Spectroscopic coherent Raman imaging of Caenorhabditis elegans reveals lipid particle diversity

Abstract

Caenorhabditis elegans serves as a model for understanding adiposity and its connections to aging. Current methodologies do not distinguish between fats serving the energy needs of the parent, akin to mammalian adiposity, from those that are distributed to the progeny, making it difficult to accurately interpret the physiological implications of fat content changes induced by external perturbations. Using spectroscopic coherent Raman imaging, we determine the protein content, chemical profiles and dynamics of lipid particles in live animals. We find fat particles in the adult intestine to be diverse, with most destined for the developing progeny. In contrast, the skin-like epidermis contains fats that are the least heterogeneous, the least dynamic and have high triglyceride content. These attributes are most consistent with stored somatic energy reservoirs. These results challenge the prevailing practice of assessing C. elegans adiposity by measurements that are dominated by the intestinal fat content.

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Fig. 1: Distinct chemical features of lipid-rich particles in C. elegans.
Fig. 2: Ratiometric BCARS imaging of C. elegans.
Fig. 3: Pulse-chase analysis of assimilated dFA.
Fig. 4: Correlation between CD:CH (2,100 cm−1/2,845 cm−1) and protein:CH (1,665–1,680 cm−1/2,845 cm−1) ratios.
Fig. 5: Characterization of lipid-rich particles in C. elegans with the ensemble machine-learning method.
Fig. 6: C. elegans hermaphrodite lipid-rich particles encompass a continuum of chemical compositions and lifetimes, which vary with tissue localization and reproductive capacity.

Data availability

The C. elegans accession codes (strains, developmental stages and sex) include the following: wild-type (Bristol N2), from L3 to gravid adult hermaphrodites; DH1390 rme-2(b1008), adult hermaphrodites; LIU1 ldrls1 [dhs-3p::dhs-3::GFP + unc-76(+)], adult hermaphrodites; CB4088 him-5(e1490), adult males; and a transgenic strain bls1(vit-2p::vit-2::GFP) expressing a fusion of YP170, adult hermaphrodites. Primary datasets generated and analyzed in this study consist of spectral image data. Each data file is in HDF5 format, and is ~5 GB in size. Approximately 50 such files were generated during the study. These data are available from the corresponding author on reasonable request. Intermediate data, such as spectral peak position and amplitude data extracted from spectral images after watershed, are also available upon request.

Code availability

Primary analysis of the coherent Raman spectra was performed using custom-built software that is publicly available and can be found at https://github.com/CCampJr/crikit2. Routine image analysis, such as watershed, was performed using Fiji ImageJ’s watershed method (v 1.52p, the latest version can be downloaded at https://imagej.net/Fiji). Statistical analysis was performed using NbClust (v 3.0), fitdistrplus (v 1.0–14) and ggplot2 (v 3.2.1) under R programming v 3.5.1, and SciKit-Learn (v 0.18.1) under Python v 3.4.

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Acknowledgements

K.A. and G.A.L. acknowledge support from NIH/NIA (R01AG046400) and BWF Innovations in Regulatory Sciences. T.C. acknowledges support from MOST-106-2119-M-001-030-MY3 of the Republic of China (Taiwan).

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K.A., T.-C.C., M.T.C. and W.-W.C. initiated the project. G.A.L., K.A. and W.-W.C. conceived and designed the study. W.-W.C. conducted all experiments. C.H.C. and W.-W.C. developed the ensemble machine-learning workflow. W.-W.C. analyzed all data with discussions and contributions from G.A.L., K.A., M.T.C., C.H.C. and T.-C.C. G.A.L. and K.A. conceived and drew Fig. 6. W.-W.C., G.A.L., K.A. and M.T.C. wrote the manuscript. All authors reviewed the manuscript.

Corresponding authors

Correspondence to Kaveh Ashrafi or Marcus T. Cicerone.

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Extended data

Extended Data Fig. 1 The analysis results after applying ensemble machine-learning method for fed and short-term starved (2–3 hr) adult hermaphrodites as well as adult male (him-5) worms.

a, The analysis results of gonad (oocytes) and skin-like epidermis near pharynx in the fed wild-type adult hermaphrodites. b, The analysis results of the intestine in the fed / short-term starved adult hermaphrodite and male (him-5) worms. c, The analysis results of gonad and epidermis in the short-term starved adult hermaphrodites. Scale bar =10 μm for (a) to (c). The experiments were repeated at least four times independently with similar results for (a) and (b), and were repeated two times independently with similar results for (c). The region of pharyngeal neurons was excluded for the analysis of skin-like epidermis near pharynx.

Extended Data Fig. 2 The effect of long-term starvation and phenformin.

a, The CARS images of control, long-term starved (18–20 hr), and phenformin-treated worms. The young adult worms that had been fed dFA since the late L4 stage (12 h) were placed on the OP50 lawns that lacked the deuterium label (for control), on the plates lacked OP50 (for 18–20 h long-term starvation), and on the OP50 lawns that lacked the deuterium label but contained phenformin (20 h treatment with final concentration = 7.5 mM), respectively. After that, the worms were imaged by BCARS. The right column is the results after applied the ensemble machine-learning method. Scale bar =10μm. The experiments were repeated at least three times independently with similar results. The region of pharyngeal neurons was excluded for the analysis of skin-like epidermis near pharynx. b, The normalized lipid content (or normalized mean 2845 cm-1 intensity) of total lipid-rich particles in the tissue (normalized to a fixed tissue area). c, The average number of the dFA-retaining observed in the tissue (normalized to a fixed tissue area). Total ~4000 particles were analyzed; the data were collected from n = 3–6 biologically independent animals for each condition, where each measurement is presented as a dot; The error bars represent the standard error of the mean.

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Chen, W., Lemieux, G.A., Camp, C.H. et al. Spectroscopic coherent Raman imaging of Caenorhabditis elegans reveals lipid particle diversity. Nat Chem Biol (2020). https://doi.org/10.1038/s41589-020-0565-2

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