Fig. 2: Three patterns of collagen dynamics during longevity quantified in vivo.

a–e COL-120 dynamics as a representative for the pattern I collagen. a Collagen COL-120 tagged with GFP (LSD1000) in the cuticular ECM furrow vanished from day 1 to day 8 on control empty vector RNAi (L4440) but was still visible at day 8 of adulthood when daf-2(RNAi) started at L4. Representative images of one independent biological trial. Scale bar = 1 µm. b Model of COL-120 mRNA, protein, and incorporated ECM reporter dynamics. c Experimental workflow and the expected outcome of photoswitching COL-120 tagged dendra2 in vivo. d Representative images of photo-switched areas of LSD1061 COL-120::Dendra of one independent trial. Scale bars = 10 µm for overlay and 1 µm for inset. e Quantification of COL-120 turnover of the mid-body area. Each dot represents 1 animal (n = 10). See Supplementary Data 5 for statistical details and raw data. f–j COL-19 is shown as a representative of the pattern II collagen. f Schematic representation of COL-19::FRET transgene. (g) Lifespan of wild-type temperature-sensitive sterile background WT (LSD2052 spe-9(hc88); COL-19::FRET) vs long-lived (LSD2053 glp-1(e2141); COL-19::FRET) at 25 °C. Light blue arrows indicate sampling days for FRET imaging. h Representative LSD2052 spe-9(hc88); COL-19::FRET images in the 3D chamber on days 2 and 12 of adulthood at 25 °C of one independent trial. The increasing FRET ratio is color-coded from dark blue to red. Scale bar = 10 µm. i FRET ratio increased from day 2 to 12 during the aging of LSD2052 spe-9(hc88); COL-19::FRET at 25°C. Data are presented as mean + SEM. P value was determined by an unpaired t-test, two-tailed. See Supplementary Data 6 for statistical details and raw data. j Long-lived LSD2053 glp-1(e2141); COL-19::FRET showed lower FRET ratio compared to WT (LSD2052 spe-9(hc88); COL-19::FRET) at day 8 of adulthood at 25°C. Data are presented as mean + SEM. P value was determined by an unpaired t-test, two-tailed. See Supplementary Data 6 for statistical details and raw data. k, l Type IV collagen EMB-9 is shown as a representative of pattern III collagen. k Representative images of photo-switched areas of NK860 EMB-9::Dendra. Scale bar = 10 µm. l Quantification of EMB-9::Dendra showed that newly synthesized EMB-9 is laid on top of the old matrix-incorporated EMB-9, which was not turnover. Each dot represents 1 animal (n = 14). See Supplementary Data 8 for statistical details and raw data.