Mechanical properties measured by atomic force microscopy define health biomarkers in ageing C. elegans

Abstract

Genetic and environmental factors are key drivers regulating organismal lifespan but how these impact healthspan is less well understood. Techniques capturing biomechanical properties of tissues on a nano-scale level are providing new insights into disease mechanisms. Here, we apply Atomic Force Microscopy (AFM) to quantitatively measure the change in biomechanical properties associated with ageing Caenorhabditis elegans in addition to capturing high-resolution topographical images of cuticle senescence. We show that distinct dietary restriction regimes and genetic pathways that increase lifespan lead to radically different healthspan outcomes. Hence, our data support the view that prolonged lifespan does not always coincide with extended healthspan. Importantly, we identify the insulin signalling pathway in C. elegans and interventions altering bacterial physiology as increasing both lifespan and healthspan. Overall, AFM provides a highly sensitive technique to measure organismal biomechanical fitness and delivers an approach to screen for health-improving conditions, an essential step towards healthy ageing.

Fig. 1. Atomic force microscopy (AFM) captures cuticle and stiffness decay of ageing C. elegans.

a Schematics show an AFM laser beam deflection system and a resulting force-indentation curve. The cantilever bends upon increasing force detected by the deflection of the laser beam reflected from the cantilever back onto a photodiode (left). Typical AFM force-indentation approach curve indicating stages of tip-sample interaction shown in blue (right). b Lifespan curve of wild-type (WT) C. elegans (n = 222). Arrows indicate time points at which AFM measurements were performed—day 1, 5, 8, 12, 15 and 18 of adulthood. c Mechanical properties as Young’s Modulus (YM; kPa) of WT C. elegans at different ages. Error bars indicate 95% confidence intervals (CI), dotted line marks mean YM at day 1. d Lifespan curve of WT C. elegans grown at 20 °C and maintained at 15 °C (blue), 20 °C (yellow) or 25 °C (red) from the L4 stage throughout their entire lifespan (n = 148, 99, 89, respectively; log rank test p < 0.001 vs 20 °C) and e mechanical properties as YM (kPa) at day 15. Error bars indicate 95% CI. Two-tailed unpaired t test for statistical comparison of WT at 20 °C to 15 °C or 25 °C. f Representative AFM cuticle topography images of ageing C. elegans at different ages. g Roughness quantification of topographical images presented as RMS roughness Rq ± standard deviation. Two-tailed unpaired t test for statistical comparison with day 1. h Three-dimension representation of AFM cuticle topography of C. elegans on day 1 and 19 of adulthood. n represented above the graph, show number of biologically independent worm samples; For lifespan measurements, n represents the number of worms scored as dead. For a summary of YM values and additional statistics for independent trials see Supplementary Data 1 and for a summary of worm lifespan trials see Supplementary Data 2. Source data are provided as a Source Data file.

Fig. 2. Reduction of the insulin signalling pathway increases lifespan, improves stiffness and reduces cuticle senescence with age.

a Lifespan curve of wild-type (WT) (blue), or daf-2 (red), daf-16 (green), daf-16;daf-2 (purple) mutant C. elegans. (n = 89, 64, 105 and 79, respectively; log rank test p < 0.001 vs WT). b Longitudinal study of mechanical properties as Young’s Modulus (YM; kPa) comparing WT (blue) and daf-2 mutant (red) C. elegans until mean lifespan (D18 for WT; D44/46 for daf-2). Error bars indicate 95% CI, dotted line marks mean YM at day 1 for WT (blue) and daf-2 (red). Two-tailed unpaired t test for statistical comparison of WT to daf-2 at chronological age day 1, 12 or at mean lifespan. c Mechanical properties as YM (kPa) of WT (blue), daf-2 (red), daf-16 (green) and daf-16;daf-2 (purple) mutant C. elegans at chronological age of 11 days. Error bars indicate 95% CI. Two-way ANOVA Tukey’s multiple comparison test for statistical comparison and interaction of terms (green). d Lifespan curve of WT (blue), glp-1 (red), daf-16 (green) and daf-16;glp-1 (purple) mutant C. elegans. (n = 55, 76, 69 and 88, respectively; log rank test p < 0.001 vs WT) and e mechanical properties as YM (kPa) at chronological age of 10 days. Two-way ANOVA Tukey’s multiple comparison test for statistical comparison and interaction of terms (green). f Mechanical properties as YM (kPa) of WT (blue), or glp-1 (red) mutant C. elegans at mean lifespan. Two-tailed unpaired t test was used for statistical comparison. g Representative AFM cuticle topography images of WT, daf-2 or glp-1 mutant at mean lifespan and h roughness quantification of topographical images presented as RMS roughness Rq ± standard deviation. Two-tailed unpaired t test for statistical comparison of WT with mutants. n represented above the graph, show number of biologically independent worm samples. For lifespan measurements, n represents the number of worms scored as dead. For a summary of YM values and additional statistics for independent trials see Supplementary Data 1 and for a summary of worm lifespan trials and statistical comparison between genotypes see Supplementary Data 2. Source data are provided as a Source Data file.

