Identification of Eps15 as Antigen Recognized by the Monoclonal Antibodies aa2 and ab52 of the Wuerzburg Hybridoma Library against Drosophila Brain

Halder, Partho; Chen, Yi‐Chun; Brauckhoff, Janine; Hofbauer, Alois; Dabauvalle, Marie-Christine; Lewandrowski, Urs; Winkler, Christiane; Sickmann, Albert; Buchner, Erich

doi:10.1371/journal.pone.0029352

Cited by 4 publications

(6 citation statements)

References 44 publications

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“…The procedure for immunostaining of cryosections has recently been described [9]. Dilutions of antibodies used: nb169 1:2; antiserotonin (Millipore AB938) 1:500; anti-GFP (Life Technologies A11122) 1:3000; DAB staining with Vectastain kit (mouse) according to manufacturer's protocol.…”

Section: Cryosectionsmentioning

confidence: 99%

Correction: Identification and Structural Characterization of Interneurons of the Drosophila Brain by Monoclonal Antibodies of the Würzburg Hybridoma Library

Blanco-Redondo¹,

Bunz²,

Halder³

et al. 2013

PLoS ONE

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Several novel synaptic proteins have been identified by monoclonal antibodies (mAbs) of the Wu ¨rzburg hybridoma library generated against homogenized Drosophila brains, e.g. cysteine string protein, synapse-associated protein of 47 kDa, and Bruchpilot. However, at present no routine technique exists to identify the antigens of mAbs of our library that label only a small number of cells in the brain. Yet these antibodies can be used to reproducibly label and thereby identify these cells by immunohistochemical staining. Here we describe the staining patterns in the Drosophila brain for ten mAbs of the Wu ¨rzburg hybridoma library. Besides revealing the neuroanatomical structure and distribution of ten different sets of cells we compare the staining patterns with those of antibodies against known antigens and GFP expression patterns driven by selected Gal4 lines employing regulatory sequences of neuronal genes. We present examples where our antibodies apparently stain the same cells in different Gal4 lines suggesting that the corresponding regulatory sequences can be exploited by the split-Gal4 technique for transgene expression exclusively in these cells. The detection of Gal4 expression in cells labeled by mAbs may also help in the identification of the antigens recognized by the antibodies which then in addition to their value for neuroanatomy will represent important tools for the characterization of the antigens. Implications and future strategies for the identification of the antigens are discussed.

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Section: Cryosectionsmentioning

confidence: 99%

Correction: Identification and Structural Characterization of Interneurons of the Drosophila Brain by Monoclonal Antibodies of the Würzburg Hybridoma Library

Blanco-Redondo¹,

Bunz²,

Halder³

et al. 2013

PLoS ONE

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show abstract

“…Also, for none of the antibodies described here a reliable Western blot signal from brain homogenate could be obtained, defeating any attempts to identify the antigens by protein purification and mass spectrometry [26], [9]. Thus the classical approaches to the identification of antigens for mAbs cannot be applied.…”

Section: Resultsmentioning

confidence: 92%

“…Hybridoma cell culture, monoclonal antibody production, and isotyping have recently been described [9].…”

Section: Methodsmentioning

confidence: 99%

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Identification and Structural Characterization of Interneurons of the Drosophila Brain by Monoclonal Antibodies of the Würzburg Hybridoma Library

et al. 2013

Self Cite

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Several novel synaptic proteins have been identified by monoclonal antibodies (mAbs) of the Würzburg hybridoma library generated against homogenized Drosophila brains, e.g. cysteine string protein, synapse-associated protein of 47 kDa, and Bruchpilot. However, at present no routine technique exists to identify the antigens of mAbs of our library that label only a small number of cells in the brain. Yet these antibodies can be used to reproducibly label and thereby identify these cells by immunohistochemical staining. Here we describe the staining patterns in the Drosophila brain for ten mAbs of the Würzburg hybridoma library. Besides revealing the neuroanatomical structure and distribution of ten different sets of cells we compare the staining patterns with those of antibodies against known antigens and GFP expression patterns driven by selected Gal4 lines employing regulatory sequences of neuronal genes. We present examples where our antibodies apparently stain the same cells in different Gal4 lines suggesting that the corresponding regulatory sequences can be exploited by the split-Gal4 technique for transgene expression exclusively in these cells. The detection of Gal4 expression in cells labeled by mAbs may also help in the identification of the antigens recognized by the antibodies which then in addition to their value for neuroanatomy will represent important tools for the characterization of the antigens. Implications and future strategies for the identification of the antigens are discussed.

