Limits...
Contribution of Orb2A stability in regulated amyloid-like oligomerization of Drosophila Orb2.

White-Grindley E, Li L, Mohammad Khan R, Ren F, Saraf A, Florens L, Si K - PLoS Biol. (2014)

Bottom Line: Mutation of LimK abolishes activity-dependent Orb2 oligomerization in the adult brain.Moreover, Tob-Orb2 association is modulated by neuronal activity and Tob activity in the mushroom body is required for stable memory formation.These observations suggest that the interplay between PP2A and Tob-LimK activity may dynamically regulate Orb2 amyloid-like oligomer formation and the stabilization of memories.

View Article: PubMed Central - PubMed

Affiliation: Stowers Institute for Medical Research, Kansas City, Missouri, United States of America.

ABSTRACT
How learned experiences persist as memory for a long time is an important question. In Drosophila the persistence of memory is dependent upon amyloid-like oligomers of the Orb2 protein. However, it is not clear how the conversion of Orb2 to the amyloid-like oligomeric state is regulated. The Orb2 has two protein isoforms, and the rare Orb2A isoform is critical for oligomerization of the ubiquitous Orb2B isoform. Here, we report the discovery of a protein network comprised of protein phosphatase 2A (PP2A), Transducer of Erb-B2 (Tob), and Lim Kinase (LimK) that controls the abundance of Orb2A. PP2A maintains Orb2A in an unphosphorylated and unstable state, whereas Tob-LimK phosphorylates and stabilizes Orb2A. Mutation of LimK abolishes activity-dependent Orb2 oligomerization in the adult brain. Moreover, Tob-Orb2 association is modulated by neuronal activity and Tob activity in the mushroom body is required for stable memory formation. These observations suggest that the interplay between PP2A and Tob-LimK activity may dynamically regulate Orb2 amyloid-like oligomer formation and the stabilization of memories.

