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Figures
- Fig. 1.
In silico structural model of the complex between rapsyn α-helical domain and DD domain homodimer of PKA-RIα. (A) The sequence of rapsyn amphipathic helix (residues 305–331) was aligned with the AKAP10 amphipathic helix (residues 628–654) via profile HMM calculated from the sequence alignment from McLaughlin (McLaughlin et al., 2011). The sequence alignment of rapsyn with several dual-specificity AKAPs is shown. The residues lining the four interaction pockets as defined by Sarma and colleagues (Sarma et al., 2010) are boxed. Rapsyn residues forming large (>0.40 nm2) surface of contact with PKA-RIα DD domain homodimer, averaged over the MD trajectory, are underlined. (B) Surface of rapsyn residues in contact with PKA-RIα DD domain homodimer. Residues with large contact surface are underlined. (C) Cartoon of the complex in water solution as determined by in silico modelling. Rapsyn helix and residues are coloured in red, DD subunits and residues are coloured in brown and silver. Residues forming large contact surface are drawn in balls and sticks and coloured by atom type. In addition, side chains of rapsyn residues are emphasised by a red surface. Inset: Helical wheel diagram of rapsyn amphipathic helix. Residues that form large surface of contact with DD domain homodimer are boxed.
- Fig. 2.
Rapsyn co-precipitates with PKA-RIα. (A) After 10 days of differentiation, C2C12 cells were harvested, and lysates prepared and incubated in the presence or absence of rapsyn antibody (AB). After immunoprecipitation, eluates were run on SDS-PAGE and then probed on western blot using antibodies against rapsyn, PKA-RIα and α-actinin (negative control). The bands show western blot signals in lysates and eluates. (B,C) T-REx293 cells were transfected with HA-tagged versions of either rapsyn (rapsyn-HA) or a rapsyn mutant lacking the α-helical domain (amino acids 299–331; rapsynΔH-HA). Lysates were prepared, immunoprecipitated with monoclonal anti-PKA-RIα antibody and subsequently probed on western blots for HA and PKA-RIα. (B) Schemes of transfected fusion constructs. (C) Western blot signals in lysates and eluates. Note that only full-length rapsyn co-precipitates with PKA-RIα. The graph on the right shows a densitometric analysis. Depicted are mean ± s.e.m. (n=3 experiments) of the eluate band intensities normalised to the lysate. **P<0.01 according to Welch test.
- Fig. 3.
BiFC shows interaction between rapsyn and PKA-RIα. T-REx293 cells were co-transfected with mCherry or RIAD–mCherry and different combinations of BiFC constructs harboring N-(VN) or C-terminals (VC) of Venus and an HA tag together with rapsyn, rapsynΔH, PKA-RIα or Δdd-PKA-RIα (for schemes see supplementary material Fig. S1). Then, cells were fixed, stained with draq5, a nuclear marker, and imaged using confocal microscopy. Subsequently, the fraction of mCherry- or RIAD–mCherry-positive cells showing BiFC signals was determined. (A,B) Fields of cells co-transfected with PKA-RIα-VN and rapsyn-VC (A) or with PKA-RIα-VN and rapsynΔH-VC (B). Overlays show BiFC signals (green), mCherry signals (red) and nuclei (blue). Scale bars: 50 μm. (C,D) Blow-ups of regions in A and B. Scale bars: 10 μm. (E) Quantification of BiFC-positive cells showing mean ± s.e.m. (n≥3 independent experiments). *P<0.05, **P<0.01 according to Welch test.
- Fig. 4.
In vivo BiFC shows interaction between rapsyn and PKA-RIα in a subsynaptic region in live mouse muscle. Mouse tibialis anterior muscles were co-transfected in different combinations as indicated with BiFC constructs harboring N-(VN) or C-terminal (VC-) fragments of Venus protein fused to rapsyn or PKA-RIα. After 10 days, muscles were imaged using in vivo confocal microscopy. NMJs were marked with BGT-AF647. Panels depict maximum z-projections of BiFC (green), BGT-AF647 (red) signals and overlays of both (yellow if colocalising) from about 20 confocal slices taken at 1.5 μm interslice interval. Scale bar: 40 μm.
