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A genetic screen identifies Tor as an interactor of VAPB in a Drosophila model of amyotrophic lateral sclerosis.

Deivasigamani S, Verma HK, Ueda R, Ratnaparkhi A, Ratnaparkhi GS - Biol Open (2014)

Bottom Line: One key modifier identified was Tor, whose knockdown reversed the large bouton phenotype associated with VAP(P58S) expression in neurons.A similar reversal was seen by over-expressing Tuberous Sclerosis Complex (Tsc1,2) that negatively regulates TOR signaling as also by reduction of S6K activity.In comparison, the small bouton phenotype associated with VAP(wt) expression was reversed with Tsc1 knock down as well as S6K-CA expression.

View Article: PubMed Central - PubMed

Affiliation: Indian Institute of Science Education and Research, Pune 411021, India.

No MeSH data available.


Related in: MedlinePlus

The Drosophila NMJ is used to screen for interaction of modifiers with VAP(P58S).Thirteen of the 103 modifiers discovered in our macro chaetae screen were tested in the larval muscle-4 NMJ for interaction with VAP(P58S). For this and subsequent figures, approximately 15 NMJs were dissected, stained (anti-HRP, red), imaged and measured for the average size of boutons (displayed in yellow at the top RHS of each figure). (A) A wild type (C155-Gal4/+) NMJ. The average bouton size is 3.987 (±0.03). Shown here and below is Z-series of a synapse rendered as maximum intensity projection. (B) Expression of UAS-VAP(P58S), using the C155-Gal4 driver increases the size of the boutons. (C–F) Knockdown of CG6048 (C), CG9172 (D), TBPH (E) and Nup75 (F) in a C155/UAS-VAP(P58S) background reverses the effect of the VAP(P58S) over-expression and rescues bouton size to wild-type levels. (G) Quantitation of bouton size (in micrometer) for the RNAi knockdown of each gene tested in a VAP(P58S) background (top panel) and in a wild type background (bottom panel). For this and subsequent NMJ figures, error bars represent standard errors of the mean (SEM). Scale bar: 5 µm. * indicates a p-value<0.01 (but >0.001), while ** indicates a p-value of <0.001.
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f03: The Drosophila NMJ is used to screen for interaction of modifiers with VAP(P58S).Thirteen of the 103 modifiers discovered in our macro chaetae screen were tested in the larval muscle-4 NMJ for interaction with VAP(P58S). For this and subsequent figures, approximately 15 NMJs were dissected, stained (anti-HRP, red), imaged and measured for the average size of boutons (displayed in yellow at the top RHS of each figure). (A) A wild type (C155-Gal4/+) NMJ. The average bouton size is 3.987 (±0.03). Shown here and below is Z-series of a synapse rendered as maximum intensity projection. (B) Expression of UAS-VAP(P58S), using the C155-Gal4 driver increases the size of the boutons. (C–F) Knockdown of CG6048 (C), CG9172 (D), TBPH (E) and Nup75 (F) in a C155/UAS-VAP(P58S) background reverses the effect of the VAP(P58S) over-expression and rescues bouton size to wild-type levels. (G) Quantitation of bouton size (in micrometer) for the RNAi knockdown of each gene tested in a VAP(P58S) background (top panel) and in a wild type background (bottom panel). For this and subsequent NMJ figures, error bars represent standard errors of the mean (SEM). Scale bar: 5 µm. * indicates a p-value<0.01 (but >0.001), while ** indicates a p-value of <0.001.

Mentions: In control C155-Gal4/+ animals, the average bouton size was 3.98±0.09 µm (n = 17); in C155-Gal4>VAP(P58S) animals, boutons are large with an average size of 4.84±0.25 µm (n = 16) (Fig. 3A,B respectively, p-value = 0.0016) without any significant change in the bouton number. Knockdown of Ada2b, CG18110, CG6048, CG9172, NaPi-T, Nup75, Ssh, TBPH and Tor suppressed the bouton phenotype observed in C155-Gal4>VAP(P58S) animals (Fig. 3C–F) while Ars2, Droj2, Karyβ-3, Prx5, Snama knock down failed to rescue or worsen the bouton size. We have interpreted the rescue of bouton size as a scaling back of the perturbation effect of VAP(P58S) on the pan neuron–glia–muscle network. Rescue of the bouton size did not seem to be additive since knockdown of these genes on their own (Fig. 3G, bottom panel), did not give rise to smaller boutons. On the contrary, the boutons were found to be marginally bigger as compared to the controls though many of them were not statistically significant except in the case of Snama and Karyβ-3, with bouton size increasing to 4.45±0.13 µm and 4.80±0.14 µm respectively (Fig. 3G). None of the genes we tested, by themselves, showed any significant increase in bouton size compared to C155-Gal4>VAP(P58S).


