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Subunits of the Drosophila actin-capping protein heterodimer regulate each other at multiple levels.

Amândio AR, Gaspar P, Whited JL, Janody F - PLoS ONE (2014)

Bottom Line: Overexpressing capping protein α and β decreases both F-actin levels and tissue growth, while expressing forms of Capping Protein that have dominant negative effects on F-actin promote tissue growth.Both subunits regulate each other's protein levels.In addition, overexpressing one of the subunit in tissues knocked-down for the other increases the mRNA and protein levels of the subunit knocked-down and compensates for its loss.

View Article: PubMed Central - PubMed

Affiliation: Instituto Gulbenkian de Ciência, Oeiras, Portugal.

ABSTRACT
The actin-Capping Protein heterodimer, composed of the α and β subunits, is a master F-actin regulator. In addition to its role in many cellular processes, Capping Protein acts as a main tumor suppressor module in Drosophila and in humans, in part, by restricting the activity of Yorkie/YAP/TAZ oncogenes. We aimed in this report to understand how both subunits regulate each other in vivo. We show that the levels and capping activities of both subunits must be tightly regulated to control F-actin levels and consequently growth of the Drosophila wing. Overexpressing capping protein α and β decreases both F-actin levels and tissue growth, while expressing forms of Capping Protein that have dominant negative effects on F-actin promote tissue growth. Both subunits regulate each other's protein levels. In addition, overexpressing one of the subunit in tissues knocked-down for the other increases the mRNA and protein levels of the subunit knocked-down and compensates for its loss. We propose that the ability of the α and β subunits to control each other's levels assures that a pool of functional heterodimer is produced in sufficient quantities to restrict the development of tumor but not in excess to sustain normal tissue growth.

