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Subunits of the Drosophila Actin-Capping Protein Heterodimer Regulate Each Other at Multiple Levels

Subunits of the Drosophila Actin-Capping Protein Heterodimer Regulate Each Other at Multiple Levels

  • Ana Rita Amândio, 
  • Pedro Gaspar, 
  • Jessica L. Whited, 
  • Florence Janody
PLOS
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Correction

7 Jul 2014: The PLOS ONE Staff (2014) Correction: Subunits of the Drosophila Actin-Capping Protein Heterodimer Regulate Each Other at Multiple Levels. PLOS ONE 9(7): e102614. https://doi.org/10.1371/journal.pone.0102614 View correction

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.

Introduction

The actin cytoskeleton controls numerous processes, including cell shape, mobility, division and intracellular transport. In normal cells, the actin cytoskeleton is tightly controlled to regulate these essential functions; however, it can be subverted by cancer cells and contributes to changes in cell growth, proliferation, stiffness, movement and invasiveness [1], [2]. Moreover, alterations in the activity or expression of actin-binding proteins (ABPs) per se, have been linked to cancer initiation and progression [2], [3], [4], [5], [6].

Among these actin regulators, the actin Capping Protein (CP) heterodimer, composed of an α and a β subunit, appears to act as a main tumor suppressor module [7], [8], [9], [10]. CP was named based on its ability to bind and cap actin filament barbed ends, inhibiting the addition and loss of actin monomers [11], [12], [13]. CP has homologs in nearly all eukaryotic cells, including vertebrates, invertebrates, plants, fungi, insects and protozoa [14]. Drosophila and organisms other than vertebrates have single genes encoding capping protein α (cpa) or β (cpb). In contrast, vertebrates contain two genes expressed somatically that encode two α subunits (α1 and α2), and one single gene that produce two β isoforms (β1 and β2) through alternative splicing [15], [16], [17]. Although the amino acid sequences of the α and β subunits are not more similar to each other than they are to other ABPs, nor they share common sequences with other proteins, they have extremely similar secondary and tertiary structures [18]. When in complex, the heterodimer resembles a mushroom with the C-terminus of each subunit forming tentacles located on the top surface of the heterodimer [19], [20]. In vitro analyses of chicken and budding yeast CP revealed that deletions or point mutations in either the α or β tentacles do not affect protein stability but reduce the capping affinity, while a complete removal of both tentacles fully abrogates the actin-binding activity [12], [20]. Thus, CP appears to cap F-actin barbed ends via the independent interaction of both tentacles with actin. In vivo, a truncated form of Drosophila cpa deleted of the C-terminal 28 amino acids has no effect on F-actin when expressed alone but promotes F-actin accumulation when co-expressed with full length cpb [21]. Similarly, a chicken β subunit containing a point mutation changing a conserved leucine to arginine at position 262, which caps actin poorly, disrupts the early steps in myofibrillogenesis of cultured myotubes and the sarcomere of mouse heart [22], [23], [24].

In yeast and Drosophila, removing either cpa or cpb induces F-actin accumulation and identical phenotypes [25], [26], [27]. In the fly, CP is required for proper differentiation of adult bristles, survival of the adult retina, determination of the oocyte and cortical integrity of nurse cells in the egg chamber [27], [28], [29], [30]. In addition, CP has a key role in restricting tissue growth. In the whole wing disc epithelium, CP-dependent F-actin regulation suppresses inappropriate tissue growth by inhibiting the activity of the Yorkie (Yki) oncogene, which mediates Hippo signalling activity [7], [9]. This function is conserved, as the α1 subunit is also required to limit the activity of the Yki orthologs YAP and TAZ in mammary epithelial cells [31]. In addition, in the distal Drosophila wing disc epithelium, CP prevents JNK-mediated apoptosis or proliferation and counteracts the oncogenic ability of Src [8], [21], [32]. Furthermore, underexpression of the human α1 subunit correlates with cancer-related death and causes a significant increase in gastric cancer cell migration and invasion in vitro, whereas its overexpression has the opposite effect [10].

We aimed in this report to understand how both subunits regulate each other in vivo to control F-actin levels and tissue growth. We show that Cpa and Cpb stabilize each other's protein levels and can stimulate the production of each other's mRNA when the level of one of the subunit is reduced. Because overexpressing CP decreases F-actin levels and tissue growth, while expressing forms of CP mutated in their actin-binding domains has opposite effects, we propose that by regulating each other, Cpa and Cpb assure that a pool of functional CP heterodimer is produced in sufficient quantities to restrict tissue growth and therein prevent tumor development but not in excess to sustain proper tissue growth.

Materials and Methods

Molecular Biology

To generate UAS-cpbL262R, site-directed mutagenesis was performed on the plasmid UAS-cpb, using the QuikChange kit (Stratagene, # 200519). The mutated plasmid was confirmed by sequencing and transgenic flies were generated by standard methods.

Fly strains and genetics

Fly stocks used were sd-Gal4 [33]; nub-Gal4 [34]; hh-Gal4 (a gift from T. Tabata); da-Gal4 [35]; UAS-cpa-IRC10, UAS-cpa-IRB4 [7]; UAS-HA-cpa89E, UAS-HA-cpaΔABD [21]; UAS-cpb7 [36]; UAS-cpb-RI45668 (Vienna Drosophila Research Center, VDRC); cpa107E [25], cpbM143 (FlyBase). To generate cpa mutant clones marked by the absence of GFP and expressing or not UAS-HA-cpa89E or UAS-HA-cpaΔABD or UAS-cpb7, w; FRT42D, cpa69E/CyO or w; FRT42D, cpa107E/CyO; UAS-HA-cpa89E/Tm6β or w; FRT42D, cpa107E/CyO; UAS-HA-cpaΔABD/Tm6β or w; FRT42D, cpa107E, UAS-cpb7/CyO males were crossed to y, w, FRT42D, ubi-GFP; T155-Gal4, UAS-flp/ST females. To generate cpb mutant clones marked by the absence of GFP, w, y; FRT40A, cpbM143/CyOy+ males were crossed to y, w, hsFLP122; FRT40A, ubi-GFP females and the progeny was heat-shocked at first and second instar larvae. All crosses were maintained at 25°C and the progeny was dissected at end of third instar larvae.

Antibody Generation

The rabbit anti-Cpa and rabbit anti-Cpb polyclonal antibodies were generated by Metabion International AG using full length Cpa or Cpb tagged with Histidine.

