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Nature methods
07, 2021;18 (7): 788-798.

Metabolomic profiling of single enlarged lysosomes.

Hongying Zhu, Qianqian Li, Tiepeng Liao, Xiang Yin, Qi Chen, Ziyi Wang, Meifang Dai, Lin Yi, Siyuan Ge, Chenjian Miao, Wenping Zeng, Lili Qu, Zhenyu Ju, Guangming Huang, Chunlei Cang, Wei Xiong
Author Information
  1. Hongying Zhu: Institute on Aging and Brain Disorders, the First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, China.
  2. Qianqian Li: Institute on Aging and Brain Disorders, the First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, China.
  3. Tiepeng Liao: Institute on Aging and Brain Disorders, the First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, China. ORCID
  4. Xiang Yin: Institute on Aging and Brain Disorders, the First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, China.
  5. Qi Chen: Institute on Aging and Brain Disorders, the First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, China.
  6. Ziyi Wang: Institute on Aging and Brain Disorders, the First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, China.
  7. Meifang Dai: Institute on Aging and Brain Disorders, the First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, China.
  8. Lin Yi: Institute on Aging and Brain Disorders, the First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, China.
  9. Siyuan Ge: Institute on Aging and Brain Disorders, the First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, China.
  10. Chenjian Miao: Institute on Aging and Brain Disorders, the First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, China.
  11. Wenping Zeng: Institute on Aging and Brain Disorders, the First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, China.
  12. Lili Qu: Institute on Aging and Brain Disorders, the First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, China.
  13. Zhenyu Ju: Key Laboratory of Regenerative Medicine of Ministry of Education, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Institute of Aging and Regenerative Medicine, Jinan University, Guangdong, China.
  14. Guangming Huang: Department of Chemistry, School of Chemistry and Materials Science, University of Science and Technology of China, Hefei, China. ORCID
  15. Chunlei Cang: Institute on Aging and Brain Disorders, the First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, China. ccang@ustc.edu.cn. ORCID
  16. Wei Xiong: Institute on Aging and Brain Disorders, the First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, China. wxiong@ustc.edu.cn. ORCID
PMID: 34127857 DOI: 10.1038/s41592-021-01182-8

Abstract

Lysosomes are critical for cellular metabolism and are heterogeneously involved in various cellular processes. The ability to measure lysosomal metabolic heterogeneity is essential for understanding their physiological roles. We therefore built a single-lysosome mass spectrometry (SLMS) platform integrating lysosomal patch-clamp recording and induced nano-electrospray ionization (nanoESI)/mass spectrometry (MS) that enables concurrent metabolic and electrophysiological profiling of individual enlarged lysosomes. The accuracy and reliability of this technique were validated by supporting previous findings, such as the transportability of lysosomal cationic amino acids transporters such as PQLC2 and the lysosomal trapping of lysosomotropic, hydrophobic weak base drugs such as lidocaine. We derived metabolites from single lysosomes in various cell types and classified lysosomes into five major subpopulations based on their chemical and biological divergence. Senescence and carcinoma altered metabolic profiles of lysosomes in a type-specific manner. Thus, SLMS can open more avenues for investigating heterogeneous lysosomal metabolic changes during physiological and pathological processes.

