The effects of <i>mts^XE2258</i>, <i>Tap42^WT</i>, and <i>Tap42^ED</i> on the viability of <i>Tap42^RNAi</i> flie ^{a, b}.

<p>a. The actual surviving ratios of F1 progeny were quantified from the following crosses:</p><p>Cross 1: +/+; pnr-Gal4/TM3, Ser ♀ x UAS-Tap42^RNAi/CyO; +/+ ♂.</p><p>Cross 2: ap-Gal4/CyO ♀ x UAS-Tap42^RNAi/CyO ♂.</p><p>Cross 3: ap-Gal4/CyO ♀ x UAS-Tap42^RNAi, mts^XE2258/CyO ♂.</p><p>Cross 4: +/+; pnr-Gal4/TM3, Ser ♀ x UAS-Tap42^RNAi/CyO; UAS-Tap42^WT/MKRS ♂.</p><p>Cross 5: +/+; pnr-Gal4/TM3, Ser ♀ x UAS-Tap42^RNAi/CyO; UAS-Tap42^ED/MKRS ♂.</p><p>Cross 6: +/+ ♀ x mts^XE2258/CyO ♂.</p><p>b. Crosses were repeated at least three times and flies that enter eclosion were counted as survivors.</p

Tap42 interacts with all three PP2A members and is required for normal wing disc development.

<p>Panel A: FLAG immunoprecipitations (FLAG IPs) were performed from extracts of <i>Drosophila</i> S2 cells expressing HA₃-Mts, HA₃-PP4, or HA₃-PPV alone or together with wildtype (FLAG₃-Tap42^WT) or mutant Tap42 (FLAG₃-Tap42^ED). The FLAG immune complexes and corresponding cell extracts (lysates) were analyzed by Western blotting using the indicated epitope tag antibodies. Panel B: Adult flies expressing <i>Tap42^RNAi</i> in the <i>ap</i> domain displayed a marked thorax cleft (red arrow, B1) and shriveled wings (B5). Expression of <i>Tap42^WT</i> in this background completely rescued both thorax (B2) and wing defects (B6). However, introduction of the <i>Tap42^ED</i> mutant in this background failed to rescue the defects and the flies lacked the scutum (B3) and formed blistered wings (B7). Expression of <i>Tap42^ED</i> alone resulted in a mild defect around the scutum (B4) and the formation of a forked wing vein (B8). Genotypes: (B1 & B5) <i>ap-Gal4/UAS-Tap42^RNAi; +/+</i>. (B2 & B6) <i>ap-Gal4/UAS-Tap42^RNAi; +/UAS-Tap42^WT</i>. (B3 & B7) <i>ap-Gal4/UAS-Tap42^RNAi; +/UAS-Tap42^ED.</i> (A4 & B4) <i>ap-Gal4/+; +/UAS-Tap42^ED</i>.</p

JNK and DPP signaling are altered in wing imaginal discs following depletion of Tap42.

<p>The activity and expression of BSK was monitored in wing imaginal discs using antibodies recognizing phospho-JNK or total JNK. The pattern of active JNK/BSK (green, A1-3) was not different between control <i>UAS-Tap42^RNAi</i> flies (A1) and flies co-expressing the <i>pnr</i> driver (A2). However, hyperphosphorylation of JNK/BSK was observed in the wing disc dorsal compartment (red arrows) along with hypophosphorylation of JNK/BSK in the ventral wing compartment when <i>Tap42^RNAi</i> was driven by <i>ap-Gal4</i> (A3). Total levels of JNK/BSK (green, B1-B3) did not change as a result of <i>Tap42</i> knockdown. <i>Dpp</i> gene expression (purple, C1-C3), as monitored by X-GAL staining of <i>dpp-LaZ</i>, in the scutellum and along the anterior/posterior boundary of the wing blade was similar in both control (C1) and <i>pnr</i>-<i>Gal4</i> driven <i>Tap42^RNAi</i> flies (C2). <i>ap-Gal4</i> driven <i>Tap42^RNAi</i> flies demonstrated decreased DPP signal in the scutellum (red arrow, C3) and expanded staining in the wing blade compartment (red dashed line, C3). Genotypes: (A1, B1, & C1) <i>UAS-Tap42^RNAi/+</i> as control. (A2, B2, & C2) <i>UAS-Tap42^RNAi/+; pnr-Gal4/+</i>. (A3, B3, & C3) <i>ap-Gal4/UAS-Tap42^RNAi; +/+</i>.</p

Tap42 is expressed in imaginal discs and primarily localized in the peripodial epithelium (PE) region.