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Related in: MedlinePlus

Overexpressing HA-cpa in wing discs knocked-down for cpb restores cpb mRNA and protein levels and suppresses apoptosis.(A–A′ and B–B′) standard confocal sections of third instar wing imaginal discs with dorsal sides up, expressing (A–A″) UAS-cpb-IR45668 and one copy of UAS-mCD8-GFP (green in A) or (B–B′) UAS-cpb-IR45668 and UAS-HA-cpa89E under sd-Gal4 control. Discs are stained with anti-activated-Caspase 3 (magenta), which monitors DRONC activation and (B–B′) anti-HA (green in B), reflecting HA-cpa89E expression. The scale bars represent 30 µm. (C) quantification of total C3 area per disc area for the two genotypes shown in A–A′ and B–B′. The means for sd>cpb-IR45668, GFP is 62.19 (n = 22); for sd>cpb-IR4566, HA-cpa89E is 26.67 (n = 32). Error bars indicate s.e.m. P<0.0001 for comparison of between both genotypes. (D and E) western blots on protein extracts from wing discs expressing two copies of UAS-mCD8-GFP (lane 1) or UAS-cpb-IR45668 and two copies of UAS-mCD8-GFP (lane 2) or UAS-cpb-IR45668 and UAS-HA-cpa89E and one copy of UAS-mCD8-GFP (lane 3) under sd-Gal4 control, blotted with (D) anti-Cpa (upper panel) and anti-H3 (lower panel) or (E) anti-Cpb (upper panel) and anti-H3 (lower panel). (F to I) graphs of (F and G) cpa or (H and I) cpb mRNA levels measured by (F and H) three or (G and I) five independent qRT-PCR in wing imaginal discs expressing (F and H) two copies of UAS-mCD8GFP (lane 1) or UAS-cpb-IR45668 and UAS-mCD8GFP (lane 2) or UAS-cpb-IR45668 and UAS-HA-cpa89E (lane 3) or (G and I) UAS-mCherry (lane 1) or UAS-HA-cpa89E under sd-Gal4 control. (F) the means for lane 1 is 0.90; for lane 2 is 1.04; for lane 3 is 9.8. P<0.033 for comparison of lane 1 with 3. (G) the means for lane is 1.07; for lane 2 is 1.88. P<0.0001 for comparison of lane 1 and 2. (H) the means for lane 1 is 0.59; for lane 2 is 0.25; for lane 3 is 0.4319. P<0.0018 for comparison of lane 1 and 2 or P<0.048 for comparison of lane 2 and 3. (I) the means for lane 1 is 0.29; for lane 2 is 0.31. n.s. indicates non-significant P value. Error bars indicate s.e.m.
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pone-0096326-g007: Overexpressing HA-cpa in wing discs knocked-down for cpb restores cpb mRNA and protein levels and suppresses apoptosis.(A–A′ and B–B′) standard confocal sections of third instar wing imaginal discs with dorsal sides up, expressing (A–A″) UAS-cpb-IR45668 and one copy of UAS-mCD8-GFP (green in A) or (B–B′) UAS-cpb-IR45668 and UAS-HA-cpa89E under sd-Gal4 control. Discs are stained with anti-activated-Caspase 3 (magenta), which monitors DRONC activation and (B–B′) anti-HA (green in B), reflecting HA-cpa89E expression. The scale bars represent 30 µm. (C) quantification of total C3 area per disc area for the two genotypes shown in A–A′ and B–B′. The means for sd>cpb-IR45668, GFP is 62.19 (n = 22); for sd>cpb-IR4566, HA-cpa89E is 26.67 (n = 32). Error bars indicate s.e.m. P<0.0001 for comparison of between both genotypes. (D and E) western blots on protein extracts from wing discs expressing two copies of UAS-mCD8-GFP (lane 1) or UAS-cpb-IR45668 and two copies of UAS-mCD8-GFP (lane 2) or UAS-cpb-IR45668 and UAS-HA-cpa89E and one copy of UAS-mCD8-GFP (lane 3) under sd-Gal4 control, blotted with (D) anti-Cpa (upper panel) and anti-H3 (lower panel) or (E) anti-Cpb (upper panel) and anti-H3 (lower panel). (F to I) graphs of (F and G) cpa or (H and I) cpb mRNA levels measured by (F and H) three or (G and I) five independent qRT-PCR in wing imaginal discs expressing (F and H) two copies of UAS-mCD8GFP (lane 1) or UAS-cpb-IR45668 and UAS-mCD8GFP (lane 2) or UAS-cpb-IR45668 and UAS-HA-cpa89E (lane 3) or (G and I) UAS-mCherry (lane 1) or UAS-HA-cpa89E under sd-Gal4 control. (F) the means for lane 1 is 0.90; for lane 2 is 1.04; for lane 3 is 9.8. P<0.033 for comparison of lane 1 with 3. (G) the means for lane is 1.07; for lane 2 is 1.88. P<0.0001 for comparison of lane 1 and 2. (H) the means for lane 1 is 0.59; for lane 2 is 0.25; for lane 3 is 0.4319. P<0.0018 for comparison of lane 1 and 2 or P<0.048 for comparison of lane 2 and 3. (I) the means for lane 1 is 0.29; for lane 2 is 0.31. n.s. indicates non-significant P value. Error bars indicate s.e.m.

Mentions: Interestingly, co-expressing cpb with HA-cpaΔABD almost fully suppressed apoptosis of wing discs knocked-down for cpa (Fig. 6 compare B–B′ with A–A′ and D; P<0.0001). This effect could be due to the stabilization and apical relocalization of HA-CpaΔABD when co-expressed with cpb (Fig. 2H–H″). However, apoptosis of sd>cpa-IR wing discs was also significantly suppressed by overexpressing cpb alone (Fig. 6C–C′ and D; P<0.0001). Conversely, expressing HA-cpa in tissues knocked-down for cpb (sd>cpb-IR) also prevented apoptosis (Fig. 7 compare B–B′ with A–A′ and C; P<0.0001).


