Saccharomyces cerevisiae PP4R3 ortholog Psy2 was identified previously in a screen for sensitivity to the DNA-damaging agent and anticancer drug cisplatin. We demonstrated that deletion of any of the PP4cs complex orthologs in S. cerevisiae elicited cisplatin hypersensitivity. Furthermore human PP4R3 complemented the yeast psy2 deletion, and Drosophila melanogaster lacking functional PP4R3 (flfl) exhibited cisplatin hypersensitivity, suggesting a highly conserved role for PP4cs in DNA damage repair. Finally we found that PP4R3 may target PP4cs to the DNA damage repair machinery at least in part via an interaction with Rad53 (CHK2).

Cell Line↗
PubMed2005

Longer lifespan, altered metabolism, and stress resistance in Drosophila from ablation of cells making insulin-like ligands.

The insulin/insulin-like growth factor-like signaling pathway, present in all multicellular organisms, regulates diverse functions including growth, development, fecundity, metabolic homeostasis, and lifespan. In flies, ligands of the insulin/insulin-like growth factor-like signaling pathway, the Drosophila insulin-like peptides, regulate growth and hemolymph carbohydrate homeostasis during development and are expressed in a stage- and tissue-specific manner. Here, we show that ablation of Drosophila insulin-like peptide-producing median neurosecretory cells in the brain leads to increased fasting glucose levels in the hemolymph of adults similar to that found in diabetic mammals. They also exhibit increased storage of lipid and carbohydrate, reduced fecundity, and reduced tolerance of heat and cold. However, the ablated flies show an extension of median and maximal lifespan and increased resistance to oxidative stress and starvation.

Animals↗
PubMed2005

The Drosophila mitochondrial ribosomal protein mRpL12 is required for Cyclin D/Cdk4-driven growth.

The Drosophila melanogaster cyclin-dependent protein kinase complex CycD/Cdk4 stimulates both cell cycle progression and cell growth (accumulation of mass). CycD/Cdk4 promotes cell cycle progression via the well-characterized RBF/E2F pathway, but our understanding of how growth is stimulated is still limited. To identify growth regulatory targets of CycD/Cdk4, we performed a loss-of-function screen for modifiers of CycD/Cdk4-induced overgrowth of the Drosophila eye. One mutation that suppressed CycD/Cdk4 was in a gene encoding the mitochondrial ribosomal protein, mRpL12. We show here that mRpL12 is required for CycD/Cdk4-induced cell growth. Cells homozygous mutant for mRpL12 have reduced mitochondrial activity, and exhibit growth defects that are very similar to those of cdk4 null cells. CycD/Cdk4 stimulates mitochondrial activity, and this is mRpL12 dependent. Hif-1 prolyl hydroxylase (Hph), another effector of CycD/Cdk4, regulates growth and is required for inhibition of the hypoxia-inducible transcription factor 1 (Hif-1). Both functions depend on mRpL12 dosage, suggesting that CycD/Cdk4, mRpL12 and Hph function together in a common pathway that controls cell growth via affecting mitochondrial activity.

Animals↗
PubMed2005

A universal method for automated gene mapping.

Small insertions or deletions (InDels) constitute a ubiquituous class of sequence polymorphisms found in eukaryotic genomes. Here, we present an automated high-throughput genotyping method that relies on the detection of fragment-length polymorphisms (FLPs) caused by InDels. The protocol utilizes standard sequencers and genotyping software. We have established genome-wide FLP maps for both Caenorhabditis elegans and Drosophila melanogaster that facilitate genetic mapping with a minimum of manual input and at comparatively low cost.

Animals↗
PubMed2005

Diet-dependent effects of the Drosophila Mnk1/Mnk2 homolog Lk6 on growth via eIF4E.

The control of cellular growth is tightly linked to the regulation of protein synthesis. A key function in translation initiation is fulfilled by the 5' cap binding eukaryotic initiation factor 4E (eIF4E), and dysregulation of eIF4E is associated with malignant transformation and tumorigenesis . In mammals, the activity of eIF4E is modulated by phosphorylation at Ser209 by mitogen-activated protein kinases (MAPK)-interacting kinases 1 and 2 (Mnk1 and Mnk2) , which themselves are activated by ERK and p38 MAPK in response to mitogens, cytokines or cellular stress . Whether phosphorylation of eIF4E at Ser209 exerts a positive or inhibitory effect on translation efficiency has remained controversial. Here we provide a genetic characterization of the Drosophila homolog of Mnk1/2, Lk6. Lk6 function is dispensable under a high protein diet, consistent with the recent finding that mice lacking both Mnk1 and Mnk2 are not growth-impaired . Interestingly, loss of Lk6 function causes a significant growth reduction when the amino acid content in the diet is reduced. Overexpression of Lk6 also results in growth inhibition in an eIF4E-dependent manner. We propose a model of eIF4E regulation that may reconcile the contradictory findings with regard to the role of phosphorylation by Mnk1/2.