<p>Panel A: Wing (A1–A3), haltere/3^rd leg (A4–A6), 2^nd leg (A7–A9), and eye imaginal discs (A10–A12) isolated from 3^rd instar larvae were immunostained for Tap42 protein expression (green) and counter-stained with the nucleic acid dye TO-PRO3 (purple). <i>UAS-Tap42^RNAi</i> control flies exhibited abundant expression of <i>Tap42</i> in the PE region of these imaginal discs (A1, A4, A7, & A10). <i>Tap42^RNAi</i> expression with the <i>pnr</i> (A2, A5, A8, & A11) and <i>ap</i> (A3, A6, A9, & A12) drivers dramatically reduced <i>Tap42</i> expression to nearly undetectable levels. Of note, <i>ap-Gal4</i>-mediated silencing of <i>Tap42</i> also disrupted the morphological patterning of the wing disc, as revealed by TO-PRO3 staining (A3). Panel B: The localization of Tap42 in the PE region was confirmed by immunofluorescence histochemistry. Immunostaining of wing discs obtained from wild type flies revealed an overlap of Ubx (red) and Tap42 (green) expression (B1). An amplified view of the merged image highlights strong Tap42 expression around the presumptive medial edge (ME) cells of the PE, which localizes near the boundary of the PE and DP (B2). Some Tap42 expression was visualized in the disc proper (DP) cells. Wing discs were counter-stained with the nucleic acid dye TO-PRO3 (blue). Genotypes: (A1, A4, A7, & A10) <i>UAS-Tap42^RNAi/+</i> as control. (A2, A5, A8, & A11) <i>UAS-Tap42^RNAi/+; pnr-Gal4/+</i>. (A3, A6, A9, & A12) <i>ap-Gal4/UAS-Tap42^RNAi; +/+</i>. (B1 & B2) wild type <i>w¹¹¹⁸</i>.</p

Silencing of <i>Tap42</i> in wing discs leads to pleiotrophic defects that include deformed thorax and wings.

<p><i>pnr-Gal4</i> and <i>ap-Gal4</i> imaginal disc drivers were used to drive expression of <i>EGFP</i> or <i>Tap42^RNAi</i> in <i>Drosophila</i>. Wing discs obtained from 3^rd instar larvae expressing EGFP (green) reveal the <i>pnr-Gal4</i> (A1) and <i>ap-Gal4</i> (A2) expression domain in wing discs. Control flies harboring the <i>UAS-Tap42^RNAi</i> construct lacked any noticeable defect in the adult thorax (B1, with head left) or wing (C1, with wing margin to left). <i>Tap42^RNAi</i> expression using the <i>pnr-Gal4</i> driver caused a marked cleft phenotype on the adult thorax (B2, red arrow) with no notable defects in fly wing (C2). Silencing the <i>Tap42</i> gene with the <i>ap-Gal4</i> driver resulted in a thorax cleft phenotype ranging in severity from mild (B3, red arrow) to severe (Fig. 6-B1) as well as drastically shriveled wings (C3). Genotypes: (A1) <i>UAS-EGFP/+; pnr-Gal4/+</i>. (A2) <i>ap-Gal4/UAS-EGFP</i>. (B1 & C1) <i>UAS-Tap42^RNAi/+</i> as control. (B2 & B3) <i>UAS-Tap42^RNAi/+; pnr-Gal4/+</i>. (C2 & C3) <i>ap-Gal4/UAS-Tap42^RNAi; +/+</i>.</p

Suppression of Tap42 expression in wing imaginal discs interrupts HH signaling, hampers mitosis, and triggers apoptosis.

