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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

Scheme for the enhancer/suppressor screen.(A) A sensitized genetic background (sca-Gal4,UAS-dVAP) was used for a screen to identify interactors of dVAP. A recombinant stable line, expressing VAP in the sca domain was found to reduce the number of 10 macro chaetae (marked by arrowheads in w1118) to about 5–6 at 25°C. At an increased VAP dosage, at 28°C, the macro chaetae reduced to 0–1. Expressing dsRNA for VAP, in a dVAP over-expression background, led to a reversal of the phenotype with macro chaetae reverting to wild type levels both at 25°C and 28°C. Numbers at the top right hand corner of each picture are average macro chaetae, counted for ten females of the corresponding genotype. (B) Primary screening was done at both 25°C and 28°C. A sca-Gal4, UAS-VAP/Cyo recombinant, stable line was generated and females from that line were crossed to males with different transgenic RNAi inserts. Genes that lead to a further decrease of macro chaetae (from 5–6) at 25°C were deemed enhancers and genes that increased number of macro chaetae (from 0) at 28°C were considered to be suppressors. 2635 genes, encompassing 4600 individual lines were used for the primary screen. 930 genes showed change in the phenotype and were categorized as modifiers. 930 genes identified in the primary screen were used for a thorough, quantitative screening, with controls, at 25°C. Macrochaetae from ten F1 females were counted and compared to the base line 5.5 macro chaetae in the master control (sca-Gal4, UAS-VAP/+). Student's t-test was used to select lines that had significantly greater macro chaetae and these were considered bona-fide suppressors. Lines that did not meet our threshold for significance (p>0.01, Average macro chaetae <7.5) were discarded. F1 females with average macro chaetae <5.5 were compared to the related RNAi control (sca-Gal4/+; UAS-RNAi/+) at 25°C. Again, a Student's t-test was used to select lines above our threshold for significance (p<0.01, average macro chaetae <4). Starting with 2635 genes in the primary screen, the final numbers for enhancers and suppressors after comparison with controls and rigorous statistical analyses was 45 and 58 respectively. These genes were shortlisted for the validation process.
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f01: Scheme for the enhancer/suppressor screen.(A) A sensitized genetic background (sca-Gal4,UAS-dVAP) was used for a screen to identify interactors of dVAP. A recombinant stable line, expressing VAP in the sca domain was found to reduce the number of 10 macro chaetae (marked by arrowheads in w1118) to about 5–6 at 25°C. At an increased VAP dosage, at 28°C, the macro chaetae reduced to 0–1. Expressing dsRNA for VAP, in a dVAP over-expression background, led to a reversal of the phenotype with macro chaetae reverting to wild type levels both at 25°C and 28°C. Numbers at the top right hand corner of each picture are average macro chaetae, counted for ten females of the corresponding genotype. (B) Primary screening was done at both 25°C and 28°C. A sca-Gal4, UAS-VAP/Cyo recombinant, stable line was generated and females from that line were crossed to males with different transgenic RNAi inserts. Genes that lead to a further decrease of macro chaetae (from 5–6) at 25°C were deemed enhancers and genes that increased number of macro chaetae (from 0) at 28°C were considered to be suppressors. 2635 genes, encompassing 4600 individual lines were used for the primary screen. 930 genes showed change in the phenotype and were categorized as modifiers. 930 genes identified in the primary screen were used for a thorough, quantitative screening, with controls, at 25°C. Macrochaetae from ten F1 females were counted and compared to the base line 5.5 macro chaetae in the master control (sca-Gal4, UAS-VAP/+). Student's t-test was used to select lines that had significantly greater macro chaetae and these were considered bona-fide suppressors. Lines that did not meet our threshold for significance (p>0.01, Average macro chaetae <7.5) were discarded. F1 females with average macro chaetae <5.5 were compared to the related RNAi control (sca-Gal4/+; UAS-RNAi/+) at 25°C. Again, a Student's t-test was used to select lines above our threshold for significance (p<0.01, average macro chaetae <4). Starting with 2635 genes in the primary screen, the final numbers for enhancers and suppressors after comparison with controls and rigorous statistical analyses was 45 and 58 respectively. These genes were shortlisted for the validation process.

Mentions: Stable expression of VAP in the sca domain leads to a dose dependent decrease in the number of macro chaetae in the dorsal thoracic region of the adult fly (Ratnaparkhi et al., 2008) (Fig. 1A). The penetrance of this phenotype is 100% with all the flies showing loss of bristles. At 25°C, the average number of macro chaetae observed in the stable recombinant lines (Sca-Gal4>UAS-VAP/Cyo) generated in our laboratory was between 5–6 macro chaetae and this number was seen to reduce further to 0–1 at 28°C. This phenotype was suppressed by co-expression of double stranded RNA (dsRNA) specific to VAP (VAP-RNAi, Fig. 1A). The loss of bristle phenotype was not significantly affected by the presence of an extra copy of UAS over-expressing RFP, eliminating the possibility of Gal4 dilution. The Sca-Gal4>UAS-VAP/Cyo is viable only as a heterozygote, and was used as a sensitized background to identify modifiers of VAP function through an RNAi based screen.