Immunohistochemistry and quantification

We performed immunocytochemistry using the procedure described in Lee and Treisman [37]. Primary antibodies used were mouse anti-Arm (N2 7A1, Developmental Studies Hybridoma Bank (DSHB); 1∶10), rat anti-DE-Cad (1∶50, CAD2, DSHB), rabbit anti-Cpa (1∶200); rabbit anti-Cpb (1∶200); mouse anti-HA (Covance 11 MMS101P; 1∶1000) and rabbit anti-Caspase 3 (Cell Signalling #9661; 1∶50). Rhodamine conjugated phalloidin (Sigma) was used at a concentration of 0.3 µM. Secondary antibodies were from Jackson Immunoresearch, used at 1∶200. Wing discs were mounted in VECTASHIELD Mounting Media (Vector Laboratories, Inc. #H-1000). Fluorescence images were obtained on a Leica SP5 confocal microscope or on a LSM 510 Zeiss confocal microscope. The NIH Image J program was used to perform measurements. Quantifications of the intensity of Caspase 3 signals were performed as described in [21]. Quantifications of the ratio of Phalloidin signal between posterior and anterior wing compartments were performed as described in [7]. To quantify the ratio of Cpa or Cpb signals between the anterior and posterior wing disc compartments, a region of interest (ROI) of 100 per 50 pixels was selected. The sum of the gray values was measured for each ROI, applied to each compartments for each disc on optical cross sections through distal wing disc epithelium comprising the apical surface. To measure wing size, wing were dissected one to two days after eclosion and imaged using the Hamamatsu Orca-ER camera attached to a Zeiss' Stereo Lumar V12 stereoscope. The total area of each wing was outlined and measured using the area measurement function. Statistical significance was calculated using a two-tailed t-test.

Western Blotting

For each genetic background, proteins were extracted from either four wing imaginal discs or four dechorionated embryos using a 2x SDS sample buffer (Sigma #S3401). Samples were frozen in liquid nitrogen, boiled for 5 minutes in 5 µl Sample Buffer 2x, spun at 13,000 g for 1 minute, loaded on a 10% SDS-PAGE gel and transferred to a PVDF membrane (Amersham Hybond-P, GE Healthcare). Proteins were visualized by immunoblotting using rabbit anti-Cpa (1∶2500) or rabbit anti-Cpb (1∶2500) or mouse anti-HA (Covance 11 MMS101P; 1∶1000) or rabbit anti-Histone H3 (Cell Signalling #9715; 1∶3000). HRP-conjugated donkey anti-mouse or donkey anti-rabbit secondary antibodies were used at 1∶5000 (Jackson ImmunoResearch Laboratories, Inc.). Blots were developed using Amersha ECL Plus Western Blotting Detection System (GE Healthcare). Densitometric analysis of signal intensity was performed using the GelQuant.NET software (biochemlabsolutions.com) and normalized with the loading control. Statistical significance was calculated using a Paired t-test.

Isolation of RNA and Real-Time qRT-PCR

Total RNAs were extracted from either 10 first instar larvae or 50 wing imaginal discs for each genetic background. Samples were homogenized in RLT buffer treated with DNase (Qiagen) at 4 degree C and total RNAs were isolated using the RNeasy mini kit (Qiagen) following manufacturer instructions. First Strand cDNA Synthesis Kit for RT-PCR (Roche) was used to produce cDNAs from 1 µg of total RNA. To quantify mRNA levels, qPCRs were carried out on reverse-transcribed total mRNA using intron-exon-specific primers (Table S1), designed using the Primer3 software [38], [39], and ensuring that efficiency is at least 90% and restricting primer dimmer formation. Real-time qPCR was performed using PerfeCTa SYBR Green FastMix (Quanta Biosciences) in 384 well skirted PCR microplates (Axygen) sealed with optically clear sealing tape (STARSTEDT) in the Applied Biosystems 7900HT Fast Real-Time PCR System. The relative amount of mRNA for each condition was calculated after normalization to the RpL32 transcript. Statistical significance was calculated using a Paired t-test with significance at P<0.05.

Results

Cpa and Cpb stabilize each other's protein levels and accumulate at Adherens Junctions

To understand how Cpa and Cpb are regulated to restrict growth of Drosophila epithelia, we generated polyclonal antibodies to each CP subunit. In lysates from embryos expressing UAS-mCD8-GFP under the control of the ubiquitous daughterless-Gal4 (da-Gal4) driver, the Cpa (Fig. 1A) and Cpb (Fig. 1B) antibodies revealed a band at around 32 and 31 kDa respectively by Western Blot. These signals were lost in embryonic extracts from homozygous cpa (Fig. 1A) or cpb mutants (Fig. 1B) respectively. Conversely, overexpressing full length cpa, tagged with HA (UAS-HA-cpa+; Fig. 1A) or cpb (UAS-cpb+; Fig. 1B) with da-Gal4, enhanced the anti-Cpa or anti-Cpb signals respectively. Similarly, Cpa levels were increased in wing disc lysates overexpressing HA-cpa under scalloped-Gal4 control (sd>HA-cpa+; Fig. 1C), while endogenous Cpb levels were similar to control sd>GFP lysates (Fig. 1D). Forcing cpb expression in this tissue also induced a significant increase in Cpb levels by Western Blot (Fig. 1D) but did not significantly affect endogenous Cpa levels (Fig. 1C). Cross-sections through wing disc epithelia expressing UAS-mCD8-GFP in the posterior compartment using the hedgehog-Gal4 (hh-Gal4) driver showed that Cpa (Fig. 1E–E′′′) and Cpb (Fig. 1F–F′′′) accumulated at the apical cell membrane and co-localized with components of Adherens Junctions, including the β-Catenin homolog Armadillo (Arm). Co-expressing cpb and mCD8-GFP in this domain strongly enhanced the anti-Cpb signals but did not affect Cpa levels (Fig. 2D–D″). Conversely, hh>HA-cpa+ wing disc epithelia displayed an apical localization of HA-Cpa, like endogenous Cpa (Fig. 1E–E″), but no change in Cpb levels (Fig. 2E–E″). Thus, the anti-Cpa and Cpb antibodies recognize specifically Cpa and Cpb respectively.

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Figure 1. Loss of cpa or cpb reduces both Cpa and Cpb protein levels.