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Tob stabilizes Orb2A and induces Orb2 oligomerization.(A) Tob enhances Orb2A stability. Orb2 stability was examined in S2 cells expressing either Orb2A by itself (top panel, Orb2A) or in conjunction with Tob (bottom panel, +Tob). Following the addition of cycloheximide (Chx), samples were taken at the given time points and then analyzed for expression levels of Orb2A by Western blot (left panels). Right panels compare protein half-lives. Half-lives were determined by plotting the percent of protein remaining after time zero and assuming first-order kinetics. The n indicates number of independent experiments performed to determine half-lives. Statistical significance was determined using an unpaired, two-tailed t test. The data are plotted as mean ± SEM. (B) Tob has little effect on Orb2B stability. (C) Both Orb2A and Orb2B significantly enhance Tob stability. (D) Overexpression of Tob increases Orb2 oligomerization. Orb2 was immunoprecipitated from adult head extracts expressing only TdTomato (Elav-Gal4: UAS-TdTom), Tob tagged to TdTomato (Elav-Gal4: UAS-TobTdTom), or Tob lacking a 28 amino acid domain critical for binding to Orb2 (Elav-Gal4: UAS-TobΔ28TdTom). (E) Overexpression of Tob increases the number and size of Orb2AEGFP puncta. (Left panel) Both proteins were expressed in the ellipsoid body of the central complex using a c547Gal4 driver. Each row represents a fly genotype: c547-Gal4:UAS-Orb2AEGFP (Orb2A only), c547-Gal4:UAS-Orb2AEGFP/UAS-TobTdtomato (Orb2A Tob), and c547-Gal4:UAS-Orb2AEGFP/UAS-TobΔ28Tdtomato (Orb2A TobΔ28). Scale bar, 25 µm. (Right panel) Higher magnification image of the boxed region in the left. Puncta were counted in the central portion of the ellipsoid body. Axiovision software was programmed to identify a continuous central region and define aggregates, indicated as white dots. Scale bar, 20 µm. Please see Figure S3 for additional images. (F) Puncta number (/100 µm2) and (G) size (average area of aggregates in µm2) were quantified. Statistical analysis was performed using an unpaired two-tailed t test (*) p≤0.05, (**) p≤0.01, and (***) p≤0.001. n, the number of flies examined for each genotype. The data are plotted as mean ± SEM. Also see Figures S2 and S3.
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pbio-1001786-g002: Tob stabilizes Orb2A and induces Orb2 oligomerization.(A) Tob enhances Orb2A stability. Orb2 stability was examined in S2 cells expressing either Orb2A by itself (top panel, Orb2A) or in conjunction with Tob (bottom panel, +Tob). Following the addition of cycloheximide (Chx), samples were taken at the given time points and then analyzed for expression levels of Orb2A by Western blot (left panels). Right panels compare protein half-lives. Half-lives were determined by plotting the percent of protein remaining after time zero and assuming first-order kinetics. The n indicates number of independent experiments performed to determine half-lives. Statistical significance was determined using an unpaired, two-tailed t test. The data are plotted as mean ± SEM. (B) Tob has little effect on Orb2B stability. (C) Both Orb2A and Orb2B significantly enhance Tob stability. (D) Overexpression of Tob increases Orb2 oligomerization. Orb2 was immunoprecipitated from adult head extracts expressing only TdTomato (Elav-Gal4: UAS-TdTom), Tob tagged to TdTomato (Elav-Gal4: UAS-TobTdTom), or Tob lacking a 28 amino acid domain critical for binding to Orb2 (Elav-Gal4: UAS-TobΔ28TdTom). (E) Overexpression of Tob increases the number and size of Orb2AEGFP puncta. (Left panel) Both proteins were expressed in the ellipsoid body of the central complex using a c547Gal4 driver. Each row represents a fly genotype: c547-Gal4:UAS-Orb2AEGFP (Orb2A only), c547-Gal4:UAS-Orb2AEGFP/UAS-TobTdtomato (Orb2A Tob), and c547-Gal4:UAS-Orb2AEGFP/UAS-TobΔ28Tdtomato (Orb2A TobΔ28). Scale bar, 25 µm. (Right panel) Higher magnification image of the boxed region in the left. Puncta were counted in the central portion of the ellipsoid body. Axiovision software was programmed to identify a continuous central region and define aggregates, indicated as white dots. Scale bar, 20 µm. Please see Figure S3 for additional images. (F) Puncta number (/100 µm2) and (G) size (average area of aggregates in µm2) were quantified. Statistical analysis was performed using an unpaired two-tailed t test (*) p≤0.05, (**) p≤0.01, and (***) p≤0.001. n, the number of flies examined for each genotype. The data are plotted as mean ± SEM. Also see Figures S2 and S3.

Mentions: The Orb2A protein is undetectable by Western analysis, and a genomic construct expressing Orb2A-EGFP suggests it is ∼100 times less abundant than Orb2B protein in the adult brain [5]. Moreover, monomeric Orb2A has a very short half-life compared to Orb2B (Figure 2A and Table S2). Taken together, these observations suggest that availability of the Orb2A protein could be an important determinant of efficient Orb2A oligomerization and/or function. In the course of our interaction studies in S2 cells, we noticed one of the candidate proteins, Tob, may influence the Orb2A protein level (Figure 1E). To determine Orb2A and Orb2B stability independent of each other, we used Drosophila S2 cells, in which Orb2 is normally not expressed and Tob is expressed at low levels. S2 cells were transfected with only HA-tagged Orb2 or coexpressed with Flag-tagged Tob. To determine half-life, total Orb2 or Tob protein levels were measured at several time points following treatment with cycloheximide (CHX), which blocks new protein synthesis. The coexpression of Tob nearly doubled the half-life of monomeric Orb2A (Figure 2A). However, Tob had no significant effect on Orb2B (Figure 2B), indicating that association with Tob does not automatically enhance half-life. Likewise, incubation with dsRNA against Tob reduced the level of Orb2A protein but not Orb2B (Figure S2A). Earlier studies with Tob family members have suggested that the stability of Tob itself can be regulated [18],[19]. We found a fourfold increase in Tob half-life in the presence of either Orb2A or Orb2B compared to Tob alone (Figure 2C and Table S2). These results suggest that not only does Tob stabilize Orb2A, but Orb2 proteins have stabilizing effects on Tob.