- Fig. 5.
Peptide encompassing rapsyn α-helical domain displaces PKA-RIα from its subsynaptic microdomain in vivo. (A–F) A peptide encompassing the rapsyn α-helix (rapsyn αHelix) or a mutant control peptide (control) was transfected (A–D) or co-transfected together with RIα-EPAC (E,F), a marker for the localisation of PKA-RIα, into mouse tibialis anterior muscles. After 10 days, muscles were extracted, sliced and stained against AChR with BGT-AF647, and with antibodies against PKA-RI (A,B) or rapsyn (C,D), or injected with BGT-AF647 and then imaged in vivo (E,F). Stained sections were analysed with confocal microscopy, and antibody staining intensities in the NMJs were determined (see Materials and Methods section for details). (A,C) Confocal slices of muscles transfected as indicated. In overlays, antibody signals are shown in green, BGT signals in red. Scale bar: 50 μm. (B,D) Cumulative plots showing the antibody staining intensities in all analysed NMJs. All NMJs right of the zero value (dotted line) were counted as PKA-RI- or rapsyn-positive. (E) Images depict maximum z-projections of 80 (upper panels) and 97 optical slices (lower panels) taken at 3 μm interslice intervals of muscles co-transfected as indicated on the left. Images show BGT (AChR), RIα-EPAC fluorescence signals or pseudo-coloured NMJ outlines. In the outlines: NMJs in white, synapses of RIα-EPAC-negative fibres; NMJs in yellow, synapses of RIα-EPAC-positive fibres with subsynaptic RIα-EPAC accumulations; NMJs in red, synapses of RIα-EPAC-positive fibres without subsynaptic RIα-EPAC accumulations. Scale bar: 200 μm. (F) Quantitative analysis showing the average amount (mean ± s.e.m.) of NMJs of RIα-EPAC-positive fibres with subsynaptic RIα-EPAC accumulations. n=4 mice; 382 NMJs (control) and 425 NMJs (αHelix) were analysed. **P<0.01 according to Welch test.
- Fig. 6.
Peptide encompassing rapsyn α-helical domain and rapsyn-ΔHelix alter AChR turnover and fragmentation of NMJs in live mouse muscle. (A,B) A peptide encompassing the rapsyn α-helix (rapsyn αHelix), a mutant control peptide (control) or rapsyn-ΔHelix was transfected into mouse tibialis anterior muscles. AChRs present on the cell surface at the time point of transfection were labelled by locally injected BGT-AF647 (‘old receptors’). After 10 days, muscles were injected with BGT-AF555 to mark AChRs now available (‘new receptors’). Then, muscles were imaged by in vivo confocal microscopy and automatically segmented and analysed. (A) Maximum z-projections of automatically segmented confocal slices taken at an interslice interval of 1.5 μm. In overlays, ‘old receptors’ are shown in green and ‘new receptors’ in red. Scale bar: 50 μm. (B) Quantitative analysis of AChR turnover and NMJ fragmentation. Shown are average values (mean ± s.e.m.) of the fractions of all pixels in NMJs with dominant ‘new receptor’ signals as a function of the numbers of fragments per NMJ (n=6 muscles). (C) Seven days after transfection, muscles were injected with 125I-BGT to pulse-label AChRs. Then, residual 125I-activity was measured at depicted time intervals. The time point of pulse labelling is t0. The graph shows the residual 125I-emission in the injected hindlimbs as a function of time after pulse labelling. Dots represent measured values (mean ± s.e.m., n=5 mice) and lines indicate two-term exponential fits. Data are normalised to the mean values measured on day 1. *P<0.05, **P<0.01 according to Welch test.
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