Protein phosphatase 2A is crucial for sarcomere organization inCaenorhabditis elegansstriated muscle

Figures

• 

  FIGURE 1:. Mapping of interaction sites of PPTR-1 and PPTR-2 for UNC-89, and of UNC-89 for PPTR-1 and PPTR-2. (A) When tested by yeast two-hybrid assays with segments that cover all of UNC-89B, PPTR-1 and PPTR-2 only interact with UNC-89 1/3 IK-Ig53-Fn2. Pink rectangles, Ig domains; green rectangles, Fn3 domains; other domains are as indicated. (B) Domain mapping using yeast two-hybrid assays indicates that the minimal region of UNC-89 that interacts with PPTR-1 is 1/3 IK-Ig53-Fn2, but that the minimal region of UNC-89 that interacts with PPTR-2 is Ig53-Fn2.

• 

  FIGURE 2:. Segments of UNC-89 interact with PPTR-1 and PPTR-2 in vitro. (A) Far-Western assay. His-tagged PPTR-1 and PPTR-2 were separated by SDS–PAGE, transferred to membrane, incubated with MBP, MBP-UNC-89 Ig53-Fn2, or MBP-UNC-89 1/3 IK-Ig53-Fn2, washed, incubated with anti–MBP-HRP, washed, and detected by ECL. Both MBP fusions of these portions of UNC-89, but not MBP itself, bind to either His-PPTR-1 or His-PPTR-2. (B) In-solution pull-down assay. The indicated proteins were incubated together, and then the His-tagged proteins were pelleted using anti-6His beads. Proteins were eluted from the beads and separated by SDS–PAGE and transferred to a membrane, and then a Western blot was performed using anti–MBP-HRP to detect any copelleting MBP or MBP-UNC-89-Ig-Fn. At the bottom is shown the blot after Ponceau S staining, and above it is the Western blot. Both His-PPTR-1 and His-PPTR-2 pull down MBP-UNC-89-Ig-Fn, but not MBP. Positions of proteins are indicated by arrows; * likely degradation products from MBP-UNC-89-Ig-Fn; ** Ig heavy chain; *** Ig light chain. The predicted molecular weights of the bacterially expressed purified proteins are as follows: His-PPTR-1, 51.5 kDa; His-PPTR-2, 60…

• 

  FIGURE 3:. Effects of loss of function mutations in pptr-1 and pptr-2 on thick filament organization in body-wall muscle. (A) Immunostaining using a monoclonal to the thick filament component MHC A of wild type, pptr-1(tm3103), or pptr-2(ok1467) grown at 20°C. In each panel, portions of several spindle-shaped body-wall muscle cells are shown. (Two representative examples of images from pptr-1(tm3103) are shown.) In wild-type muscle, the A-bands are straight and parallel. Although the deletion allele pptr-2(ok1467) shows normal thick-filament organization, the deletion allele pptr-1(tm3103) shows disorganization of thick filaments (two representative examples are shown). (B) Immunostaining using anti-MHC A of missense allele pptr-1(abc19), deletion allele pptr-2(ok1467), and the double mutant, pptr-1(abc19) pptr-2(ok1467), grown at 15°C. Two representative examples of images from pptr-1(abc19) pptr-2(ok1467) animals are shown. Note that both single mutants have normal A-band organization, but the double mutant shows some A-band disorganization (two examples shown). The double mutant pptr-1(abc19) pptr-2(ok1467) is 90% sterile at 20°C, but only 75% sterile at 15°C. Therefore, it was easier…

• 

  FIGURE 4:. Immunolocalization of PPTR-1 and PPTR-2 to sarcomeres of body-wall muscle. (A) Schematic representation of PPTR-1 and PPTR-2 polypeptides, each having a single domain, the B56 domain (shown in red). Blue bars indicate the unique C-termini used as immunogens to generate rabbit antibodies. (B) Western blots demonstrate that antibodies to PPTR-1 or PPTR-2 detect expected-size proteins from the wild type but not from the respective deletion alleles. The numbers on the left side of each blot show the position of molecular weight markers in kDa. (C) By immunofluorescence microscopy, anti–PPTR-1 localizes to sarcomeric I-bands, and anti–PPTR-2 localizes to sarcomeric dense bodies and M-lines. Portions of a single body-wall muscle cell are shown in each row, costained with antibodies to UNC-89, which localizes to M-lines. Arrowheads mark the positions of dense bodies, and arrows mark the positions of M-lines. In the two-color image at bottom right, white indicates colocalization of PPTR-2 with UNC-89 at M-lines. Scale bar, 10 μm.

