In Vivo Analysis of Proteomes and Interactomes Using Parallel Affinity Capture (iPAC) Coupled to Mass Spectrometry

Rees, Johanna S.; Lowe, Nick; Armean, Irina M.; Roote, John; Johnson, Glynnis; Drummond, Emma; Spriggs, Helen F.; Ryder, Edward; Russell, Steven; Johnston, Daniel St; Lilley, Kathryn S.

doi:10.1074/mcp.m110.002386

Cited by 73 publications

(89 citation statements)

References 29 publications

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“…8). We used a gene trap that generates a YFPNotch fusion protein that is functional and contain the YFP in the extra-cellular domain (Rees et al, 2011). The combination with an antibody directed against NICD revealed that the enlarged endosomes are positive for NECD and NICD and therefore probably contain the full-length receptor and also Dl (Fig.…”

Section: Loss Of Lgd Function In the Follicle Epithelium Results In Amentioning

confidence: 99%

Activation of Notch in lgd mutant cells requires the fusion of late endosomes with the lysosome

Schneider

Troost

Grawe

et al. 2013

Journal of Cell Science

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SummaryThe tumour suppressor Lethal (2) giant discs (Lgd) is a regulator of endosomal trafficking of the Notch signalling receptor as well as other transmembrane proteins in Drosophila. The loss of its function results in an uncontrolled ligand-independent activation of the Notch signalling receptor. Here, we investigated the consequences of loss of lgd function and the requirements for the activation of Notch. We show that the activation of Notch in lgd cells is independent of Kuz and dependent on c-secretase. We found that the lgd cells have a defect that delays degradation of transmembrane proteins, which are residents of the plasma membrane. Furthermore, our results show that the activation of Notch in lgd cells occurs in the lysosome. By contrast, the pathway is activated at an earlier phase in mutants of the gene that encodes the ESCRT-III component Shrub, which is an interaction partner of Lgd. We further show that activation of Notch appears to be a general consequence of loss of lgd function. In addition, electron microscopy of lgd cells revealed that they contain enlarged multi-vesicular bodies. The presented results further elucidate the mechanism of uncontrolled Notch activation upon derailed endocytosis.

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Section: Loss Of Lgd Function In the Follicle Epithelium Results In Amentioning

confidence: 99%

Activation of Notch in lgd mutant cells requires the fusion of late endosomes with the lysosome

Schneider

Troost

Grawe

et al. 2013

Journal of Cell Science

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“…1) 14 . We focused on IR25a, a member of a divergent subfamily of ionotropic glutamate receptors and verified the interaction by co-immunoprecipitation after overexpressing IR25a and NOCTE in all clock cells using tim-gal4 (Extended Data Fig.…”

Section: Main Textmentioning

confidence: 99%

“…Co-immunoprecipitation experiments were performed as described 14 . For each protein purification, 200-300 mg wet-weight of heads from gmr-gal4 flies expressing UAS-nocte-flag (FSNS and FSNH transgenics were used in 2 independent experiments) or gmr-gal4 alone (negative controls) were collected on dry ice and manually homogenized with a 2 ml Dounce homogenizer (Fisher) in 1 ml of extraction buffer (final protein concentration 5mg/ml extraction buffer) containing 50 mM Tris, pH 7.5, 125 mM NaCl, 1.5 mM MgCl2, 1mM EDTA, 5% Glycerol, 0.4% NP-40, and 0.1% Tween 20.…”

Section: Co-immunoprecipitationmentioning

confidence: 99%

“…Briefly, eluates from the tagged line and untagged control flies, were processed as described 14 . The only deviation from the method described was that peptides were applied to a 180 μm x 20 mm (5 μm particle size) C18 trap column (Waters UPLC Trap Symmetry) coupled to a nanoAcquity UPLC system (Waters) using 0.1% formic acid in water (Buffer A) at a flow rate of 10 μl/min.…”

Section: Co-immunoprecipitationmentioning

confidence: 99%

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Drosophila Ionotropic Receptor 25a mediates circadian clock resetting by temperature