Contribution of Orb2A stability in regulated amyloid-like oligomerization of Drosophila Orb2.

White-Grindley E, Li L, Mohammad Khan R, Ren F, Saraf A, Florens L, Si K - PLoS Biol. (2014)

Tob stabilizes Orb2A and induces Orb2 oligomerization.(A) Tob enhances Orb2A stability. Orb2 stability was examined in S2 cells expressing either Orb2A by itself (top panel, Orb2A) or in conjunction with Tob (bottom panel, +Tob). Following the addition of cycloheximide (Chx), samples were taken at the given time points and then analyzed for expression levels of Orb2A by Western blot (left panels). Right panels compare protein half-lives. Half-lives were determined by plotting the percent of protein remaining after time zero and assuming first-order kinetics. The n indicates number of independent experiments performed to determine half-lives. Statistical significance was determined using an unpaired, two-tailed t test. The data are plotted as mean ± SEM. (B) Tob has little effect on Orb2B stability. (C) Both Orb2A and Orb2B significantly enhance Tob stability. (D) Overexpression of Tob increases Orb2 oligomerization. Orb2 was immunoprecipitated from adult head extracts expressing only TdTomato (Elav-Gal4: UAS-TdTom), Tob tagged to TdTomato (Elav-Gal4: UAS-TobTdTom), or Tob lacking a 28 amino acid domain critical for binding to Orb2 (Elav-Gal4: UAS-TobΔ28TdTom). (E) Overexpression of Tob increases the number and size of Orb2AEGFP puncta. (Left panel) Both proteins were expressed in the ellipsoid body of the central complex using a c547Gal4 driver. Each row represents a fly genotype: c547-Gal4:UAS-Orb2AEGFP (Orb2A only), c547-Gal4:UAS-Orb2AEGFP/UAS-TobTdtomato (Orb2A Tob), and c547-Gal4:UAS-Orb2AEGFP/UAS-TobΔ28Tdtomato (Orb2A TobΔ28). Scale bar, 25 µm. (Right panel) Higher magnification image of the boxed region in the left. Puncta were counted in the central portion of the ellipsoid body. Axiovision software was programmed to identify a continuous central region and define aggregates, indicated as white dots. Scale bar, 20 µm. Please see Figure S3 for additional images. (F) Puncta number (/100 µm2) and (G) size (average area of aggregates in µm2) were quantified. Statistical analysis was performed using an unpaired two-tailed t test (*) p≤0.05, (**) p≤0.01, and (***) p≤0.001. n, the number of flies examined for each genotype. The data are plotted as mean ± SEM. Also see Figures S2 and S3.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3921104&req=5