Subunits of the Drosophila actin-capping protein heterodimer regulate each other at multiple levels.

Amândio AR, Gaspar P, Whited JL, Janody F - PLoS ONE (2014)

Overexpressing HA-cpa in wing discs knocked-down for cpb restores cpb mRNA and protein levels and suppresses apoptosis.(A–A′ and B–B′) standard confocal sections of third instar wing imaginal discs with dorsal sides up, expressing (A–A″) UAS-cpb-IR45668 and one copy of UAS-mCD8-GFP (green in A) or (B–B′) UAS-cpb-IR45668 and UAS-HA-cpa89E under sd-Gal4 control. Discs are stained with anti-activated-Caspase 3 (magenta), which monitors DRONC activation and (B–B′) anti-HA (green in B), reflecting HA-cpa89E expression. The scale bars represent 30 µm. (C) quantification of total C3 area per disc area for the two genotypes shown in A–A′ and B–B′. The means for sd>cpb-IR45668, GFP is 62.19 (n = 22); for sd>cpb-IR4566, HA-cpa89E is 26.67 (n = 32). Error bars indicate s.e.m. P<0.0001 for comparison of between both genotypes. (D and E) western blots on protein extracts from wing discs expressing two copies of UAS-mCD8-GFP (lane 1) or UAS-cpb-IR45668 and two copies of UAS-mCD8-GFP (lane 2) or UAS-cpb-IR45668 and UAS-HA-cpa89E and one copy of UAS-mCD8-GFP (lane 3) under sd-Gal4 control, blotted with (D) anti-Cpa (upper panel) and anti-H3 (lower panel) or (E) anti-Cpb (upper panel) and anti-H3 (lower panel). (F to I) graphs of (F and G) cpa or (H and I) cpb mRNA levels measured by (F and H) three or (G and I) five independent qRT-PCR in wing imaginal discs expressing (F and H) two copies of UAS-mCD8GFP (lane 1) or UAS-cpb-IR45668 and UAS-mCD8GFP (lane 2) or UAS-cpb-IR45668 and UAS-HA-cpa89E (lane 3) or (G and I) UAS-mCherry (lane 1) or UAS-HA-cpa89E under sd-Gal4 control. (F) the means for lane 1 is 0.90; for lane 2 is 1.04; for lane 3 is 9.8. P<0.033 for comparison of lane 1 with 3. (G) the means for lane is 1.07; for lane 2 is 1.88. P<0.0001 for comparison of lane 1 and 2. (H) the means for lane 1 is 0.59; for lane 2 is 0.25; for lane 3 is 0.4319. P<0.0018 for comparison of lane 1 and 2 or P<0.048 for comparison of lane 2 and 3. (I) the means for lane 1 is 0.29; for lane 2 is 0.31. n.s. indicates non-significant P value. Error bars indicate s.e.m.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4008575&req=5