Amino Acid Sequence↗
PubMed2005

Susi, a negative regulator of Drosophila PI3-kinase.

The Phosphatidylinositol-3 kinase/Protein Kinase B (PI3K/PKB) signaling pathway controls growth, metabolism, and lifespan in animals, and deregulation of its activity is associated with diabetes and cancer in humans. Here, we describe Susi, a coiled-coil domain protein that acts as a negative regulator of insulin signaling in Drosophila. Whereas loss of Susi function increases body size, overexpression of Susi reduces growth. We provide genetic evidence that Susi negatively regulates dPI3K activity. Susi directly binds to dP60, the regulatory subunit of dPI3K. Since Susi has no overt similarity to known inhibitors of PI3K/PKB signaling, it defines a novel mechanism by which this signaling cascade is kept in check. The fact that Susi is expressed in a circadian rhythm, with highest levels during the night, suggests that Susi attenuates insulin signaling during the fasting period.

Animals↗
PubMed2004

Integration with the human genome of peptide sequences obtained by high-throughput mass spectrometry.

A crucial aim upon the completion of the human genome is the verification and functional annotation of all predicted genes and their protein products. Here we describe the mapping of peptides derived from accurate interpretations of protein tandem mass spectrometry (MS) data to eukaryotic genomes and the generation of an expandable resource for integration of data from many diverse proteomics experiments. Furthermore, we demonstrate that peptide identifications obtained from high-throughput proteomics can be integrated on a large scale with the human genome. This resource could serve as an expandable repository for MS-derived proteome information.

Amino Acid Sequence↗
PubMed2004

Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex.

Mammalian target of rapamycin (mTOR) is a central regulator of protein synthesis whose activity is modulated by a variety of signals. Energy depletion and hypoxia result in mTOR inhibition. While energy depletion inhibits mTOR through a process involving the activation of AMP-activated protein kinase (AMPK) by LKB1 and subsequent phosphorylation of TSC2, the mechanism of mTOR inhibition by hypoxia is not known. Here we show that mTOR inhibition by hypoxia requires the TSC1/TSC2 tumor suppressor complex and the hypoxia-inducible gene REDD1/RTP801. Disruption of the TSC1/TSC2 complex through loss of TSC1 or TSC2 blocks the effects of hypoxia on mTOR, as measured by changes in the mTOR targets S6K and 4E-BP1, and results in abnormal accumulation of Hypoxia-inducible factor (HIF). In contrast to energy depletion, mTOR inhibition by hypoxia does not require AMPK or LKB1. Down-regulation of mTOR activity by hypoxia requires de novo mRNA synthesis and correlates with increased expression of the hypoxia-inducible REDD1 gene. Disruption of REDD1 abrogates the hypoxia-induced inhibition of mTOR, and REDD1 overexpression is sufficient to down-regulate S6K phosphorylation in a TSC1/TSC2-dependent manner. Inhibition of mTOR function by hypoxia is likely to be important for tumor suppression as TSC2-deficient cells maintain abnormally high levels of cell proliferation under hypoxia.

3T3 Cells↗
PubMed2004

The hypoxia-induced paralogs Scylla and Charybdis inhibit growth by down-regulating S6K activity upstream of TSC in Drosophila.

Diverse extrinsic and intrinsic cues must be integrated within a developing organism to ensure appropriate growth at the cellular and organismal level. In Drosophila, the insulin receptor/TOR/S6K signaling network plays a fundamental role in the control of metabolism and cell growth. Here we show that scylla and charybdis, two homologous genes identified as growth suppressors in an EP (enhancer/promoter) overexpression screen, act as negative regulators of growth. The simultaneous loss of both genes generates flies that are more susceptible to reduced oxygen concentrations (hypoxia) and that show mild overgrowth phenotypes. Conversely, scylla or charybdis overactivation reduces growth. Growth inhibition is associated with a reduction in S6K but not PKB/Akt activity. Together, genetic and biochemical analysis places Scylla/Charybdis downstream of PKB and upstream of TSC. Furthermore, we show that scylla and charybdis are induced under hypoxic conditions and that scylla is a target of Drosophila HIF-1 (hypoxia-inducible factor-1) like its mammalian counterpart RTP801/REDD1, thus establishing a potential cross-talk between growth and oxygen sensing.