(A) western blot on protein extracts from embryos expressing UAS-mCD8GFP (lane 1) or UAS-HA-cpa89E (lane 2) under da-Gal4 control or homozygote mutant for the cpa69E allele (lane 3), blotted with anti-Cpa (upper panel) and anti-H3 (lower panel). (B) western blot on protein extracts from embryos expressing UAS-mCD8GFP (lane 1) or UAS-cpb7 (lane 2) under da-Gal4 control or homozygote mutant for the cpbM143 allele (lane 3), blotted with anti-Cpb (upper panel) and anti-H3 (lower panel). (C and D) western blots on protein extracts from wing imaginal discs expressing UAS-mCD8GFP (lane 1) or UAS-HA-cpa89E (lane 2) or UAS-cpa-IRC10 (lane 3) or UAS-cpb7 (lane 4) or UAS-cpb-IR45668 (lane 5) under sd-Gal4 control, blotted with (C) anti-Cpa (upper panel) and anti-H3 (lower panel) or (D) anti-Cpb (upper panel) and anti-H3 (lower panel). (E–E″ to J–J′) optical cross sections through distal wing disc epithelium of third instar larvae with apical side up in which hh-Gal4 drives (E–E″ and F–F″) UAS-mCD8-GFP (green in E and F) and (G–G′ and H–H′) UAS-cpa-IRC10 or (I–I′ and J–J′) UAS-cpb-IR45668. Discs are stained with (E–E″, G–G′ and I–I′) anti-Cpa (magenta) or (F–F″, H–H′ and J–J′) anti-Cpb (magenta) and (E–E″ and F–F″) anti-Arm. The arrows in G′, H′, I′ and J′ mark the limits of the posterior compartment boundary. The scale bars represent 15 µm. (K and L) graphs of (K) cpa or (L) cpb mRNA levels measured by five independent qRT-PCR in wing imaginal discs expressing UAS-mCD8GFP (lane 1) or UAS-cpa-IRC10 (lane 2) or UAS-cpb-IR45668 (lane 3) under sd-Gal4 control. (K) the mean for sd>GFP is 1.084; for sd>cpa-IRC10 is 0.4328; for sd>cpb-IR45668 is 1.155. P<0.0027 for comparison of lane 1 and 2. (L) the mean for sd>GFP is 0.6210; for sd>cpa-IRC10 is 0.5037; for sd>cpb-IR45668 is 0.2375. P<0.0049 for comparison of lane 1 and 3. n.s. indicates a non-significant P. Error bars indicate s.e.m.

https://doi.org/10.1371/journal.pone.0096326.g001

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Figure 2. Increasing the levels of individual CP subunits alone has no effect on the endogenous levels of the other subunit, while co-expressing HA-cpa or HA-cpaΔABD and cpb enhance synergistically the levels of both subunits.

(A) model by which Cpa and Cpb cap F-actin barbed ends via the interaction of α and β tentacles with actin. (B) western blot on protein extracts from wing discs expressing UAS-mCD8-GFP (lane 1) or UAS-mCD8-GFP and UAS-HA-cpa89E (lane 2) or UAS-HA-cpa89E and UAS-cpb7 (lane 3) or UAS-mCD8-GFP and UAS-HA-cpaΔABD (lane 4) or UAS-HA-cpaΔABD and UAS-cpb7 (lane 5) under sd-Gal4 control, blotted with anti-HA (middle panel) and anti-H3 (lower panel). The means for lane 1 is 0, for lane 2 is 0.6250, for lane 3 is 2, for lane 4 is 0.0667, for lane 5 is 1.300. Error bars indicate s.e.m.. P<0.0092 for comparison of lanes 2 and 3 and of lanes 4 and 5. (B) western blot on protein extracts from wing discs expressing UAS-mCD8-GFP (lane 1) or UAS-mCD8-GFP and UAS-cpb7 (lane 2) or UAS-cpb7 and UAS-HA-cpa89E (lane 3) under sd-Gal4 control, blotted with anti-Cpb (middle panel) and anti-H3 (lower panel). The upper panels in B and C represent a quantification of relative (B) HA or (C) Cpb intensity signals for each genetic combination, measured by 4 independent blots. The means for lane 1 is 0.1088, for lane 2 is 0.5699, for lane 3 is 0.7982. Error bars indicate s.e.m. P<0.0182 for comparison of lanes 2 and 3. (D–D″ to I–I″) optical cross sections through distal epithelia of third instar wing imaginal discs with apical sides up and posterior sides to the left in which hh-Gal4 drives (D–D″) UAS-cpb7 and one copy of UAS-mCD8-GFP (green in D) or (E–E″) UAS-HA-cpa89E and two copies of UAS-mCD8-GFP (green in E) or (F–F″) UAS-HA-cpa89E, UAS-cpb7 and one copy of UAS-mCD8-GFP (green in F) or (G–G″) UAS-HA-cpaΔABD and two copies of UAS-mCD8-GFP (green in G) or (H–H″) UAS-HA-cpaΔABD, UAS-cpb7 and one copy of UAS-mCD8-GFP (green in H) or (I–I″) UAS-cpbL262R, UAS-HA-cpaΔABD and one copy of UAS-mCD8-GFP (green in I). Discs are stained with anti-Cpb (Cyan blue) and (D–D″) anti-Cpa (magenta) or (E–E″ to I–I″) anti-HA (magenta), which reveals (E–E″ and F–F″) HA-cpa89E or (G–G″ to I–I″) HA-cpaΔABD expression. The scale bars represent 15 µm.

https://doi.org/10.1371/journal.pone.0096326.g002

Strikingly, Cpa levels were strongly reduced not only in wing disc extracts expressing double-stranded RNAs (dsRNA) for cpa under sd-Gal4 control (sd>cpa-IR) but also in discs knocked-down for cpb (sd>cpb-IR; Fig. 1C). In the converse experiment, the amount of Cpb was also strongly reduced in both sd>cpb-IR and sd>cpa-IR wing disc extracts (Fig. 1D). Similarly, knocking down cpa (Fig. 1G–G′ and H–H′) or cpb (Fig. 1I–I′ and J–J′) in the posterior wing disc compartment with hh-Gal4 significantly reduced the apical accumulation of both Cpa and Cpb when compared to anterior compartments used as internal controls. Moreover, both Cpa and Cpb levels were also strongly reduced in lysates from first instar larvae homozygote mutant for cpa or cpb (Fig. S1A) and in clones mutant for cpa or cpb (Fig. S1B–B″ to E–E″). To verify that the cpa dsRNA did not affect cpb mRNA and vice versa, we performed quantitative RT-PCR (qRT-PCR) experiments on wing imaginal discs knocked down for cpa or cpb. As expected, sd>cpa-IR or sd>cpb-IR wing discs showed a significant reduction of cpa (Fig. 1K, 2.5±0.43 folds) or cpb mRNA (Fig. 1L, 2.6±0.41 folds) levels respectively, relative to control sd>GFP. However, cpa mRNA levels were not significantly affected by a reduction in cpb (Fig. 1K), nor were cpb mRNA levels reduced in wing discs knocked-down for cpa (Fig. 1L). Similarly, a reduction in cpa or cpb levels had no effect on cpb or cpa mRNA levels, respectively, in first instar larvae expressing cpa-IR or cpb-IR under da-Gal4 control (Fig. S1F and G). Taken together, we conclude that Cpa and Cpb accumulate at apical cell membrane and enhance each other's protein levels.