pbio-1001786-g002: Tob stabilizes Orb2A and induces Orb2 oligomerization.(A) Tob enhances Orb2A stability. Orb2 stability was examined in S2 cells expressing either Orb2A by itself (top panel, Orb2A) or in conjunction with Tob (bottom panel, +Tob). Following the addition of cycloheximide (Chx), samples were taken at the given time points and then analyzed for expression levels of Orb2A by Western blot (left panels). Right panels compare protein half-lives. Half-lives were determined by plotting the percent of protein remaining after time zero and assuming first-order kinetics. The n indicates number of independent experiments performed to determine half-lives. Statistical significance was determined using an unpaired, two-tailed t test. The data are plotted as mean ± SEM. (B) Tob has little effect on Orb2B stability. (C) Both Orb2A and Orb2B significantly enhance Tob stability. (D) Overexpression of Tob increases Orb2 oligomerization. Orb2 was immunoprecipitated from adult head extracts expressing only TdTomato (Elav-Gal4: UAS-TdTom), Tob tagged to TdTomato (Elav-Gal4: UAS-TobTdTom), or Tob lacking a 28 amino acid domain critical for binding to Orb2 (Elav-Gal4: UAS-TobΔ28TdTom). (E) Overexpression of Tob increases the number and size of Orb2AEGFP puncta. (Left panel) Both proteins were expressed in the ellipsoid body of the central complex using a c547Gal4 driver. Each row represents a fly genotype: c547-Gal4:UAS-Orb2AEGFP (Orb2A only), c547-Gal4:UAS-Orb2AEGFP/UAS-TobTdtomato (Orb2A Tob), and c547-Gal4:UAS-Orb2AEGFP/UAS-TobΔ28Tdtomato (Orb2A TobΔ28). Scale bar, 25 µm. (Right panel) Higher magnification image of the boxed region in the left. Puncta were counted in the central portion of the ellipsoid body. Axiovision software was programmed to identify a continuous central region and define aggregates, indicated as white dots. Scale bar, 20 µm. Please see Figure S3 for additional images. (F) Puncta number (/100 µm2) and (G) size (average area of aggregates in µm2) were quantified. Statistical analysis was performed using an unpaired two-tailed t test (*) p≤0.05, (**) p≤0.01, and (***) p≤0.001. n, the number of flies examined for each genotype. The data are plotted as mean ± SEM. Also see Figures S2 and S3.
Mentions: The Orb2A protein is undetectable by Western analysis, and a genomic construct expressing Orb2A-EGFP suggests it is ∼100 times less abundant than Orb2B protein in the adult brain [5]. Moreover, monomeric Orb2A has a very short half-life compared to Orb2B (Figure 2A and Table S2). Taken together, these observations suggest that availability of the Orb2A protein could be an important determinant of efficient Orb2A oligomerization and/or function. In the course of our interaction studies in S2 cells, we noticed one of the candidate proteins, Tob, may influence the Orb2A protein level (Figure 1E). To determine Orb2A and Orb2B stability independent of each other, we used Drosophila S2 cells, in which Orb2 is normally not expressed and Tob is expressed at low levels. S2 cells were transfected with only HA-tagged Orb2 or coexpressed with Flag-tagged Tob. To determine half-life, total Orb2 or Tob protein levels were measured at several time points following treatment with cycloheximide (CHX), which blocks new protein synthesis. The coexpression of Tob nearly doubled the half-life of monomeric Orb2A (Figure 2A). However, Tob had no significant effect on Orb2B (Figure 2B), indicating that association with Tob does not automatically enhance half-life. Likewise, incubation with dsRNA against Tob reduced the level of Orb2A protein but not Orb2B (Figure S2A). Earlier studies with Tob family members have suggested that the stability of Tob itself can be regulated [18],[19]. We found a fourfold increase in Tob half-life in the presence of either Orb2A or Orb2B compared to Tob alone (Figure 2C and Table S2). These results suggest that not only does Tob stabilize Orb2A, but Orb2 proteins have stabilizing effects on Tob.

Bottom Line: Mutation of LimK abolishes activity-dependent Orb2 oligomerization in the adult brain.Moreover, Tob-Orb2 association is modulated by neuronal activity and Tob activity in the mushroom body is required for stable memory formation.These observations suggest that the interplay between PP2A and Tob-LimK activity may dynamically regulate Orb2 amyloid-like oligomer formation and the stabilization of memories.

View Article: PubMed Central - PubMed

Affiliation: Stowers Institute for Medical Research, Kansas City, Missouri, United States of America.

ABSTRACT
How learned experiences persist as memory for a long time is an important question. In Drosophila the persistence of memory is dependent upon amyloid-like oligomers of the Orb2 protein. However, it is not clear how the conversion of Orb2 to the amyloid-like oligomeric state is regulated. The Orb2 has two protein isoforms, and the rare Orb2A isoform is critical for oligomerization of the ubiquitous Orb2B isoform. Here, we report the discovery of a protein network comprised of protein phosphatase 2A (PP2A), Transducer of Erb-B2 (Tob), and Lim Kinase (LimK) that controls the abundance of Orb2A. PP2A maintains Orb2A in an unphosphorylated and unstable state, whereas Tob-LimK phosphorylates and stabilizes Orb2A. Mutation of LimK abolishes activity-dependent Orb2 oligomerization in the adult brain. Moreover, Tob-Orb2 association is modulated by neuronal activity and Tob activity in the mushroom body is required for stable memory formation. These observations suggest that the interplay between PP2A and Tob-LimK activity may dynamically regulate Orb2 amyloid-like oligomer formation and the stabilization of memories.

Show MeSH
Related in: MedlinePlus