A genetic screen identifies Tor as an interactor of VAPB in a Drosophila model of amyotrophic lateral sclerosis.

Deivasigamani S, Verma HK, Ueda R, Ratnaparkhi A, Ratnaparkhi GS - Biol Open (2014)

The Drosophila NMJ is used to screen for interaction of modifiers with VAP(P58S).Thirteen of the 103 modifiers discovered in our macro chaetae screen were tested in the larval muscle-4 NMJ for interaction with VAP(P58S). For this and subsequent figures, approximately 15 NMJs were dissected, stained (anti-HRP, red), imaged and measured for the average size of boutons (displayed in yellow at the top RHS of each figure). (A) A wild type (C155-Gal4/+) NMJ. The average bouton size is 3.987 (±0.03). Shown here and below is Z-series of a synapse rendered as maximum intensity projection. (B) Expression of UAS-VAP(P58S), using the C155-Gal4 driver increases the size of the boutons. (C–F) Knockdown of CG6048 (C), CG9172 (D), TBPH (E) and Nup75 (F) in a C155/UAS-VAP(P58S) background reverses the effect of the VAP(P58S) over-expression and rescues bouton size to wild-type levels. (G) Quantitation of bouton size (in micrometer) for the RNAi knockdown of each gene tested in a VAP(P58S) background (top panel) and in a wild type background (bottom panel). For this and subsequent NMJ figures, error bars represent standard errors of the mean (SEM). Scale bar: 5 µm. * indicates a p-value<0.01 (but >0.001), while ** indicates a p-value of <0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4232771&req=5

f03: The Drosophila NMJ is used to screen for interaction of modifiers with VAP(P58S).Thirteen of the 103 modifiers discovered in our macro chaetae screen were tested in the larval muscle-4 NMJ for interaction with VAP(P58S). For this and subsequent figures, approximately 15 NMJs were dissected, stained (anti-HRP, red), imaged and measured for the average size of boutons (displayed in yellow at the top RHS of each figure). (A) A wild type (C155-Gal4/+) NMJ. The average bouton size is 3.987 (±0.03). Shown here and below is Z-series of a synapse rendered as maximum intensity projection. (B) Expression of UAS-VAP(P58S), using the C155-Gal4 driver increases the size of the boutons. (C–F) Knockdown of CG6048 (C), CG9172 (D), TBPH (E) and Nup75 (F) in a C155/UAS-VAP(P58S) background reverses the effect of the VAP(P58S) over-expression and rescues bouton size to wild-type levels. (G) Quantitation of bouton size (in micrometer) for the RNAi knockdown of each gene tested in a VAP(P58S) background (top panel) and in a wild type background (bottom panel). For this and subsequent NMJ figures, error bars represent standard errors of the mean (SEM). Scale bar: 5 µm. * indicates a p-value<0.01 (but >0.001), while ** indicates a p-value of <0.001.
Mentions: In control C155-Gal4/+ animals, the average bouton size was 3.98±0.09 µm (n = 17); in C155-Gal4>VAP(P58S) animals, boutons are large with an average size of 4.84±0.25 µm (n = 16) (Fig. 3A,B respectively, p-value = 0.0016) without any significant change in the bouton number. Knockdown of Ada2b, CG18110, CG6048, CG9172, NaPi-T, Nup75, Ssh, TBPH and Tor suppressed the bouton phenotype observed in C155-Gal4>VAP(P58S) animals (Fig. 3C–F) while Ars2, Droj2, Karyβ-3, Prx5, Snama knock down failed to rescue or worsen the bouton size. We have interpreted the rescue of bouton size as a scaling back of the perturbation effect of VAP(P58S) on the pan neuron–glia–muscle network. Rescue of the bouton size did not seem to be additive since knockdown of these genes on their own (Fig. 3G, bottom panel), did not give rise to smaller boutons. On the contrary, the boutons were found to be marginally bigger as compared to the controls though many of them were not statistically significant except in the case of Snama and Karyβ-3, with bouton size increasing to 4.45±0.13 µm and 4.80±0.14 µm respectively (Fig. 3G). None of the genes we tested, by themselves, showed any significant increase in bouton size compared to C155-Gal4>VAP(P58S).

Bottom Line: One key modifier identified was Tor, whose knockdown reversed the large bouton phenotype associated with VAP(P58S) expression in neurons.A similar reversal was seen by over-expressing Tuberous Sclerosis Complex (Tsc1,2) that negatively regulates TOR signaling as also by reduction of S6K activity.In comparison, the small bouton phenotype associated with VAP(wt) expression was reversed with Tsc1 knock down as well as S6K-CA expression.

View Article: PubMed Central - PubMed

Affiliation: Indian Institute of Science Education and Research, Pune 411021, India.

No MeSH data available.


Related in: MedlinePlus