Subcellular localization of Hairless protein shows a major focus of activity within the nucleus

Maier, Dieter; Nagel, Anja C.; Johannes, Bernd; Preiss, Anette

doi:10.1016/s0925-4773(99)00208-7

Cited by 47 publications

(70 citation statements)

References 19 publications

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“…Fly stocks were obtained from the Bloomington Stock Center and the Vienna Drosophila RNAi Center (VDRC), or alternative published sources as noted: P{EPgy2}Eaat1 EY20741 ; Df(2L)30A-C (here denoted Eaat1 Df ); UAS-mCD8::RFP; UAS-Eaat1; UAS-Pros RNAi (VDRC ID 101477); Eaat1-Gal4 (Rival et al, 2004); htl-Gal4 (Shishido et al, 1997); Repo-Gal4 (Sepp and Auld, 2003); tub-Gal80 ts (McGuire et al, 2003); UAS-N ICD (Go et al, 1998); UAS-Hairless (Maier et al, 1999); UAS-Tom (Lai et al, 2000); UAS-Eaat1::GFP (Rival et al, 2004); UAS-mCD8::GFP (Lee and Luo, 1999); UAS-nuclearGFP (van Meyel et al, 2003); Su(H)-lacZ (Griffiths et al, 2007); and fng L73 and fng 13 (Irvine and Wieschaus, 1994;Correia et al, 2003). To create CG31235-nuclear green fluorescent protein (nGFP) and CG31235-Gal4, we PCR amplified 3004 base pairs (bp) of regulatory sequences located immediately upstream of CG31235 using the primers 5Ј-CATAGTGCATTGGTGAGGTGT-3Ј and 5Ј-TACTGGGTGCGCGTTAGGTC-3Ј.…”

Section: Methodsmentioning

confidence: 99%

DrosophilaGlial Glutamate Transporter Eaat1 Is Regulated by Fringe-Mediated Notch Signaling and Is Essential for Larval Locomotion

Stacey¹,

Muraro²,

Peco³

et al. 2010

J. Neurosci.

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In the mammalian CNS, glial cells expressing excitatory amino acid transporters (EAATs) tightly regulate extracellular glutamate levels to control neurotransmission and protect neurons from excitotoxic damage. Dysregulated EAAT expression is associated with several CNS pathologies in humans, yet mechanisms of EAAT regulation and the importance of glutamate transport for CNS development and function in vivo remain incompletely understood. Drosophila is an advanced genetic model with only a single high-affinity glutamate transporter termed Eaat1. We found that Eaat1 expression in CNS glia is regulated by the glycosyltransferase Fringe, which promotes neuron-to-glia signaling through the Delta-Notch ligand-receptor pair during embryogenesis. We made Eaat1 loss-of-function mutations and found that homozygous larvae could not perform the rhythmic peristaltic contractions required for crawling. We found no evidence for excitotoxic cell death or overt defects in the development of neurons and glia, and the crawling defect could be induced by postembryonic inactivation of Eaat1. Eaat1 fully rescued locomotor activity when expressed in only a limited subpopulation of glial cells situated near potential glutamatergic synapses within the CNS neuropil. Eaat1 mutants had deficits in the frequency, amplitude, and kinetics of synaptic currents in motor neurons whose rhythmic patterns of activity may be regulated by glutamatergic neurotransmission among premotor interneurons; similar results were seen with pharmacological manipulations of glutamate transport. Our findings indicate that Eaat1 expression is promoted by Fringe-mediated neuron-glial communication during development and suggest that Eaat1 plays an essential role in regulating CNS neural circuits that control locomotion in Drosophila.

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Section: Methodsmentioning

confidence: 99%

DrosophilaGlial Glutamate Transporter Eaat1 Is Regulated by Fringe-Mediated Notch Signaling and Is Essential for Larval Locomotion

Stacey¹,

Muraro²,

Peco³

et al. 2010

J. Neurosci.

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“…WT proteins were extracted from S2 cell culture, staged embryos, dissected imaginal discs or ovaries, or whole larvae (4,6). Cytoplasmic and nuclear extracts were prepared from 0-to 3-h-old Drosophila embryos.…”

Section: Methodsmentioning

confidence: 99%

“…Ab stainings of imaginal tissues and Western blots were performed as described (4,6,8). Production of central Hairless domain A-GST fusion protein was as described (4).…”

Section: Methodsmentioning

confidence: 99%

“…Antisera directed against Hairless protein fragments A and NTH were raised in rabbits (pAB Productions, Munich). Anti-A Abs detect WT protein preferentially in the cytoplasm and only overexpressed protein in the nucleus (4,6). Anti-␤-galactosidase and anti-firefly luciferase Abs were purchased from Promega and Chemicon, respectively; anti-phospho-Histone H3 was obtained from Upstate Biotechnology (Lake Placid, NY).…”

Section: Methodsmentioning

confidence: 99%

“…In agreement with its role as a general Notch antagonist, H protein is ubiquitously expressed throughout development. Like its target Su(H), H protein is cytoplasmic and nuclear with a major focus of activity within the nucleus (3,4). The antagonistic activity of H appears largely dose sensitive.…”

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Two isoforms of the Notch antagonist Hairless are produced by differential translation initiation

Maier

Nagel

Preiss

2002

Proc. Natl. Acad. Sci. U.S.A.

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The Notch-signaling pathway controls cellular differentiation, including proliferation and cell death in all higher metazoans (including flies and men show that the smaller isoform derives from an internal ribosome entry site (IRES) within the ORF. The IRES is active in a heterologous assay and contains an essential, conserved structural element. The two Hairless isoforms have residual activity in vivo which is, however, reduced compared to a combination of both, which implies that both protein isoforms are necessary for WT function. In larval tissues, translation of the two isoforms is cell-cycle regulated: whereas the H p150 isoform is translated during interphase, H p120 is enriched during mitosis. Thus, the presence of either H isoform throughout the cell cycle allows efficient inhibition of Notch-regulated cell proliferation.

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Genetic screen for modifiers of the rough eye phenotype resulting from overexpression of the notch antagonist hairless in drosophila

Schreiber¹,

Preiss²,

Nagel³

et al. 2002

Genesis

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Hairless was identified as antagonist in the Notch signaling pathway based on genetic interactions. Molecularly, Hairless inhibits Notch target gene activation by directly binding to the Notch signal transducer Su(H). Additional functional domains apart from the Su(H) binding domain, however, suggest additional roles for the Hairless protein. To further our understanding of Hairless functions, we have performed a genetic screen for modifiers of a rough eye phenotype caused by overexpression of Hairless during eye development. A number of enhancers were identified that comprise mutations in components of Notch- and EGFR-signaling pathways, some unknown genes and the gene rugose. Mutant alleles of rugose display manifold genetic interactions with mutants in Notch and EGFR signaling pathway components. Accordingly, the rugose eye phenotype is rescued by Hairless and enhanced by Delta. Molecularly, interactions might occur at the protein level because rugose appears not to be a direct transcriptional target of Notch.

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Subcellular localization of Hairless protein shows a major focus of activity within the nucleus

Cited by 47 publications

References 19 publications

DrosophilaGlial Glutamate Transporter Eaat1 Is Regulated by Fringe-Mediated Notch Signaling and Is Essential for Larval Locomotion

DrosophilaGlial Glutamate Transporter Eaat1 Is Regulated by Fringe-Mediated Notch Signaling and Is Essential for Larval Locomotion

Two isoforms of the Notch antagonist Hairless are produced by differential translation initiation

Genetic screen for modifiers of the rough eye phenotype resulting from overexpression of the notch antagonist hairless in drosophila

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