Cpa and Cpb levels are rate limited to form a functional heterodimer

The Capping Protein α and β subunits form a functional heterodimer, which caps F-actin barbed ends via the interaction of the α and β tentacles with actin (Fig. 1A and [11], [12], [13], [20]). To confirm that the stabilization of Cpa and Cpb's protein levels by each other promotes the formation of a functional heterodimer, we first tested if co-expressing cpb and HA-cpa would enhance the levels of both subunits by comparing the levels of HA-Cpa and Cpb when overexpressed alone or together, ensuring that each genetic combination contained the same number of UAS transgenes. Indeed, by Western Blot (Fig. 2B, P<0.0092) and in wing disc epithelia (Fig. 2 compare F–F″ with E–E″), HA levels were strongly enhanced when HA-cpa was co-expressed with cpb. Similarly, the co-expression of HA-cpa and cpb strongly increased Cpb levels compared to wing disc lysates overexpressing cpb alone (Fig. 2C). Overexpressed HA-cpa and cpb appeared to form a functional heterodimer as their co-expression in the posterior wing disc compartment with hh-Gal4 decreased the apical F-actin ratio between both compartments compared to hh>GFP control (Fig. 3F, P<0.0001). In contrast, overexpressing either HA-cpa or cpb alone has no effect on F-actin levels [21]. We conclude that the levels of endogenous Cpa and Cpb available are rate limited to form a functional heterodimer.

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Figure 3. Overexpressing HA-cpa suppresses the apical F-actin accumulation of cpbL262R-expressing wing discs, whereas HA-cpaΔABD expression has the opposite effect.

(A–A″ to E–E″) standard confocal sections of the apical cell membrane of third instar wing imaginal discs with dorsal sides up and posterior sides to the left, expressing (A–A′) one copy of UAS-mCD8-GFP (green in A) or (B–B″) UAS-cpb7 and one copy of UAS-mCD8-GFP (green in B) or (C–C″) UAS-cpbL262R and two copies of UAS-mCD8-GFP (green in C) or (D–D″) UAS-cpbL262R, UAS-HA-cpa89E and one copy of UAS-mCD8-GFP (green in D) or (E–E″) UAS-cpbL262R, UAS-HA-cpaΔABD and one copy of UAS-mCD8-GFP (green in E) under hh-Gal4 control. Discs are stained with Phalloidin (white) to mark F-actin and (B–B″ to E–E″) anti-Cpb (cyan blue). The yellow lines outline the anterior-posterior compartment boundary. The scale bars represent 30 µm. (F) Mean intensity of the ratio of Phalloidin signal between posterior and anterior wing compartments of hh-Gal4 driving two copies of UAS-mCD8-GFP (lane 1) or UAS-HA-cpa89E, UAS-cpb7 and one copy of UAS-mCD8-GFP (lane 2) or UAS-HA-cpaΔABD, UAS-cpb7 and one copy of UAS-mCD8-GFP (lane 3) or UAS-cpb7 and one copy of UAS-mCD8-GFP (lane 4) or UAS-cpbL262R and two copies of UAS-mCD8-GFP (lane 5) or UAS-cpbL262R, UAS-HA-cpa89E and one copy of UAS-mCD8-GFP (lane 6) or UAS-cpbL262R, UAS-HA-cpaΔABD and one copy of UAS-mCD8-GFP (lane 7). The mean for lane 1 is 0.922 (n = 12) for lane 2 is 0.775 (n = 8), for lane 3 is 1.435 (n = 10), for lane 4 is 0.977 (n = 10), for lane 5 is 1.175 (n = 16), for lane 6 is.0.937 (n = 14), for 7 is 2.348 (n = 6). Error bars indicate s.e.m.. *** indicate P<0.0001.

https://doi.org/10.1371/journal.pone.0096326.g003

Forms of CP mutated in α or β tentacle counteract the ability of wild type CP to restrict F-actin accumulation

Surprisingly, expressing an HA-tagged form of Cpa deleted of the α tentacle (UAS-HA-cpaΔABD) has no significant effect on F-actin when expressed alone [21] but triggered apical F-actin accumulation when co-expressed with cpb (Fig. 3F, P<0.0001 and [21]), indicating that HA-CpaΔABD affects F-actin only in the presence of overexpressed cpb. We therefore tested if the co-expression of cpb would also enhance the levels of HA-CpaΔABD. In contrast to full length HA-Cpa, which accumulated apically (Fig. 2E–E″), HA-CpaΔABD localized uniformly along the apical-basal axis in the posterior compartment of hh>HA-cpaΔABD wing discs (Fig. 2G–G″). Strikingly, co-expressing cpb not only enhanced strongly HA-CpaΔABD levels as assessed by Western Blot (Fig. 2B, P<0.0002), but also relocalized HA-CpaΔABD at the apical cell membrane (Fig. 2H–H″). Thus, forcing Cpb levels enhances the levels of HA-CpaΔABD and promotes its apical localization.

The heterodimer formed between HA-CpaΔABD and Cpb appears to have reduced capping activity and may be recruited to F-actin barbed ends, preventing the binding of wild type CP. If so, we would expect that a form of Cpb truncated of its β tentacle would also promote F-actin accumulation in the presence of endogenous CP. To test this possibility, we expressed a form of cpb mutated in the highly conserved Leucine 262 (UAS-cpbL262R), which has been proposed to directly interact with actin [12]. While overexpressing full length cpb had no significant effect on F-actin (Fig. 3 compare B–B″ with A–A′ and F), hh>cpbL262R wing discs accumulated apical F-actin in the posterior compartment (Fig. 3C–C″ and F, P<0.0001). However, co-expressing full length HA-cpa in these tissues suppressed the apical F-actin accumulation due to the presence CpbL262R (Fig. 3D–D″ and F, P<0.0001). Thus, forcing Cpa levels tethers the effects of CpbL262R on F-actin. In contrast, F-actin accumulation was strongly enhanced when cpbL262R was co-expressed with HA-cpaΔABD (Fig. 3E–E″ and F, P<0.0001). Moreover, CpbL262R, like full length Cpb, enhances HA-CpaΔABD levels and triggered its relocalization to the apical cell membrane (Fig. 2I–I″). We conclude that forms of CP with reduced capping activity inhibit wild type CP to restrict F-actin accumulation, most likely by tethering barbed ends, preventing the recruitment of wild type CP.

CP and forms of CP with dominant negative effects on F-actin have opposite effects on tissue growth

Decreasing or increasing CP levels has opposite effects on F-actin levels (Fig. 3F and [25]). Because loss of CP induces overgrowth of the wing disc epithelium by promoting Yki activity [7], [9], we asked of overexpressing cpa and cpb has an opposite effect on tissue growth. Indeed, overexpressing full length HA-cpa and cpb in the wing primordium using the nubbin-Gal4 (nub-Gal4) driver significantly reduced the size of the adult wing (Fig. 4A, compare nub>GFP control wing in green to nub>cpa+, cpb+ wing in magenta and F; P<0.0151), but does not affect cell survival [21]. Thus, tight CP levels are critical to control tissue growth.

