Fig. 1: F-actin accumulates in aged Drosophila brains and correlates with health.

a Immunostaining of brains at 10x magnification from young (10-day-old) and aged (30-day-old and 45-day-old, as indicated) Canton S flies, showing F-actin fluorescence intensity (red channel, phalloidin). Scale bar is 100 µm. b Quantification of mean phalloidin fluorescence intensity in brains as shown in a. d10 n = 7, d30 n = 6, and d45 n = 5 flies, as indicated. *p (d10 vs. d30) = 0.0158, *p (d10 vs. d45) = 0.0263; one-way ANOVA, Tukey’s multiple comparisons test. c Immunostaining of brains at 63x magnification from young (10-day-old) and aged (45-day-old) flies, showing F-actin (red channel, phalloidin) and nuclear DNA (blue channel, DAPI). These findings were repeated in more than 15 independent experiments. Scale bar is 20 µm. Accompanying diagram indicates brain region where imaging was conducted. d Immunostaining of brains from young (10-day-old) and aged (30-day-old) elavGS > UAS-Act5c-GFP flies, showing F-actin (red channel, phalloidin), Act5c-GFP (green channel), and nuclear DNA (blue channel, To-Pro-3). These findings were repeated in 3 independent experiments. Scale bar is 20 µm. e Immunostaining of brains at 63x magnification from young (10-day-old) and aged (30-day-old and 45-day-old, as indicated) Canton S flies, showing F-actin-rich rods (red channel, phalloidin). Scale bar is 5 µm. Accompanying diagram indicates brain region where imaging was conducted. f Quantification of F-actin-rich rods per 1 mm² area of brain optic lobes as shown in e. d10 n = 8, d30 n = 9, d45 n = 8 flies, as indicated. ***p (d10 vs d30 and d10 vs d45) < 0.0001; one-way ANOVA, Tukey’s multiple comparisons test. (g) Quantification of F-actin protein by ELISA using head homogenates from young (10-day-old) and aged (30-day-old and 45-day-old, as indicated) Canton S flies. n = 4 homogenates generated with 5 brains each per condition. *p = 0.0192, **p = 0.0053, unpaired two-tailed t-tests. h Survival curves of Canton S flies given a rich diet (5.0% yeast extract) versus those undergoing dietary restriction (DR, 0.5% yeast extract) from day 4 post eclosion onwards. ***p = 0.001; log-rank test; n > 140 flies. i Immunostaining of brains from Canton S flies aged day 21 post eclosion provided a rich or restricted (DR) diet, as in (h), showing F-actin-rich rods (red channel, phalloidin). Scale bar is 5 µm. j Quantification of F-actin-rich rods by phalloidin stain per 1 mm² area of brains as shown in i. DR n = 6, Rich n = 5 flies, as indicated. *p = 0.0225, unpaired two-tailed t-test. k Survival curves of white Dahomey flies given 10 µM rapamycin or vehicle from day 4 post eclosion onwards. ***p < 0.0001; log-rank test; n > 250 flies per condition. l Immunostaining of brains from young (10-day-old) and aged (45-day-old) white Dahomey flies given 10 µM rapamycin or vehicle, showing F-actin-rich rods (red channel, phalloidin). Scale bar is 5 µm. m Quantification of F-actin-rich rods by phalloidin stain per 1 mm² area of brains as shown in l. d10 n = 6, d44 Rapa - = 6, d44 Rapa + = 9 flies, as indicated. **p = 0.0023., ***p = 0.0001; one-way ANOVA, Tukey’s multiple comparisons test. Data are presented as scatter plots overlaying mean values +/- SEM.