Chen

Buhl

et al. 2015

Nature

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Summary:Circadian clocks are endogenous timers adjusting behaviour and physiology with the solar day 1 .Synchronized circadian clocks improve fitness 2 and are crucial for our physical and mental wellbeing 3 . Visual and non-visual photoreceptors are responsible for synchronizing circadian clocks to light 4,5 , but clock-resetting is also achieved by alternating day and night temperatures with only 2°-4°C difference [6][7][8] . This temperature sensitivity is remarkable considering that the circadian clock period (~24 h) is largely independent of surrounding ambient temperatures 1,8 .Here we show that Drosophila Ionotropic Receptor 25a (IR25a) is required for behavioural synchronization to low-amplitude temperature cycles. This channel is expressed in sensory neurons of internal stretch receptors previously implicated in temperature synchronization of the circadian clock 9 . IR25a is required for temperature-synchronized clock protein oscillations in subsets of central clock neurons. Extracellular leg nerve recordings reveal temperature -and IR25a-dependent sensory responses, and IR25a mis-expression confers temperature-dependent firing of heterologous neurons. We propose that IR25a is part of an input pathway to the circadian clock that detects small temperature differences. This pathway operates in the absence of known 'hot' and 'cold' sensors in the Drosophila antenna 10,11 , revealing the existence of novel periphery-to-brain temperature signalling channels. Main Text:In Drosophila, daily activity rhythms are controlled by a network of ~150 clock neurons expressing the clock genes period (per) and timeless (tim). These encode repressor proteins that 3 negatively feedback on their own promoters resulting in 24 h oscillations of clock molecules.Temperature cycles (TC) synchronise molecular clocks present in peripheral appendages in a tissue-autonomous manner 9,12 , while synchronization of clock neurons in the brain largely depends on peripheral temperature receptors located in the chordotonal organs (ChO) and the ChO-expressed gene nocte 9,12,13 .To discover novel factors involved in temperature entrainment, we identified NOCTEinteracting proteins by co-immunoprecipitation and mass-spectrometry (Extended Data Tab. 1) 14 . We focused on IR25a, a member of a divergent subfamily of ionotropic glutamate receptors and verified the interaction by co-immunoprecipitation after overexpressing IR25a and NOCTE in all clock cells using tim-gal4 (Extended Data Fig. 1a). IR25a is expressed in different populations of sensory neurons, including those in the antenna and labellum [15][16][17] . In the olfactory system IR25a acts as a co-receptor with different odour-sensing IRs 15 .To investigate if IR25a is co-expressed with nocte in ChO we analysed IR25a expression in femur and antennal ChO using an IR25a-gal4 line 15 (Extended Data Fig. 2a). IR25a-gal4 driven mCD8-GFP labelled subsets of ChO neurons in the femur, overlapping substantially with nompC-QF driven QUAS-Tomato signals (Fig. 1 a-c). nompC-QF is expressed in larv...

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“…The following fly strains were used: repo-GAL4 (Sepp et al, 2001); spg-GAL4 (Schwabe et al, 2005); UAS-mCD8::GFP (Lee and Luo, 1999); UAS-mCD8::RFP (a gift from Dr Elizabeth Gavis, Princeton University); UAS-Dicer2 (Dietzl et al, 2007); mys 1 (Bunch et al, 1992); FRT19A,tubP-GAL80,hsFLP,w* ( Lee and Luo, 1999); repo-FLP (Stork et al, 2008); Gli-lacZ (Auld et al, 1995); Perlecan::GFP; NrxIV::GFP; ILK::GFP; αPS1::YFP and αPS2::YFP (Rees et al, 2011); talin::GFP (a gift from Dr Guy Tanentzapf, University of British Columbia). The UAS-RNAi strains used were: UAS-βPS-RNAi(GD) (GD15002), UAS-talin-RNAi(GD) (GD12050), UAS-αPS2-RNAi(GD) (GD44885), UAS-αPS3-RNAi(GD) (GD4891), UAS-NrxIV-RNAi(GD) (GD2436) (Dietzl et al, 2007); UAS-βPS-RNAi(R1) (1560R-1), UAS-αPS2-RNAi(R1) (9623R-2), UAS-αPS3-RNAi(R1) (8095R-1) and UAS-talin-RNAi(R1) (6831R-1) from the National Institute of Genetics, Japan.…”

Section: Fly Strains and Geneticsmentioning

confidence: 99%

Loss of focal adhesions in glia disrupts both glial and photoreceptor axon migration in the Drosophila visual system

et al. 2014

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Many aspects of glial development are regulated by extracellular signals, including those from the extracellular matrix (ECM). Signals from the ECM are received by cell surface receptors, including the integrin family. Previously, we have shown that Drosophila integrins form adhesion complexes with Integrin-linked kinase and talin in the peripheral nerve glia and have conserved roles in glial sheath formation. However, integrin function in other aspects of glial development is unclear. The Drosophila eye imaginal disc (ED) and optic stalk (OS) complex is an excellent model with which to study glial migration, differentiation and glia-neuron interactions. We studied the roles of the integrin complexes in these glial developmental processes during OS/eye development. The common beta subunit βPS and two alpha subunits, αPS2 and αPS3, are located in puncta at both glia-glia and glia-ECM interfaces. Depletion of βPS integrin and talin by RNAi impaired the migration and distribution of glia within the OS resulting in morphological defects. Reduction of integrin or talin in the glia also disrupted photoreceptor axon outgrowth leading to axon stalling in the OS and ED. The neuronal defects were correlated with a disruption of the carpet glia tube paired with invasion of glia into the core of the OS and the formation of a glial cap. Our results suggest that integrin-mediated extracellular signals are important for multiple aspects of glial development and non-autonomously affect axonal migration during Drosophila eye development.

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In Vivo Analysis of Proteomes and Interactomes Using Parallel Affinity Capture (iPAC) Coupled to Mass Spectrometry

Cited by 73 publications

References 29 publications

Activation of Notch in lgd mutant cells requires the fusion of late endosomes with the lysosome

Activation of Notch in lgd mutant cells requires the fusion of late endosomes with the lysosome

Drosophila Ionotropic Receptor 25a mediates circadian clock resetting by temperature

Loss of focal adhesions in glia disrupts both glial and photoreceptor axon migration in the Drosophila visual system

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