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Figure 4. Overexpressing full length HA-cpa and cpb prevents wing growth, while ectopic expression of HA-cpaΔABD and/or cpbL262R has the opposite effect.

(A, B, C, D and E) merge between adult wings expressing in green UAS-mCD8GFP under nub-Gal4 control and in magenta (A) UAS-HA-cpa89E and UAS-cpb7 or (B) UAS-HA-cpaΔABD and UAS-cpb7 or (C) UAS-cpbL262R and one copy of UAS-mCD8-GFP or (D) UAS-cpbL262R and UAS-HA-cpa89E or (E) UAS-cpbL262R and UAS-HA-cpaΔABD under nub-Gal4 control. (A′, B′, C′ D′ and E′) magnification of hairs on adult wings for the genotypes shown in A, B, C, D and E. (F) quantification of relative wing size normalized to nub>GFP control for nub-Gal4 driving UAS-mCD8-GFP (lane 1) or UAS-HA-cpa89E and UAS-cpb7 (lane 2) or UAS-HA-cpaΔABD and UAS-cpb7 (lane 3) or UAS-cpbL262R and one copy of UAS-mCD8-GFP (lane 4) or UAS-cpbL262R and UAS-HA-cpa89E (lane 5) or UAS-cpbL262R and UAS-HA-cpaΔABD (lane 6). The mean for lane 1 is 1(n = 32), for lane 2 is 0.9702 (n = 12), for lane 3 is 1.119 (n = 13), for lane 4 is 1.061 (n = 24), for lane 5 is 1.015 (n = 13), for lane 6 is 1.051 (n = 13). Error bars indicate s.e.m.. P<0.015 for comparison of lanes 1 and 2. P<0.0001 for comparison of lanes 1 and 3 or 4 or 6 and for comparison of lane 4 and 5.

https://doi.org/10.1371/journal.pone.0096326.g004

To determine if CP controls tissue growth via F-actin regulation, we analyzed the effect of expressing forms of cpa and cpb that have dominant negative effects on F-actin on wing growth. Expressing HA-cpaΔABD and cpb (Fig. 4B and F, P<0.0001) or cpbL262R alone (Fig. 4C and F, P<0.0001) or combined with HA-cpaΔABD (Fig. 4E and F, P<0.0001) under nub-Gal4 control, not only promoted apical F-actin accumulation (Fig. 3), but also enhanced significantly the growth of adult wings. Strikingly, expressing HA-cpa suppressed the overgrowth of nub>cpbL262R wings (Fig. 4D and F, P<0.0001), indicating that the effect of CpbL262R on F-actin and tissue growth is dependent on the levels of full length Cpa. Because altering the levels or activity of CP did not affect the density of wing hairs (Fig. 4A′, B′, C′ D′ and E′), which develop from one single cell, the CP-dependent growth defects most likely result from changes in proliferation rate rather than alteration of cell size. We conclude that a CP-dependent reduction of F-actin levels correlates with tissue undergrowth, while a CP-dependent increase in F-actin levels is associated with tissue overgrowth.

The α tentacle is not absolutely required to form a functional heterodimer

Because the heterodimer formed between HA-CpaΔABD and Cpb appears to be recruited at F-actin barbed ends, we tested if HA-CpaΔABD can partially compensate for the loss of endogenous Cpa. Expressing cpa-IR under sd-Gal4 control induced the activation of Caspase 3 in numerous cells in the distal wing disc epithelium (Fig. 5A–A′). Apoptosis was almost fully suppressed by overexpressing full length HA-cpa (Fig. 5B–B′ and G; P<0.0001). Expressing HA-cpaΔABD also significantly prevented apoptosis of sd>cpa-IR wing discs, although to a much weaker extent than HA-cpa (Fig. 5C–C′ and G; P<0.0005). These effects were not only due to titration of the cpa dsRNAs by the overexpressed cpa constructs as HA-cpa (Fig.5E–E″ and H) or HA-cpaΔABD (Fig. 5F–F″ and H; P<0.0048) also rescued apoptosis of clones mutant for a cpa allele. Expressing HA-cpa or HA-cpaΔABD in sd>cpa-IR wing discs also partially restored Cpa (Fig. 5I) and Cpb (Fig. 5J) levels, as assessed by Western blot. Quantification of the ratio of Cpb signals between the posterior and anterior compartments of wing discs expressing cpa-IR under hh-Gal4 control showed that knocking-down cpa reduced Cpb levels in the posterior compartment compared to hh>GFP control (Fig. 5K). This decrease in Cpb levels was significantly alleviated by the presence of HA-CpaΔABD (Fig. 5K P<0.0085). We conclude that in the absence of wild type Cpa, CpaΔABD s capable of forming a functional heterodimer with Cpb, which prevents apoptosis.

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Figure 5. Expressing HA-cpa or HA-cpaΔABD suppresses apoptosis and restores Cpb levels of wing discs knocked-down for cpa.