<p>Panel A: Isolated wing imaginal discs were immunostained with antibodies recognizing Tap42 (green) and multiple components in the HH signaling pathway, including Ptc, Smo, and Ci (red). Control wing discs displayed strong Tap42 (A1) expression and the expected expression pattern for Ptc (B1), Smo (C1), and Ci (D1). Suppression of <i>Tap42</i> with the <i>pnr-Gal4</i> or ap<i>-Gal4</i> driver effectively reduced Tap42 levels in wing discs (A2 & A3). While the levels of the HH receptor Ptc were unaffected by Tap42 silencing (B3), the expression of other downstream components of HH signaling, Smo (C3) and Ci (D3), were abrogated. Suppression of Tap42 with the <i>pnr-Gal4</i> driver did not alter the expression pattern of HH signaling as shown in B2 (Ptc), C2 (Smo) and D2 (Ci). Genotypes: (A1, B1, C1, & D1) <i>UAS-Tap42^RNAi/+</i> as control. (A2, B2, C2, & D2) <i>UAS-Tap42^RNAi/+; pnr-Gal4/+</i>. (A3, B3, C3, & D3) <i>ap-Gal4/UAS-Tap42^RNAi; +/+</i>.</p

Subcellular distribution of PP2A subunits.

<p>(<b>A</b>) NIH3T3 cells were transiently transfected with pCA2-6myc-PP2A/Aα or pCMV-HA-PP2Acα-YC, and expression of the exogenous Aα and Cα subunits was assessed by indirect immunofluorescence using anti-Myc tag and anti-HA antibodies, respectively, in conjunction with Cy3-conjugated secondary antibody. (<b>B</b>) Diagrams of B55α, B55β, and the B55βαβ chimera mutant are shown. (<b>C</b>) NIH3T3 cells were transiently transfected with pcDNA3.1/Zeo(+)-B55α-HA, pcDNA3.1/Zeo(+)-B55β-HA, pcDNA3.1/Zeo(+)-B55βαβ-HA, pcDNA3.1/Zeo(+)-B55β2-HA, pcDNA3.1/Zeo(+)- B55δ-HA, or pcDNA3.1/Zeo(+)-B56γ3-HA. Expression of various exogenous B isoforms was assessed by indirect immunofluorescence using the anti-HA antibody and Cy3-conjugated secondary antibody. DAPI was applied for staining of nuclei. Scale bars: 20 µm. Cells with different distribution patterns were scored as follows: predominantly nuclear (N>C), homogenously distributed in both nucleus and cytoplasm (N∼C), and predominantly cytoplasmic (N</p

BiFC analysis enables visualization of association between two subunits of PP2A in cells.

<p>(<b>A</b>) Design of BiFC analysis of dimeric interactions between PP2A subunits is shown. Fluorescence is regained when reconstitution of YFP from two fragments of YFP takes place due to an interaction between PP2A subunits fused to the fragments. (<b>B</b>) Equal amounts of BiFC expression constructs encoding YN-Aα and PP2Acα-YC were co-transfected into NIH3T3 cells. YFP signals due to BiFC of YN-Aα and PP2Acα-YC were measured by fluorescence microscopy. (<b>C</b>) Equal amounts of BiFC expression constructs encoding Aα-YC and YN-B55α, YN-B55β1, YN-B55β2, YN-B55βαβ, or YN-B55δ, or constructs encoding YC-Aα and YN-B56γ3 were transfected into NIH3T3 cells. YFP signals due to BiFC of Aα-YC and YN-B were measured by fluorescence microscopy. (<b>D</b>) Equal amounts of BiFC expression constructs encoding PP2Acα-YC and YN-B55β1 or YN-B56γ3 with or without equal amounts of pCA2-6myc-PP2A/Aα were co-transfected into NIH3T3 cells, and 24 h after transfection, YFP signals due to BiFC of PP2Acα-YC and YN-B55β1 or YN-B56γ3 were measured by direct fluorescence microscopy and expression of 6myc-PP2A/Aα was confirmed by indirect immunofluorescence using anti-Myc tag antibody and Cy3-conjugated secondary antibody. DAPI was applied for staining of nuclei. Scale bars: 20 µm. Graphs show quantitative analysis of distribution of BiFC signals in cells from one of at least two independent experiments with similar results, and at least 100 cells were assessed from several random fields. Cells with different distribution patterns of BiFC signals were scored as described earlier.</p