Amino Acid Sequence↗
PubMed2004

A reverse genetic screen in Drosophila using a deletion-inducing mutagen.

We report the use of the cross-linking drug hexamethylphosphoramide (HMPA), which introduces small deletions, as a mutagen suitable for reverse genetics in the model organism Drosophila melanogaster. A compatible mutation-detection method based on resolution of PCR fragment-length polymorphisms on standard DNA sequencers is implemented. As the spectrum of HMPA-induced mutations is similar in a variety of organisms, it should be possible to transfer this mutagenesis and detection procedure to other model systems.

Animals↗
PubMed2004

An efficient method to generate chromosomal rearrangements by targeted DNA double-strand breaks in Drosophila melanogaster.

Homologous recombination (HR) is an indispensable tool to modify the genome of yeast and mammals. More recently HR is also being used for gene targeting in Drosophila. Here we show that HR can be used efficiently to engineer chromosomal rearrangements such as pericentric and paracentric inversions and translocations in Drosophila. Two chromosomal double-strand breaks (DSBs), introduced by the rare-cutting I-SceI endonuclease on two different mobile elements sharing homologous sequences, are sufficient to promote rearrangements at a frequency of 1% to 4%. Such rearrangements, once generated by HR, can be reverted by Cre recombinase. However, Cre-mediated recombination efficiency drops with increasing distance between recombination sites, unlike HR. We therefore speculate that physical constraints on chromosomal movement are modulated during DSB repair, to facilitate the homology search throughout the genome.

Animals↗
PubMed2004

Long-lived Drosophila with overexpressed dFOXO in adult fat body.

Reduced activity of the insulin/insulin-like growth factor signaling (IIS) pathway increases life-span in diverse organisms. We investigated the timing of the effect of reduced IIS on life-span and the role of a potential target tissue, the fat body. We overexpressed dFOXO, a downstream effector of IIS, in the adult Drosophila fat body, which increased life-span and reduced fecundity of females but had no effect on male life-span. The role of FOXO transcription factors and the adipose tissue are therefore evolutionarily conserved in the regulation of aging, and reduction of IIS in the adult is sufficient to mediate its effects on life-span and fecundity.

Animals↗
PubMed2004

Targeted expression of the class II phosphoinositide 3-kinase in Drosophila melanogaster reveals lipid kinase-dependent effects on patterning and interactions with receptor signaling pathways.

Phosphoinositide 3-kinases (PI3Ks) can be divided into three distinct classes (I, II, and III) on the basis of their domain structures and the lipid signals that they generate. Functions have been assigned to the class I and class III enzymes but have not been established for the class II PI3Ks. We have obtained the first evidence for a biological function for a class II PI3K by expressing this enzyme during Drosophila melanogaster development and by using deficiencies that remove the endogenous gene. Wild-type and catalytically inactive PI3K_68D transgenes have opposite effects on the number of sensory bristles and on wing venation phenotypes induced by modified epidermal growth factor (EGF) receptor signaling. These results indicate that the endogenous PI3K_68D may act antagonistically to the EGF receptor-stimulated Ras-mitogen-activated protein kinase pathway and downstream of, or parallel to, the Notch receptor. A class II polyproline motif in PI3K_68D can bind the Drk adaptor protein in vitro, primarily via the N-terminal SH3 domain of Drk. Drk may thus be important for the localization of PI3K_68D, allowing it to modify signaling pathways downstream of cell surface receptors. The phenotypes obtained are markedly distinct from those generated by expression of the Drosophila class I PI3K, which affects growth but not pattern formation.

Animals↗
PubMed2004

The DrosDel collection: a set of P-element insertions for generating custom chromosomal aberrations in Drosophila melanogaster.

We describe a collection of P-element insertions that have considerable utility for generating custom chromosomal aberrations in Drosophila melanogaster. We have mobilized a pair of engineered P elements, p[RS3] and p[RS5], to collect 3243 lines unambiguously mapped to the Drosophila genome sequence. The collection contains, on average, an element every 35 kb. We demonstrate the utility of the collection for generating custom chromosomal deletions that have their end points mapped, with base-pair resolution, to the genome sequence. The collection was generated in an isogenic strain, thus affording a uniform background for screens where sensitivity to genetic background is high. The entire collection, along with a computational and genetic toolbox for designing and generating custom deletions, is publicly available. Using the collection it is theoretically possible to generate >12,000 deletions between 1 bp and 1 Mb in size by simple eye color selection. In addition, a further 37,000 deletions, selectable by molecular screening, may be generated. We are now using the collection to generate a second-generation deficiency kit that is precisely mapped to the genome sequence.

Animals↗