(A–A′ to F–F′) standard confocal sections of third instar wing imaginal discs with dorsal sides up. (A–A′ to C–C′) sd-Gal4 driving (A–A′) UAS-cpa-IRC10 and two copies of UAS-mCD8-GFP (green in A) or (B–B′) UAS-cpa-IRC10, UAS-HA-cpa89E and one copy of UAS-mCD8-GFP (green in B) or (C–C′) UAS-cpa-IRC10, UAS-HA-cpaΔABD and one copy of UAS-mCD8-GFP (green in C). (D–D″ to F–F″) T155-Gal4; UAS-flp induced cpa107E mutant clones marked by the absence of GFP (green) and expressing (E–E″) UAS-HA-cpa89E or (F–F″) UAS-HA-cpaΔABD in the whole wing disc epithelium. Discs are stained with anti-activated-Caspase 3 (magenta), which monitors DRONC activation and (D–D″ to F–F″) anti-DE-Cad (cyan blue). The scale bars represent 30 µm. (G) quantification of total C3 area per disc area for the three genotypes shown in A–A′ to C–C′. The mean for sd>cpa-IRC10, 2XGFP is 92.4 (n = 23); for sd>cpa-IRC10, HA-cpa89E, 1XGFP is 0.7 (n = 10); for sd>cpa-IRC10, HA-cpaΔABD, 1XGFP is 51.4 (n = 20). Error bars indicate s.e.m. P<0.0001 for comparison of lane 1 and 2. P<0.0005 for comparison of lane 1 and 3. (H) quantification of total C3 area per disc area for the three genotypes shown in D–D″ to F–F″. The means for T155>flp; cpa107E is 9.228 (n = 18); for T155>flp; cpa107E; UAS-HA-cpa89E is 0.608 (n = 12); for T155>flp; cpa107E; UAS-HA-cpaΔABD is 4.329 (n = 17). Error bars indicate s.e.m. P<0.0001 for comparison of T155>flp; cpa107E and T155>flp; cpa107E; UAS-HA-cpa89E and P<0.0048 for comparison of T155>flp; cpa107E and T155>flp; cpa107E; UAS-HA-cpaΔABD. (I and J) western blots on protein extracts from wing discs expressing two copies of UAS-mCD8-GFP (lane 1) or UAS-cpa-IRC10 and two copies of UAS-mCD8-GFP (lane 2) or UAS-cpa-IRC10 and UAS-HA-cpa89E and one copy of UAS-mCD8-GFP (lane 3) or UAS-cpa-IRC10 and UAS-HA-cpaΔABD and one copy of UAS-mCD8-GFP (lane 4) under sd-Gal4 control, blotted with (I) anti-Cpa (upper panel) and anti-H3 (lower panel) or (J) anti-Cpb (upper panel) and anti-H3 (lower panel). (K) mean intensity of the ratio of Cpb intensity signals between posterior and anterior wing compartments of hh-Gal4 driving two copies of UAS-mCD8-GFP (lane 1) or UAS-cpa-IRC10 and two copies of UAS-mCD8-GFP (lane 2) or UAS-cpa-IRC10 and UAS-HA-cpaΔABD and one copy of UAS-mCD8-GFP (lane 3). The mean for lane 1 is 1.064 (n = 20), for lane 2 is 0.822 (n = 17), for lane 3 is 0.883 (n = 24). Error bars indicate s.e.m.. P<0.0001 for comparison of lanes 1 and 2 or 3 or P<0.0085 for comparison of lanes 2 and 3.

https://doi.org/10.1371/journal.pone.0096326.g005

Cpb compensates for a reduction in cpa by enhancing cpa mRNA levels and vice versa

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).

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Figure 6. Overexpressing cpb in wing discs knocked-down for cpa, restores cpa mRNA and protein levels and suppresses apoptosis.

(A–A′ to C–C′) standard confocal sections of third instar wing imaginal discs with dorsal sides up, expressing (A–A″) UAS-cpa-IRC10 and two copies of UAS-mCD8-GFP (green in A) or (B–B′) UAS-cpa-IRC10, UAS-HA-cpaΔABD and UAS-cpb7 or (C–C′) UAS-cpa-IRC10, UAS-cpb7 and one copy of UAS-mCD8-GFP (green in C) under sd-Gal4 control. Discs are stained with anti-activated-Caspase 3 (magenta), which monitors DRONC activation and (B–B′) Phalloidin (cyan blue in B) to underline wing disc shape. The scale bars represent 30 µm. (D) quantification of total C3 area per disc area for the genotypes sd>cpa-IRC10, 2XGFP (lane 1); sd>cpa-IRC10, HA-cpaΔABD, cpb7 (lane 2) and sd>cpa-IRC10, cpb7, 1XGFP (lane 3). The means for lane 1 is 92.4 (n = 23); for lane 2 is 10.61 (n = 19); for lane 3 is 32.9 (n = 20). Error bars indicate s.e.m. P<0.0001 for comparison of lane 1 and 2 or 3 or lane 2 and 3. (E) quantification of total C3 area per disc area for wing discs containing T155>flp; cpa107E mutant clones (lane 1) or T155>flp; cpa107E mutant clones expressing UAS-cpb7 (lane 2). The means for lane 1 is 10.80 (n = 26); for lane 2 is 13.77 (n = 20). n.s. indicates non-significant P value. (F and G) western blots on protein extracts from wing discs expressing two copies of UAS-mCD8-GFP (lane 1) or UAS-cpa-IRC10 and two copies of UAS-mCD8-GFP (lane 2) or UAS-cpa-IRC10, UAS-cpb7 and one copy of UAS-mCD8-GFP (lane 3) under sd-Gal4 control, blotted with (F) anti-Cpa (upper panel) and anti-H3 (lower panel) or (G) anti-Cpb (upper panel) and anti-H3 (lower panel). Panels derive from the same experiment shown in Figure 5E and F and blots were processed in parallel (see Figure S2 showing the whole experiment). (H) mean intensity of the ratio of Cpa signals between posterior and anterior wing compartments of hh-Gal4 driving two copies of UAS-mCD8-GFP (lane 1) or UAS-cpb7 and one copy of UAS-mCD8-GFP (lane 2) or UAS-cpa-IRC10 and two copies of UAS-mCD8-GFP (lane 3) or UAS-cpa-IRC10 and UAS-cpb7 and one copy of UAS-mCD8-GFP (lane 4). The mean for lane 1 is 0.959 (n = 15), for lane 2 is 0.970 (n = 20), for lane 3 is 0.716 (n = 21), for lane 4 is 0.776 (n = 20). Error bars indicate s.e.m.. P<0.0001 for comparison of lanes 1 and 3 or 4 or P<0.01 for comparison of lanes 3 and 4. (I to L) graph of (I and J) cpa or (K and L) cpb mRNA levels measured by five independent qRT-PCR in wing imaginal discs expressing (I and K) two copies of UAS-mCD8GFP (lane 1) or UAS-cpa-IRC10 and UAS-mCD8GFP (lane 2) or UAS-cpa-IRC10 and UAS-cpb7 (lane 3) or (J and L) UAS-mCherry (lane 1) or UAS-cpb7 (lane 2) under sd-Gal4 control. (I) the means for lane 1 is 1.084; for lane 2 is 0.4328; for lane 3 is 1.086. P<0.0027 for comparison of lane 1 with 2 or P<0.0003 for comparison of lane 2 with 3. (J) the means for lane 1 is 1.07; for lane 2 is 0.824. n.s. indicates non-significant P value. (K) the means for lane 1 is 0.621; for lane 2 is 0.5031; for lane 3 is 3.735. P<0.0001 for comparison of lane 3 with 1 or 2. (L) the means for lane 1 is 0.292; for lane 2 is 1.961. P<0.0001 for comparison of lane 1 and 2. Error bars indicate s.e.m.

https://doi.org/10.1371/journal.pone.0096326.g006

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Figure 7. 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.