Visualization of the PP2A trimeric holoenzyme complexes Aα/B56γ3/Cα and Aα/B55β2/Cα in cells by BiFC-FRET.

<p>(<b>A</b>) The design of BiFC-FRET analysis of Aα/B56γ3/Cα and Aα/B55β2/Cα is shown. YFP, which serves as a FRET acceptor, is reconstituted via BiFC of YC-Aα/YN-B56γ3 or PP2Acα-YC/YN-Aα, in ternary complex Aα/B56γ3/Cα or Aα/B55β2/Cα, respectively. FRET occurs when CFP-PP2Acα or B55β2-CFP, which serves as a FRET donor, associates with YC-Aα/YN-B56γ3 or YN-Aα/PP2Acα-YC BiFC complex, respectively. (<b>B</b>) NIH3T3 cells were transiently transfected with constructs encoding YC-Aα, CFP-PP2Acα and YN-vector or YN-B56γ3, in the presence of pCMV5-SMT_WT, pCMV5-SMT_MUT, or empty vector. Images of expression of CFP-PP2Acα, YFP (due to BiFC of YC-Aα and YN-B56γ3), and FRET between CFP and YFP were acquired by indicated filters. Corrected FRET images were generated and FRET intensity was assessed by AxioVision (Zeiss), as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116074#s2" target="_blank">Methods and Materials</a>”. Representative images are shown. Scale bars, 20 µm. Quantified data from one of two independent experiments with similar results are shown. Mean (±s.d.) of corrected FRET maximum intensity values of individual cells from at least 15 cells were measured for each group. (<b>C</b>) NIH3T3 cells were transiently transfected with constructs encoding PP2Acα-YC and YN-vector or YN-Aα, and B55β2-CFP in the presence of pCMV5-SMT_WT, pCMV5-SMT_MUT, or empty vector, or transfected with equal amounts of constructs harboring PP2Acα-YC, YN-Aα, and B55β2_MUT-CFP. Representative images of expression of B55β2-CFP and B55β2_MUT-CFP, YFP (due to BiFC of association of PP2Acα-YC and YN-Aα), and FRET between CFP and YFP were acquired by indicated filters as described above. Scale bars: 20 µm. Corrected FRET images were generated and FRET intensity was assessed as described earlier. Quantified data from one of two independent experiments with similar results are shown. At least 10 cells were measured for each group.</p

Visualization of the α4/PP2Ac complex by BiFC.

<p>(<b>A</b>) BiFC expression constructs encoding YN-α4_WT or YN-α4_MUT and YC-PP2Acα were transfected into NIH3T3 cells. YFP signals due to BiFC of YN-α4_WT or BiFC of YN-α4_MUT and YC-PP2Acα were measured by fluorescence microscopy. Expression of Myc-tagged YN-α4_WT or YN-α4_MUT was confirmed using anti-Myc antibody in conjunction with Cy3-conjugated secondary antibody by indirect immunofluorescence microscopy. DAPI was applied for staining of nuclei. Scale bars: 20 µm. Cells with different distribution patterns were scored as described earlier. Quantified data from one of at least two independent experiments with similar results are shown. At least 150 cells were counted for each group. (<b>B</b>) BiFC expression constructs encoding YN-α4_WT or YN-α4_MUT and YC-PP2Acα were transfected into NIH3T3 cells. Cell lysates were collected 24 h post-transfection and immunoprecipitations were performed using anti-Myc tag antibody. The cell lysates and anti-Myc tag immunocomplexes were then analyzed by SDS-PAGE and Western blotting using the indicated antibodies.</p