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)

Scheme for the enhancer/suppressor screen.(A) A sensitized genetic background (sca-Gal4,UAS-dVAP) was used for a screen to identify interactors of dVAP. A recombinant stable line, expressing VAP in the sca domain was found to reduce the number of 10 macro chaetae (marked by arrowheads in w1118) to about 5–6 at 25°C. At an increased VAP dosage, at 28°C, the macro chaetae reduced to 0–1. Expressing dsRNA for VAP, in a dVAP over-expression background, led to a reversal of the phenotype with macro chaetae reverting to wild type levels both at 25°C and 28°C. Numbers at the top right hand corner of each picture are average macro chaetae, counted for ten females of the corresponding genotype. (B) Primary screening was done at both 25°C and 28°C. A sca-Gal4, UAS-VAP/Cyo recombinant, stable line was generated and females from that line were crossed to males with different transgenic RNAi inserts. Genes that lead to a further decrease of macro chaetae (from 5–6) at 25°C were deemed enhancers and genes that increased number of macro chaetae (from 0) at 28°C were considered to be suppressors. 2635 genes, encompassing 4600 individual lines were used for the primary screen. 930 genes showed change in the phenotype and were categorized as modifiers. 930 genes identified in the primary screen were used for a thorough, quantitative screening, with controls, at 25°C. Macrochaetae from ten F1 females were counted and compared to the base line 5.5 macro chaetae in the master control (sca-Gal4, UAS-VAP/+). Student's t-test was used to select lines that had significantly greater macro chaetae and these were considered bona-fide suppressors. Lines that did not meet our threshold for significance (p>0.01, Average macro chaetae <7.5) were discarded. F1 females with average macro chaetae <5.5 were compared to the related RNAi control (sca-Gal4/+; UAS-RNAi/+) at 25°C. Again, a Student's t-test was used to select lines above our threshold for significance (p<0.01, average macro chaetae <4). Starting with 2635 genes in the primary screen, the final numbers for enhancers and suppressors after comparison with controls and rigorous statistical analyses was 45 and 58 respectively. These genes were shortlisted for the validation process.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232771&req=5

f01: Scheme for the enhancer/suppressor screen.(A) A sensitized genetic background (sca-Gal4,UAS-dVAP) was used for a screen to identify interactors of dVAP. A recombinant stable line, expressing VAP in the sca domain was found to reduce the number of 10 macro chaetae (marked by arrowheads in w1118) to about 5–6 at 25°C. At an increased VAP dosage, at 28°C, the macro chaetae reduced to 0–1. Expressing dsRNA for VAP, in a dVAP over-expression background, led to a reversal of the phenotype with macro chaetae reverting to wild type levels both at 25°C and 28°C. Numbers at the top right hand corner of each picture are average macro chaetae, counted for ten females of the corresponding genotype. (B) Primary screening was done at both 25°C and 28°C. A sca-Gal4, UAS-VAP/Cyo recombinant, stable line was generated and females from that line were crossed to males with different transgenic RNAi inserts. Genes that lead to a further decrease of macro chaetae (from 5–6) at 25°C were deemed enhancers and genes that increased number of macro chaetae (from 0) at 28°C were considered to be suppressors. 2635 genes, encompassing 4600 individual lines were used for the primary screen. 930 genes showed change in the phenotype and were categorized as modifiers. 930 genes identified in the primary screen were used for a thorough, quantitative screening, with controls, at 25°C. Macrochaetae from ten F1 females were counted and compared to the base line 5.5 macro chaetae in the master control (sca-Gal4, UAS-VAP/+). Student's t-test was used to select lines that had significantly greater macro chaetae and these were considered bona-fide suppressors. Lines that did not meet our threshold for significance (p>0.01, Average macro chaetae <7.5) were discarded. F1 females with average macro chaetae <5.5 were compared to the related RNAi control (sca-Gal4/+; UAS-RNAi/+) at 25°C. Again, a Student's t-test was used to select lines above our threshold for significance (p<0.01, average macro chaetae <4). Starting with 2635 genes in the primary screen, the final numbers for enhancers and suppressors after comparison with controls and rigorous statistical analyses was 45 and 58 respectively. These genes were shortlisted for the validation process.
Mentions: Stable expression of VAP in the sca domain leads to a dose dependent decrease in the number of macro chaetae in the dorsal thoracic region of the adult fly (Ratnaparkhi et al., 2008) (Fig. 1A). The penetrance of this phenotype is 100% with all the flies showing loss of bristles. At 25°C, the average number of macro chaetae observed in the stable recombinant lines (Sca-Gal4>UAS-VAP/Cyo) generated in our laboratory was between 5–6 macro chaetae and this number was seen to reduce further to 0–1 at 28°C. This phenotype was suppressed by co-expression of double stranded RNA (dsRNA) specific to VAP (VAP-RNAi, Fig. 1A). The loss of bristle phenotype was not significantly affected by the presence of an extra copy of UAS over-expressing RFP, eliminating the possibility of Gal4 dilution. The Sca-Gal4>UAS-VAP/Cyo is viable only as a heterozygote, and was used as a sensitized background to identify modifiers of VAP function through an RNAi based screen.

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