https://doi.org/10.1371/journal.pone.0096326.g007

To understand the mechanisms by which Cpa and Cpb compensate for each other's function, we tested the effect of overexpressing cpb on Cpa levels in cpa-depleted tissues. As previously observed, by Western Blots, Cpa (Fig. 6F) and Cpb (Fig. 6G) levels were strongly reduced in wing disc extracts knocked-down for cpa. Forcing cpb levels in these tissues enhanced the levels of both Cpa (Fig. 6F and Fig. S2) and Cpb (Fig. 6G and Fig. S2). We quantified this effect by measuring the ratio of Cpa signals between the posterior and anterior compartments of hh>cpa-IR-expressing wing discs, in the presence or absence of overexpressed cpb. While in control hh>GFP tissues this ratio was 0.95, knocking down cpa reduced this ratio to 1,34 folds (Fig. 6H; P<0.0001). This effect was significantly alleviated by the overexpression of cpb (Fig. 6H; P<0.01). In contrast, overexpressing cpb in control hh>GFP wing discs did not affect Cpa levels (Fig. 6H), indicating that Cpb enhances Cpa levels only when cells contain reduced Cpa levels. By Western Blots, HA-cpa also enhanced both Cpa (Fig. 7D) and Cpb (Fig. 7E) levels when expressed in tissues knocked-down for cpb. Thus, Cpa compensates for a reduction in cpb by stimulating the production of Cpb, and vice versa.

Using qRT-PCR, we next analyzed if overexpressing either subunits affects the mRNA levels of the other. After normalization to the RpL32 transcript used as an internal control, we observed that whereas cpa (Fig. 6I, P<0.0027) but not cpb (Fig. 6K) mRNA levels were strongly reduced in wing discs knocked-down for cpa (sd>cpa-IR), forcing cpb levels in these tissues fully restored cpa mRNA to wild type levels (Fig. 6I; P<0.0003). In contrast, in wing discs that contained endogenous cpa and cpb, overexpressing cpb, which strongly enhanced cpb mRNA levels (Fig. 6L), had no significant effect on cpa mRNA levels (Fig. 6J). Thus, Cpb stimulates the production or stabilization of cpa mRNA only when Cpa levels are reduced. In the converse experiment, overexpressing HA-cpa in sd>cpb-depleted wing discs enhanced the levels of both cpa (Fig. 7F) and cpb (Fig. 7H; P<0.0018) mRNA. However, in wing discs that contained endogenous cpa and cpb, only cpa mRNA levels were strongly increased (Fig. 7G and I). The ability of Cpb to suppress apoptosis of cpa-depleted wing discs was due to the increase in cpa mRNA and protein levels as clones mutant for a cpa allele showed similar apoptotic levels in the absence or presence of overexpressing cpb (Fig. 6E). We conclude that Cpa compensates for a reduction in cpb by increasing cpb mRNA levels and vice versa.

Discussion

Cpa and Cpb regulate each other at multiple levels

Our data argue that in Drosophila, different pools of Cpa and/or Cpb co-exist, and they regulate each other at various levels. One level of regulation involves their reciprocal stabilization of their protein levels. First, in Drosophila, like in yeast, the loss of one CP subunit reduces the protein levels of the other subunit ([26] and Fig. 1) but does not affect its mRNA levels (Fig. 1 and Fig. S1). Second, co-expressing cpa and cpb in Drosophila tissues enhances synergistically the levels of both subunits relative to the levels of each subunit overexpressed alone (Fig. 2). Third, large quantities of soluble active chicken CP can be produced in bacteria only when both subunits are co-expressed [40]. Cpa and Cpb may stabilize each other's protein levels via direct protein-protein interactions [19]. The tight interaction between both subunits may prevent the recruitment of E3 ubiquitin ligases that would otherwise target individual CP subunits for degradation by the 26S proteasome. As an heterodimer, CP has been shown to bind to the fast polymerizing ends of actin filaments, preventing further addition of actin monomers [41], [42] and to restrict F-actin accumulation in Drosophila tissues [25], [27]. In addition, Cpa and Cpb appear to show some function on their own as overexpressing cpb rescues apoptosis of wing discs knocked-down for cpa and vice versa (Fig. 6 and 7). Overexpression of cpb alone is also sufficient to enhance the retinal defects of flies knocked down for the Cbl-interacting protein cindr [43] and to rescue the migration and F-actin polarization defects of Drosophila border cells mutant for warts [44]. Because individual chicken CP subunits expressed in bacteria are mainly deposited into insoluble cytoplasmic inclusion bodies but can be renaturated as active heterodimers [45], individual subunit may exist in the cell as pools of insoluble monomers. The molecular mechanism by which individual CP subunit compensates for each other's function remains to be determined. Several observations argue that this mechanism involves the production of the subunit knocked-down by the other subunit via an increase of its mRNA levels (Fig. 6 and 7). CP has been observed in the nuclei of chicken retinal and kidney epithelial cells in culture, in Madin-Darby canine kidney (MDCK) cells, in Xenopus laevis oocytes and bovine lens epithelial cells in culture [46], [47]. Whether Cpa and Cpb influence each other's transcription in the nucleus is an interesting possibility to be tested. The protein-mRNA feedbacks between Cpa and Cpb may guarantee that a pool of functional heterodimer is present to limit F-actin polymerization. However, a CP-dependent negative feedback mechanism must exist that restricts the production of CP in excess, as forcing the expression of one of the subunit in tissues that contain endogenous CP does not enhance the mRNA and protein levels of the other subunit (Fig. 6 and 7). Because the loss of one subunit has no effect on the mRNA levels of the other subunit (Fig. 1 and Fig. S1), the CP-dependent negative feedback may act by limiting the ability of individual subunits to stimulate the production of each other's mRNAs. Thus, in addition to regulate each other's protein levels, individual CP subunit stimulates each other's mRNA production up to an optimal physiological threshold of functional heterodimers. Further experiments are necessary to elucidate the protein-mRNA feedback loop mechanisms, which operate between both subunits.