Fig. 3. AFM identifies differences in stiffness and cuticle senescence with age among diverse dietary restriction regimens.

a Lifespan curves of wild-type (WT) (blue), or eat-2 (green) and phm-2 (purple) feeding impaired mutant C. elegans (n = 98, 104 and 125, respectively; log rank test p < 0.001 vs fully-fed WT) and b longitudinal study of mechanical properties as Young’s Modulus (YM; kPa) until mean lifespan (D18 for WT; D23 for eat-2 and phm-2). Error bars indicate 95% CI, dotted lines mark mean YM at day 1 for WT (blue), eat-2 (green) or phm-2 (purple). Two-tailed unpaired t test for statistical comparison of WT with eat-2 or phm-2 at chronological age day 12 and at mean lifespan, WT at mean lifespan to day 1 (blue), phm-2 at mean lifespan to day 1 (purple), and eat-2 at mean lifespan to day 1 (green). c Lifespan curve of untreated (0 mm) (blue) and metformin-treated (50 mm) (red) WT C. elegans grown on sensitive OP50 (full line) or grown on metformin-resistant OP50-MR (dotted line). (n = 107, 142, 119 and 175, respectively; log rank test p < 0.001, p = 0.0883 and p = 0.0004 vs OP50 0 mm) and d mechanical properties as YM (kPa) at chronological age of 16 days. Error bars indicate 95% CI. Two-way ANOVA Tukey’s multiple comparison test for statistical comparison and interaction of terms (green). e Longitudinal study of mechanical properties as YM (kPa) of untreated (0 mm) (blue) and metformin-treated (50 mm) (red) WT C. elegans until mean lifespan (D18 for WT; D24 for metformin-treated). Error bars indicate 95% CI, dotted lines mark mean YM at day 1 for untreated (blue) and metformin-treated (red). Two-tailed unpaired t test for statistical comparison of untreated to metformin-treated at chronological age day 10 and mean lifespan. f Representative AFM cuticle topography images of WT, eat-2 and phm-2 mutant worms, and WT under bacterial deprivation or metformin-treated at mean lifespan. g Roughness quantification of topographical images presented as RMS roughness Rq ± standard deviation. Two-tailed unpaired t test for statistical comparison of WT with mutants/conditions. n represented above the graph, show number of biologically independent worm samples. For lifespan measurements, n represents the number of worms scored as dead. For a summary of YM values and additional statistics for independent trials, see Supplementary Data 1 and for a summary of worm lifespan trials and statistical comparison between genotypes see Supplementary Data 2. Source data are provided as a Source Data file.

Fig. 4. Bacterial physiology impact stiffness and cuticle senescence with age.

