Calpains Mediate Integrin Attachment Complex Maintenance of Adult Muscle in Caenorhabditis elegans
Figure 8
Calpains are important for maintenance of adult C. elegans muscle.
A) Animals expressing a full length translational fusion of gfp to myo-3 (myosin heavy chain A) were age synchronised at L1 stage and grown to young adulthood at 16°C (t = 0 h). Adult animals were then transferred to NGM RNAi plates [87] seeded with bacteria expressing dsRNA against genes indicated for a further 72 h to mid-adulthood. 20 random animals were picked and scored for identical defects in sarcomere structure in at least two muscles within the animal and this was repeated for 5 independent RNAi treatments (n = 100 animals per condition/time point). Displayed is the percentage of animals where torn or collapsed arrays of sarcomeres were observed (average ± SEM). Example images for each treatment can be found in Figure S5. **Significant difference from control (t = 72 h, (P<0.001)). B) Animals expressing GFP labelled mitochondria and nuclei were grown, treated and analysed as in A with the exception that mitochondrial structure was scored. Displayed is the percentage of animals where moderate fragmentation of the mitochondrial network was observed (average ± SEM). Example images for each treatment can be found in Figure S5. *, **Significant difference from control (t = 72 h, (P<0.01, P<0.001)). C) Animals expressing GFP labelled attachment complexes (UNC-95::GFP) were grown, treated and analysed as in A with the exception that attachment complex structure was scored. Displayed is the percentage of animals where torn or collapsed arrays of sarcomeres were observed were observed (average ± SEM). Example images for each treatment can be found in Figure S5. **Significant difference from control (t = 72 h, (P<0.001)). D) Wild-type, unc-112ts, and unc-112ts; dim-1(gk54) were age synchronised at L1 stage and grown to young adulthood at 16°C (t = 0 h). Adult animals were then transferred to 25°C and grown for a further 72 h to mid-adulthood. Some unc-112ts animals were also placed on calpain inhibitor II drug plates (5 µg/ml) at t = 0 h and cultured on drug plates for a further 72 h. 30 animals were picked for western blot analysis of DEB-1 levels at t = 0 h, and at 72 h. All experiments were performed at least three times. Displayed are representative western blots for each condition and a graph of the initial DEB-1 remaining at 72 h (average ± SEM for three independent experiments). **Significant difference from all other conditions (P<0.001). All significance values are from two way repeated measures ANOVA.