Computational protocol: The formation of complex acetylcholine receptor clusters requires MuSK kinase activity and structural information from the MuSK extracellular domain

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Protocol publication

[…] Myoblasts were cultured and differentiated according to . Briefly, a 10 μg/ml solution of EHS laminin (Roche) in L-15 medium (Sigma-Aldrich) containing 0.2% NaHCO3 and 100 × glutamine was used to coat poly-l-ornithine-coated dishes overnight at 37 °C. Before plating of cells laminin was aspirated and cells were maintained at 33 °C and 5% CO2. After 24 h the growth media was replaced with differentiation medium (DM) and the temperature was increased to 37 °C to induce differentiation. After inducing fusion, cultures were maintained in DM for 1–6 days to access the prevalence of simple to complex aggregates over time as a function of PF days. 1 μg/ml TTX and 100 × FUDR was added to block the twitching of myotubes and to inhibit the growth of myoblasts. AChR clusters were stained on each consecutive PF day with 2 ng/ml of Alexa 594-coupled α-BGT, washed, fixed, mounted and imaged with 63 ×/1.4–0.60 oil objective on a Leica inverted fluorescence microscope (DMIRB) equipped with a cooled MicroMax CCD camera. Cells were photographed at equivalent exposure times using Metamorph imaging software. Multiple forms of AChR aggregates formed per PF day were classified as microclusters, plaque, perforated, C-shaped and pretzel as described previously by . Total number of aggregates of each individual PF day was summed up as 100% and the distribution of each form of clusters in individual PF day was plotted as a percentage of the combined total. The prevalence of morphology from 2 to 6 independent experiments of each cell line was analyzed and compared to MuSK/WT. For comparison of the size, all observed aneural AChR clusters of MuSK/WT and MuSK/KA-2 were categorized into plaque, perforated, C-shaped and pretzel. The area and length (feret diameter) of each cluster, bigger than 5 μm were measured by using free hand tool (ImageJ software). […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Application Microscopic phenotype analysis
Diseases Asbestosis
Chemicals Acetylcholine