a Lifespan curve of wild-type (WT) C. elegans on untreated control bacteria (blue), heat-treated (green) or UV-treated bacteria (purple) (n = 90, 85 and 106, respectively; log rank test p < 0.001 vs untreated control bacteria) and b longitudinal study of mechanical properties as Young’s Modulus (YM; kPa) until mean lifespan (D18 for WT, D24 for heat-treated, D28 for UV-treated). Error bars indicate 95% CI, dotted lines mark mean YM at day 1 for WT (blue), heat-treated (green) or UV-treated bacteria (purple). Two-tailed unpaired t test for statistical comparison of WT to treatments at chronological age day 10 and mean lifespan. c Lifespan curve of untreated (0 µg/ml) (blue) and trimethoprim (Tmp)-treated (3 µg/ml) (red) WT C. elegans grown on sensitive OP50 (line) or grown on trimethoprim-resistant OP50-TmpR overexpressing a dihydrofolate reductase cassette (dotted line) (n = 83, 70, 100 and 91, respectively; log rank test p < 0.001, p = 0.4094 and p = 0.0206 vs OP50 0 µg/ml) and d mechanical properties as YM (kPa) at chronological age of 16 days. Error bars indicate 95% CI. Two-way ANOVA Tukey’s multiple comparison test for statistical comparison and interaction of terms (green). e Mechanical properties as YM (kPa) at mean lifespan (D18 for WT, D33 for Tmp-treated). Error bars indicate 95% CI. Two-tailed unpaired t test for statistical comparison. f Lifespan curve of WT C. elegans grown on OP50 (blue) or OP50 mutants of the TCA cycle genes gltA (yellow) or sucA (red) (n = 111, 113 and 101, respectively; log rank test p < 0.001 vs OP50), and g mechanical properties as YM (kPa) at chronological age of 14 days or h at mean lifespan (D18 for OP50, D23 for OP50ΔgltA and D26 for OP50ΔsucA). Error bars indicate 95% CI. Two-tailed unpaired t test for statistical comparison of OP50 to bacterial mutants. i Representative AFM cuticle topography images of WT C. elegans at mean lifespan grown on untreated control, heat-treated, UV-treated, carbenicilin-treated (50 µg/ml) and trimethoprim-treated (3 µg/ml) OP50 bacteria or on OP50ΔgltA and OP50ΔsucA bacterial mutants. j Roughness quantification of topographical images presented as RMS roughness Rq ± standard deviation. Two-tailed unpaired t test for statistical comparison of WT with conditions. n represented above the graph, show number of biologically independent worm samples. For lifespan measurements, n represents the number of worms scored as dead. For a summary of YM values and additional statistics for independent trials, see Supplementary Data 1 and for a summary of worm lifespan trials and statistical comparison between genotypes see Supplementary Data 2. Source data are provided as a Source Data file.

Fig. 5. Microbial diet effects on stiffness and cuticle senescence with age are regulated by host nutrient sensors.

a Lifespan curve of wild-type (WT) C. elegans grown on control E. coli OP50 (blue), C. aquatica DA2123 (yellow) and B. subtilis PY79 (black) bacteria (n = 256, 250 and 292, respectively; log rank test p < 0.001 vs OP50) and b longitudinal study of mechanical properties as Young’s Modulus (YM; kPa) until mean lifespan (D18 for OP50, D16 for C. aquatica and D21 for B. subtilis). Error bars indicate 95% CI, dotted lines mark mean YM at day 1 for OP50 (blue), C. aquatica (yellow) and B. subtilis (black). Two-tailed unpaired t test for statistical comparison of OP50 with other bacteria at chronological age day 12 and at mean lifespan. c Representative AFM cuticle topography images of WT C. elegans grown on E. coli, C. aquatica or B. subtilis at mean lifespan and d roughness quantification of topographical images presented as RMS roughness Rq ± standard deviation. Two-tailed unpaired t test for statistical comparison of OP50 to other bacteria. e Lifespan curve of daf-16 mutant C. elegans grown on E. coli (blue), C. aquatica (yellow) or B. subtilis (black) (n = 94, 120 and 128, respectively; log rank test p = 0.9265 and p = 0.3676 vs daf-16 on E. coli). f Mechanical properties as YM (kPa) of WT or daf-16 mutant C. elegans grown on control E. coli (blue), C. aquatica (yellow) or B. subtilis (black) at chronological age of 9 days. Error bars indicate 95% CI. Two-way ANOVA Tukey’s multiple comparison test for statistical comparison and interaction of terms (green). g Lifespan curve of nhr-49 mutant C. elegans grown on E. coli (blue), C. aquatica (yellow) or B. subtilis (black) (n = 65, 55 and 71, respectively; log rank test p < 0.0001 vs nhr-49 on E. coli). h Mechanical properties as YM (kPa) of WT or nhr-49 mutant C. elegans grown on control E. coli (blue), C. aquatica (yellow) or B. subtilis (black) at chronological age of 9 days. Error bars indicate 95% CI. Two-way ANOVA Tukey’s multiple comparison test for statistical comparison and interaction of terms (green). n represented above the graph, show number of biologically independent worm samples. For lifespan measurements, n represents the number of worms scored as dead. For a summary of YM values and additional statistics for independent trials, see Supplementary Data 1 and for a summary of worm lifespan trials and statistical comparison between genotypes see Supplementary Data 2. Source data are provided as a Source Data file.