Role of Myosin Va in the Plasticity of the Vertebrate Neuromuscular Junction In Vivo

Background: Myosin Va is a motor protein involved in vesicular transport and its absence leads to movement disorders in humans (Griscelli and Elejalde syndromes) and rodents (e.g. dilute lethal phenotype in mice). We examined the role of myosin Va in the postsynaptic plasticity of the vertebrate neuromuscular junction (NMJ). Methodology/Principal Findings: Dilute lethal mice showed a good correlation between the propensity for seizures, and fragmentation and size reduction of NMJs. In an aneural C2C12 myoblast cell culture, expression of a dominant-negative fragment of myosin Va led to the accumulation of punctate structures containing the NMJ marker protein, rapsyn-GFP, in perinuclear clusters. In mouse hindlimb muscle, endogenous myosin Va co-precipitated with surface-exposed or internalised acetylcholine receptors and was markedly enriched in close proximity to the NMJ upon immunofluorescence. In vivo microscopy of exogenous full length myosin Va as well as a cargo-binding fragment of myosin Va showed localisation to the NMJ in wildtype mouse muscles. Furthermore, local interference with myosin Va function in live wildtype mouse muscles led to fragmentation and size reduction of NMJs, exclusion of rapsyn-GFP from NMJs, reduced persistence of acetylcholine receptors in NMJs and an increased amount of punctate structures bearing internalised NMJ proteins. Conclusions/Significance: In summary, our data show a crucial role of myosin Va for the plasticity of live vertebrate neuromuscular junctions and suggest its involvement in the recycling of internalised acetylcholine receptors back to th

Lifetime of AChRs is reduced in dystrophic muscles and correlates with synaptic integrity and subsynaptic enrichment of myosin Va and PKA-RI.

<p>A: Tibialis anterior muscles of wildtype (wt) and mdx mice were pulse-labeled with ¹²⁵I-BGT on day 0. Subsequently, residual ¹²⁵I-emission was measured repetitively in the live animals at indicated time points from these muscles in situ. For the duration of measurements (10 min), mice were anaesthetized with isofluorane. Data represent mean ± SEM (n = 5 mice). Welch-test revealed significant differences between wildtype and mdx values, ** p<0.01. B–F: Tibialis anterior muscles were injected with BGT-AF647 (old receptors). Ten days later, muscles were exposed, injected with BGT-AF555 (new receptors), and then monitored with in vivo confocal microscopy (B–D). Subsequently, muscles were sliced, immunostained, and analyzed with confocal microscopy (E–F). B: Representative maximum z-projections of wildtype and mdx NMJs as indicated. Old and new receptor signals are shown in green and red, respectively. Pixels with similar intensities of both dyes appear in yellow. Scale bar, 50 µm. C: Graph showing the fraction of pixels with new receptor signals dominating over old receptor signals in individual NMJs as a function of the number of fragments per NMJ. Data represent mean ± SEM (n = 4 wildtype muscles, n = 8 mdx muscles. 109 and 191 NMJs were analyzed for wildtype and mdx, respectively). Significance was tested with Welch test, ** p<0.01. D: Graph depicts all individual values of the fractions of pixels with new receptor signals dominating over old receptor signals in NMJs. Values were grouped in NMJs with less than 3 fragments and NMJs with 3 or more fragments. Red lines indicate medians. Note the large variance in mdx. Same data set as in C. E–F: Correlations of subsynaptic myosin Va (E) and PKA-RI enrichment (F) with the apparent turnover of AChRs. Tibialis anterior muscles used for in vivo imaging (B–D) were sliced transversally and immunostained for myosin Va or PKA-RI. Ratio of old and new receptors and the accumulation of myosin Va or PKA-RI were determined for each synapse (n = 5 muscles; 651 and 340 NMJs were quantified for myosin Va and PKA-RI, respectively). Significance was tested with Welch test, ** p<0.01.</p

Notexin treatment transiently reduces the subsynaptic accumulation of PKA-RI and myosin Va.

<p>EDL muscles were injected with Notexin to induce muscle degeneration. 6, 10, and 30 days after treatment muscles were resected and sliced transversally. <b>A:</b> Slices were stained with wheat germ agglutinin-AlexaFluor488 for plasma membranes (green), and with DRAQ5 for nuclei (red). Images show confocal sections through muscles harvested 6, 10, or 30 days after Notexin, as indicated. Scale bar, 100 µm. <b>B–D:</b> Slices were stained with BGT-AF647 (NMJs) and with antibodies against PKA-RI (B), myosin Va (C), and utrophin (D). Confocal images were taken and then analyzed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040860#s4" target="_blank">Methods</a> section. Scatter plots (3 left columns) show diameters of all analyzed fibers as a function of their subsynaptic enrichment of immunofluorescence. Vertical dotted lines indicate the separation between immuno-negative (left halves of plots) and immuno-positive NMJs (right halves of plots). Horizontal dotted lines indicate the separation between fibers smaller and larger than 40 µm in diameter. Column graphs (right) summarize data in scatter plots and depict the fractions of immuno-positive NMJs obtained 6, 10, and 30 days after Notexin treatment. Data are mean ± SEM (n = 6, 4, and 4 muscles for 6, 10, and 30 day time points). White and grey columns represent values for fibers smaller and larger than 40 µm in diameter, respectively.</p

Response to cAMP agonists differs between wildtype and mdx synapses. Tibialis anterior muscles of wildtype and mdx mice were transfected with RIα-EPAC.

<p>Ten days later, muscles were injected with BGT-AF647 to stain NMJs and monitored with in vivo confocal (A–D, G–J) or two-photon (E–F, K–M) microscopy. Scale bars depict 50 µm. <b>A and G:</b> Shown are single mdx NMJs with normal (A1) or fragmented morphology (A2, G1, G2). <b>B and H:</b> BGT-AF647 fluorescence signals. Boxed regions are shown enlarged in A and G. <b>C and I:</b> RIα-EPAC fluorescence signals in the same field as in B and H. <b>D and J:</b> Overlays of B and C (D) and H and I (J). BGT-AF647 and RIα-EPAC signals are in red and green, respectively. <b>E and K:</b> Same field as in B and H showing FRET-ratios in pseudo-colors before application of CGRP or NE (indicated). <b>F and L:</b> Same field as in E and K showing FRET-ratio in pseudo-colors after application of CGRP or NE (indicated). <b>M:</b> Quantification of several experiments. Shown is the percentage of increase in CFP/YFP ratio values (F(485 nm)/F(535 nm)) compared to basal upon application of 50 µl of either 10 µM CGRP or 10 µM NE as indicated. Data represent mean ± SEM (n = 10 and n = 14 wildtype NMJs for CGRP and NE, respectively; n = 13 and n = 9 mdx NMJs for CGRP and NE, respectively).</p