• 

  FIGURE 5:. In an unc-89 mutant expressing UNC-89 protein that lacks the PPTR-2 binding region, there are reduced levels of PPTR-2 at M-lines. (A) Each row shows a portion of a single body-wall muscle cell immunostained with anti–PPTR-2 and anti–PAT-6 (α-parvin) of wild type and unc-89(tm752). PAT-6 is a marker for dense bodies (arrowheads) and M-lines (arrows). (B) Intensity profiles, created by Zeiss ZEN software, of the PPTR-2 images shown in A. The images were rotated to improve viewability. Note that in unc-89(tm752) as compared with wild type, the level of PPTR-2 at M-lines is reduced, but the level of PPTR-2 at dense bodies is unchanged. Scale bar, 10 μm.

• 

  FIGURE 6:. PP2A holoenzyme in C. elegans. There is a single catalytic subunit, C, encoded by the gene let-92, a single scaffolding subunit, A, encoded by paa-1, and at least the following regulatory subunits and their encoded genes: B by sur-6, B’ by pptr-1 or B’ by pptr-2, B” by rsa-1, and B’’’ by cash-1.

• 

  FIGURE 7:. Disorganization of sarcomeres in loss-of-function mutants for PP2A B (SUR-6), B” (RSA-1), and B”’(CASH-1) subunits. Immunofluorescence images of portions of several body-wall muscle cells reacted with antibodies to MHC A from wild type, the temperature-sensitive mutant sur-6(or550), the maternal-effect lethal mutants rsa-1(dd10) and rsa-1(dd13), and cash-1(RNAi). There is clear disorganization of A-bands in sur-6(or550) and cash-1(RNAi). In the case of the rsa-1 mutants, although there are normal straight parallel A-bands, these A-bands appear as doublets, seen at higher resolution in Figures 9 and 10. Scale bar, 20 μm.

• 

  FIGURE 8:. Split or doublet MHC A localization is also found in MHC B myosin head mutants. Immunolocalization of UNC-95 (marker for M-lines and dense bodies) and MHC A in muscle of wild type, rsa-1, and special unc-54 mutants. Insets show higher-magnification views. Whereas wild type shows single tight MHC A localization, both rsa-1 mutants and unc-54 mutants show closely spaced doublet MHC A localization. unc-54(s74), unc-54(s95), and unc-54(st134) are rare missense mutations in the head domain of MHC B. Scale bar, 10 μm.

• 

  FIGURE 9:. Superresolution images of MHC A staining in wild type, rsa-1, and unc-54 mutants. Nematodes with the indicated genotypes were immunostained with anti-MHC A and imaged with N-SIM. A z-series, planes 0.2 μm apart, was taken, and then 3D rendering was used to create the views of the middle of the A-bands where MHC A resides. For each strain, views from opposite sides of a body-wall muscle cell are shown. Note the single line in wild type and the closely spaced double line in rsa-1 and unc-54. Scale bar, 3 μm.

• 

  FIGURE 10:. MHC B localization in wild type, rsa-1, and unc-54 mutants. Nematodes of the indicated genotypes were immunostained with anti-MHC A and anti-MHC B and imaged by confocal microscopy. Because both antibodies were mouse monoclonals, it was not practical to conduct costaining. MHC B localization broadly to the A-bands, with a gap in the middle, is the same in the wild type and the mutants. In the two mutants, the double-line MHC A localization is narrower than the localization of MHC B. Scale bar, 10 μm.

• 

  FIGURE 11:. Muscle phenotypes for loss of function for the PP2A catalytic subunit (LET-92) and for the PP2A scaffolding subunit (PAA-1). RNAi was performed beginning from the L1 stage and continuing into adulthood. (A) RNAi for let-92 or paa-1 results in paralyzed folded-over or “jack-knifed” adults. Scale bar, 500 μm. (B) RNAi for let-92 or paa-1 results in highly disorganized A-bands and in some cells, detachment of the myofilament lattice from the muscle cell/basement membrane (indicated by yellow arrows). Four representative examples are shown for each gene. Scale bar, 20 μm.

• 

  FIGURE 12:. Sarcomeric localization of additional PP2A components. Wild-type animals were immunostained with antibodies to SUR-6, PAA-1, or RSA-1, together with anti–PAT-6 (α-parvin) to mark M-lines (arrows) and dense bodies (arrowheads). As shown, SUR-6 localizes to I-bands, PAA-1 localizes to I-bands, and RSA-1 localizes to M-lines and I-bands. Scale bar, 10 μm.