show abstract

“…Preparations of fly heads for cryo-sectioning requires removal of proboscis and air sacks below the brain, fixation for 3 h in fresh 4% buffered paraformaldehyde, freeze protection in 25% sucrose in Ringer's solution (128 mmol l −1 NaCl, 4.7 mmol l −1 KCl, 1.7 mmol l −1 CaCl, 0.7 mmol l −1 Na 2 HPO 4 , 0.35 mol l −1 KH 2 PO 4 , pH 7.4) overnight, and sectioning at 30 µm thickness using a cryostat microtome. Staining for fluorescence microscopy has been described previously (Halder et al, 2011;. Image stacks were taken at 2 µm z-steps using an Olympus Fluoview FV1000 confocal microscope equipped with an Olympus UPLSAPO 20× (air) objective.…”

Section: Western Blotting and Immunohistochemistrymentioning

confidence: 99%

Implications of the Sap47 null mutation for synapsin phosphorylation, longevity, climbing, and behavioural plasticity in adult Drosophila

Blanco-Redondo

Nuwal

Kneitz

et al. 2019

Journal of Experimental Biology

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The Sap47 gene of Drosophila melanogaster encodes a highly abundant 47 kDa synaptic vesicle-associated protein. Sap47 null mutants show defects in synaptic plasticity and larval olfactory associative learning but the molecular function of Sap47 at the synapse is unknown. We demonstrate that Sap47 modulates the phosphorylation of another highly abundant conserved presynaptic protein, synapsin. Site-specific phosphorylation of Drosophila synapsin has repeatedly been shown to be important for behavioural plasticity but it was not known where these phospho-synapsin isoforms are localized in the brain. Here, we report the distribution of serine-6-phosphorylated synapsin in the adult brain and show that it is highly enriched in rings of synapses in the ellipsoid body and in large synapses near the lateral triangle. The effects of knockout of Sap47 or synapsin on olfactory associative learning/memory support the hypothesis that both proteins operate in the same molecular pathway. We therefore asked if this might also be true for other aspects of their function. We show that knockout of Sap47 but not synapsin reduces lifespan, whereas knockout of Sap47 and synapsin, either individually or together, affects climbing proficiency, as well as plasticity in circadian rhythms and sleep. Furthermore, electrophysiological assessment of synaptic properties at the larval neuromuscular junction (NMJ) reveals increased spontaneous synaptic vesicle fusion and reduced paired pulse facilitation in Sap47 and synapsin single and double mutants. Our results imply that Sap47 and synapsin cooperate non-uniformly in the control of synaptic properties in different behaviourally relevant neuronal networks of the fruitfly.

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Identification of Eps15 as Antigen Recognized by the Monoclonal Antibodies aa2 and ab52 of the Wuerzburg Hybridoma Library against Drosophila Brain

Cited by 4 publications

References 44 publications

Correction: Identification and Structural Characterization of Interneurons of the Drosophila Brain by Monoclonal Antibodies of the Würzburg Hybridoma Library

Correction: Identification and Structural Characterization of Interneurons of the Drosophila Brain by Monoclonal Antibodies of the Würzburg Hybridoma Library

Identification and Structural Characterization of Interneurons of the Drosophila Brain by Monoclonal Antibodies of the Würzburg Hybridoma Library

Implications of the Sap47 null mutation for synapsin phosphorylation, longevity, climbing, and behavioural plasticity in adult Drosophila

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