Capping activity of the CP heterodimer at actin filament barbed ends

Our observations argue that in vivo the actin-binding domain of Cpa is not absolutely required to form a functional CP heterodimer, as HA-CpaΔABD partially compensates for the loss of endogenous Cpa (Fig. 5). Consistent with our observations, in actin assembly assays, a mutant form of the chicken α subunit that lacks the α tentacle is able to cap F-actin [12]. Nevertheless, the α tentacle may favor the interaction and therefore stabilization of the α subunit by the β. This possibility is consistent with the observation that HA-CpaΔABD is found in the cell at much lower levels than full length HA-Cpa (Fig. 2) despite both transgenes being inserted at the same locus in the fly genome and therefore likely expressed at similar levels [21]. Consistent with this hypothesis, Arginine 259 of the chicken α1 tentacle forms side-chain hydrogen bonds with three residues of the β subunit, all residues being conserved across isoforms and species [19]. Moreover, in vitro, a truncated form of the chicken α1 subunit, consisting only of the C-terminal domain, retains the ability to form a heterodimer [48]. The reduced ability of HA-CpaΔABD to interact with Cpb may explain its inability to fully suppress apoptosis of Cpa-depleted tissues (Fig. 5) and to affect F-actin levels when overexpressed alone [21]. However, several observations indicate that the α and β tentacles also enable full capping activity in vivo. First, in actin assembly assays, the C-terminus of the chicken α1 and β1 subunits are required for high-affinity capping [12]. Second, in the presence of endogenous CP, stabilizing HA-CpaΔABD levels by forcing cpb expression does not reduce F-actin levels, as does overexpressed HA-cpa/cpb, but instead, promotes F-actin accumulation (Fig. 3 and [21]). Third, replacing leucine 262 of the chicken β subunit has no effect on protein stability and global structure but decreases the capping affinity significantly [12], [20]. Fourth, identical mutations in the β orthologs induces F-actin accumulation in Drosophila tissues (Fig. 3) and disrupts the sarcomere of mouse heart [24]. Thus, we propose that the heterodimers formed between HA-CpaΔABD and Cpb or between CpbL262R and Cpa are recruited to F-actin barbed ends and cap actin filaments less efficiently than wild type CP. The low capping activity of the HA-CpaΔABD/Cpb heterodimer is sufficient to partially compensate for the loss of Cpa. However, in the presence of endogenous CP, the HA-CpaΔABD/Cpb heterodimers compete with wild type Cpa/Cpb heterodimers for binding the barbed ends of F-actin, which can lead to defects in F-actin.

Tight regulation of CP levels is critical to control tissue growth

CP appears to act as a gatekeeper, which limits the development of cancer-related processes. Loss of the α subunit promotes Yki/YAP/TAZ-dependent proliferation in Drosophila epithelia and in human cells [9], [31], causes a significantly increase in gastric cancer cell migration and is associated with cancer-related death [10]. In contrast, increasing CP levels has opposite effects: it reduces tissue growth (Fig. 4) and prevents Src-mediated tumour development in Drosophila [21], and significantly restricts gastric cancer cell migration [10]. Several of our observations argue that the function of CP on tissue growth involves its F-actin capping activity. First expressing cpbL262R, which contains a single point mutation affecting the capping activity [23], induces F-actin accumulation (Fig. 3) and wing overgrowth (Fig. 4). Moreover, CP-dependent F-actin accumulation correlates with tissue overgrowth, whereas tissue undergrowth is associated with a CP-dependent reduction in F-actin (Fig. 3 and 4). Consistent with these observations, other actin regulators have been shown to control Yki/YAP/TAZ dependent tissue growth [7], [9], [31]. Thus, a reduction or an increase of CP levels has deleterious consequences on tissue growth, implying that it must be tightly regulated. This may be achieved in part by the ability of Cpa and Cpb to stimulate or limit the production of each other in conditions of lower or higher CP levels respectively, assuring that a pool of functional CP heterodimer is produced in sufficient quantities in the cell to prevent cancer development but not in excess to sustain proper tissue growth.

Supporting Information

Figure S1.

Reducing cpa or cpb levels reduces both Cpa and Cpb protein levels. (A) western blot on protein extracts from first instar larvae, either white minus (lane 1) or homozygote mutant for cpa69E (lane 2) or homozygote mutant for cpbM143 (lane 3), blotted with (upper panel) anti-Cpa (upper bands) and anti-Cpb (lower band) and (lower panel) anti-H3. (B–B″ to E–E″) standard confocal sections of third instar wing imaginal discs, containing (B–B″ and C–C″) T155-Gal4; UAS-flp induced cpa69E mutant clones marked by the absence of GFP (green) or (D–D″ and E–E″) heat shocked-induced cpbM143 mutant clones marked by the absence of GFP (green). Discs are stained with (B–B″ and E–E″) anti-Cpa (magenta) or (C–C″ and E–E″) anti-Cpb (magenta). The scale bars represent 15 µm. (F and G) graphs of (F) cpa or (G) cpb mRNA levels measured by three independent qRT-PCR in first instar larvae expressing UAS-mCD8-GFP (lane 1) or UAS-cpa-IRC10 (lane 2) or UAS-cpb-IR45668 (lane 3) under da-Gal4 control. (F) The means for lane 1 is 7.04; for lane 2 is 1.13; for lane 3 is 5.91. Error bars indicate s.e.m.. P<0.015 for comparison of lane 1 and 2. (F) The means for lane 1 is 1.97; for lane 2 is 1.96; for lane 3 is 0.46. Error bars indicate s.e.m.. P<0.021 for comparison of lane 1 and 3. n.s. indicates non-significant P values.

https://doi.org/10.1371/journal.pone.0096326.s001

(TIF)

Figure S2.

Expressing HA-cpa or HA-cpaΔABD or cpb in wing discs knocked down for cpa restores Cpa and Cpb levels. Western blots on protein extracts from wing discs expressing two copies of UAS-mCD8-GFP (lane 1) or UAS-cpa-IRC10 and two copies of UAS-mCD8-GFP (lane 2) or UAS-cpa-IRC10 and UAS-HA-cpa89E and one copy of UAS-mCD8-GFP (lane 3) or UAS-cpa-IRC10 and UAS-HA-cpaΔABD and one copy of UAS-mCD8-GFP (lane 4) or UAS-cpa-IRC10 and UAS-HA-cpaABD, which contains the last 28 amino acids of the Cpa C-terminus and one copy of UAS-mCD8-GFP (lane 5) or UAS-cpa-IRC10, UAS-cpb7 and one copy of UAS-mCD8-GFP (lane 6) under sd-Gal4 control, blotted with (A) anti-Cpa (upper panel) and anti-H3 (lower panel) or (B) anti-Cpb (upper panel) and anti-H3 (lower panel).

https://doi.org/10.1371/journal.pone.0096326.s002

(TIF)

Table S1.

Intron-exon-specific primers used to quantify cpa, cpb and RpL32 mRNA levels by qRT-PCR.

https://doi.org/10.1371/journal.pone.0096326.s003

(DOCX)

Acknowledgments

We thank the Bloomington Drosophila Stock Center, the National Institute of Genetics and the Developmental Studies Hybridoma Bank for fly stocks. We are grateful to Barbara Jezowska, Beatriz García Fernández, Francesca Vasconcelos, Sonia Rosa, Vania Neves and Claire Brombey for technical support and advice on qRT-PCR. The manuscript was improved by the critical comments of Claudine Chaouiya and Alekos Athanasiadis.

Author Contributions

Conceived and designed the experiments: ARA PG JLW FJ. Performed the experiments: ARA PG JLW FJ. Analyzed the data: ARA PG FJ. Wrote the paper: FJ.

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