Extended Data Fig. 10: Lowering hyperactivated RAC signalling prevents alterations in actin networks induced by increased EPS-8 levels.
From: Rewiring of the ubiquitinated proteome determines ageing in C. elegans
a, Myosin heavy chain tagged to GFP indicates destabilization of myosin filaments in muscle cells during ageing, whereas knockdown of eps-8 during adulthood maintains organization of the myosin network. Scale bar, 20 μm. Representative of three independent experiments. b, Data are mean ± s.e.m. thrashing movements over a 30-s period on day 1 (n = 30 worms per condition, three independent experiments), day 3 (n = 30 worms per condition, three independent experiments) and day 10 (n = 45 worms per condition, three independent experiments) of adulthood. Knockdown of eps-8 after development ameliorates the age-associated decline in motility (day 1 vector RNAi versus day 1 eps-8 RNAi, P = 0.2127; day 3 Vector RNAi versus day 3 eps-8 RNAi, P < 0.0001; day 10 Vector RNAi versus day 10 eps-8 RNAi, P < 0.0001). P values were determined by two-sided t-test. c, Images of transgenic worms expressing LifeAct::mRuby in the epidermis. Scale bar, 20 μm. Representative of two independent experiments. d, Images of transgenic worms expressing LifeAct::mRuby in the intestine. Scale bar, 100 μm. Representative of two independent experiments. e, Filter trap analysis with an antibody to β-actin in wild-type worms. Knockdown of eps-8 during adulthood reduces the amounts of actin aggregates in aged worms. Representative of six independent experiments. f, Filter trap analysis with an antibody to β-actin. Muscle-specific knockdown of eps-8 during adulthood reduces the amounts of actin aggregates in aged worms. Representative of three independent experiments. g, Neuronal-specific knockdown of eps-8 results in decreased actin aggregates during ageing. Representative of three independent experiments. h, Epidermal-specific knockdown of eps-8 does not decrease age-related actin aggregation. Representative of three independent experiments. i, Intestinal-specific knockdown of eps-8 does not decrease age-related actin aggregation. Representative of three independent experiments. j, Knockdown of either mig-2 or rac-2 during adulthood ameliorates the age-associated destabilization of actin filaments in muscle cells. RNAi treatment was initiated at day 1 of adulthood. Scale bar, 20 μm. Representative of two independent experiments. k, Knockdown of either mig-2 or rac-2 during adulthood ameliorates the age-associated destabilization of myosin filaments in muscle cells. Scale bar, 20 μm. Representative of two independent experiments. l, Filter trap with an antibody to β-actin in worms expressing endogenous wild-type EPS-8 or mutant Ub-less EPS-8 at day 3 of adulthood. Representative of three independent experiments. In all the experiments, RNAi was initiated at day 1 of adulthood.