pone-0096326-g007: Overexpressing HA-cpa in wing discs knocked-down for cpb restores cpb mRNA and protein levels and suppresses apoptosis.(A–A′ and B–B′) standard confocal sections of third instar wing imaginal discs with dorsal sides up, expressing (A–A″) UAS-cpb-IR45668 and one copy of UAS-mCD8-GFP (green in A) or (B–B′) UAS-cpb-IR45668 and UAS-HA-cpa89E under sd-Gal4 control. Discs are stained with anti-activated-Caspase 3 (magenta), which monitors DRONC activation and (B–B′) anti-HA (green in B), reflecting HA-cpa89E expression. The scale bars represent 30 µm. (C) quantification of total C3 area per disc area for the two genotypes shown in A–A′ and B–B′. The means for sd>cpb-IR45668, GFP is 62.19 (n = 22); for sd>cpb-IR4566, HA-cpa89E is 26.67 (n = 32). Error bars indicate s.e.m. P<0.0001 for comparison of between both genotypes. (D and E) western blots on protein extracts from wing discs expressing two copies of UAS-mCD8-GFP (lane 1) or UAS-cpb-IR45668 and two copies of UAS-mCD8-GFP (lane 2) or UAS-cpb-IR45668 and UAS-HA-cpa89E and one copy of UAS-mCD8-GFP (lane 3) under sd-Gal4 control, blotted with (D) anti-Cpa (upper panel) and anti-H3 (lower panel) or (E) anti-Cpb (upper panel) and anti-H3 (lower panel). (F to I) graphs of (F and G) cpa or (H and I) cpb mRNA levels measured by (F and H) three or (G and I) five independent qRT-PCR in wing imaginal discs expressing (F and H) two copies of UAS-mCD8GFP (lane 1) or UAS-cpb-IR45668 and UAS-mCD8GFP (lane 2) or UAS-cpb-IR45668 and UAS-HA-cpa89E (lane 3) or (G and I) UAS-mCherry (lane 1) or UAS-HA-cpa89E under sd-Gal4 control. (F) the means for lane 1 is 0.90; for lane 2 is 1.04; for lane 3 is 9.8. P<0.033 for comparison of lane 1 with 3. (G) the means for lane is 1.07; for lane 2 is 1.88. P<0.0001 for comparison of lane 1 and 2. (H) the means for lane 1 is 0.59; for lane 2 is 0.25; for lane 3 is 0.4319. P<0.0018 for comparison of lane 1 and 2 or P<0.048 for comparison of lane 2 and 3. (I) the means for lane 1 is 0.29; for lane 2 is 0.31. n.s. indicates non-significant P value. Error bars indicate s.e.m.
Mentions: Interestingly, co-expressing cpb with HA-cpaΔABD almost fully suppressed apoptosis of wing discs knocked-down for cpa (Fig. 6 compare B–B′ with A–A′ and D; P<0.0001). This effect could be due to the stabilization and apical relocalization of HA-CpaΔABD when co-expressed with cpb (Fig. 2H–H″). However, apoptosis of sd>cpa-IR wing discs was also significantly suppressed by overexpressing cpb alone (Fig. 6C–C′ and D; P<0.0001). Conversely, expressing HA-cpa in tissues knocked-down for cpb (sd>cpb-IR) also prevented apoptosis (Fig. 7 compare B–B′ with A–A′ and C; P<0.0001).

Bottom Line: Overexpressing capping protein α and β decreases both F-actin levels and tissue growth, while expressing forms of Capping Protein that have dominant negative effects on F-actin promote tissue growth.Both subunits regulate each other's protein levels.In addition, overexpressing one of the subunit in tissues knocked-down for the other increases the mRNA and protein levels of the subunit knocked-down and compensates for its loss.

View Article: PubMed Central - PubMed

Affiliation: Instituto Gulbenkian de Ciência, Oeiras, Portugal.

ABSTRACT
The actin-Capping Protein heterodimer, composed of the α and β subunits, is a master F-actin regulator. In addition to its role in many cellular processes, Capping Protein acts as a main tumor suppressor module in Drosophila and in humans, in part, by restricting the activity of Yorkie/YAP/TAZ oncogenes. We aimed in this report to understand how both subunits regulate each other in vivo. We show that the levels and capping activities of both subunits must be tightly regulated to control F-actin levels and consequently growth of the Drosophila wing. Overexpressing capping protein α and β decreases both F-actin levels and tissue growth, while expressing forms of Capping Protein that have dominant negative effects on F-actin promote tissue growth. Both subunits regulate each other's protein levels. In addition, overexpressing one of the subunit in tissues knocked-down for the other increases the mRNA and protein levels of the subunit knocked-down and compensates for its loss. We propose that the ability of the α and β subunits to control each other's levels assures that a pool of functional heterodimer is produced in sufficient quantities to restrict the development of tumor but not in excess to sustain normal tissue growth.

Show MeSH
Related in: MedlinePlus