References

  1. Saftig, P. & Klumperman, J. Lysosome biogenesis and lysosomal membrane proteins: trafficking meets function. Nat. Rev. Mol. Cell Biol. 10, 623–635 (2009). [PMID: 19672277]
  2. Lawrence, R. E. & Zoncu, R. The lysosome as a cellular centre for signalling, metabolism and quality control. Nat. Cell Biol. 21, 133–142 (2019). [PMID: 30602725]
  3. Ravikumar, B. et al. Regulation of mammalian autophagy in physiology and pathophysiology. Physiol. Rev. 90, 1383–1435 (2010). [DOI: 10.1152/physrev.00030.2009]
  4. Saxton, R. A. & Sabatini, D. M. mTOR signaling in growth, metabolism, and disease. Cell 168, 960–976 (2017). [PMID: 28283069]
  5. Platt, F. M., d′Azzo, A., Davidson, B. L., Neufeld, E. F. & Tifft, C. J. Lysosomal storage diseases. Nat. Rev. Dis. Primers 4, 27–51 (2018). [PMID: 30275469]
  6. Mellman, I. Organelles observed: lysosomes. Science 244, 853–854 (1989). [PMID: 17802262]
  7. Zhao, H. et al. Characterize collective lysosome heterogeneous dynamics in live cell with a space- and time-resolved method. Anal. Chem. 90, 9138–9147 (2018). [PMID: 29996056]
  8. Kelly, B. M., Waheed, A., Etten, R. V. & Chang, P. L. Heterogeneity of lysosomes in human fibroblasts. Mol. Cell. Biochem. 87, 171–183 (1989). [PMID: 2770720]
  9. Luzio, J. P., Hackmann, Y., Dieckmann, N. M. & Griffiths, G. M. The biogenesis of lysosomes and lysosome-related organelles. Cold Spring Harb. Perspect. Biol. 6, a016840 (2014). [PMID: 25183830]
  10. Blott, E. J. & Griffiths, G. M. Secretory lysosomes. Nat. Rev. Mol. Cell Biol. 3, 122–131 (2002). [PMID: 11836514]
  11. Flannagan, R. S., Jaumouille, V. & Grinstein, S. The cell biology of phagocytosis. Annu. Rev. Pathol. 7, 61–98 (2012). [PMID: 21910624]
  12. Truschel, S. T. et al. Age-related endolysosome dysfunction in the rat urothelium. PLoS ONE 13, e0198817 (2018). [PMID: 29883476]
  13. Sasaki, T. et al. Autolysosome biogenesis and developmental senescence are regulated by both Spns1 and v-ATPase. Autophagy 13, 386–403 (2017). [PMID: 27875093]
  14. Winckler, B. et al. The endolysosomal system and proteostasis: from development to degeneration. J. Neurosci. 38, 9364–9374 (2018). [PMID: 30381428]
  15. Cang, C. et al. mTOR regulates lysosomal ATP-sensitive two-pore Na channels to adapt to metabolic state. Cell 152, 778–790 (2013). [PMID: 23394946]
  16. Cang, C., Aranda, K., Seo, Y.-j, Gasnier, B. & Ren, D. TMEM175 Is an organelle K channel regulating lysosomal function. Cell 162, 1101–1112 (2015). [PMID: 26317472]
  17. Warnes, G. Flow cytometric assays for the study of autophagy. Methods 82, 21–28 (2015). [PMID: 25846398]
  18. Dolman, N. J., Chambers, K. M., Mandavilli, B., Batchelor, R. H. & Janes, M. S. Tools and techniques to measure mitophagy using fluorescence microscopy. Autophagy 9, 1653–1662 (2013). [PMID: 24121704]
  19. Ashford, T. P. & Porter, K. R. Cytoplasmic components in hepatic cell lysosomes. J. Cell Biol. 12, 198–202 (1962). [PMID: 13862833]
  20. Zhu, H. et al. Single-neuron identification of chemical constituents, physiological changes, and metabolism using mass spectrometry. Proc. Natl Acad. Sci. USA 114, 2586–2591 (2017). [PMID: 28223513]
  21. Zhu, H. et al. Moderate UV exposure enhances learning and memory by promoting a novel glutamate biosynthetic pathway in the brain. Cell 173, 1716–1727 (2018). [PMID: 29779945]
  22. Gradov, O. ‘MS-patch-clamp’ or the possibility of mass spectrometry hybridization with patch-clamp setups for single cell metabolomics and channelomics. Adv. Biochem. 3, 66–71 (2015). [DOI: 10.11648/j.ab.20150306.11]
  23. Aerts, J. T. et al. Patch clamp electrophysiology and capillary electrophoresis–mass spectrometry metabolomics for single cell characterization. Anal. Chem. 86, 3203–3208 (2014). [PMID: 24559180]
  24. Huang, G., Li, G. & Cooks, R. G. Induced nanoelectrospray ionization for matrix-tolerant and high-throughput mass spectrometry. Angew. Chem. Int. Ed. Engl. 50, 9907–9910 (2011). [PMID: 21898729]
  25. Cang, C., Bekele, B. & Ren, D. The voltage-gated sodium channel TPC1 confers endolysosomal excitability. Nat. Chem. Biol. 10, 463–469 (2014). [PMID: 24776928]
  26. Chen, C. C. et al. Patch-clamp technique to characterize ion channels in enlarged individual endolysosomes. Nat. Protoc. 12, 1639–1658 (2017). [PMID: 28726848]
  27. Yao, H. et al. Label-free mass cytometry for unveiling cellular metabolic heterogeneity. Anal. Chem. 91, 9777–9783 (2019). [PMID: 31242386]
  28. Abu-Remaileh, M. et al. Lysosomal metabolomics reveals V-ATPase- and mTOR-dependent regulation of amino acid efflux from lysosomes. Science 358, 807–814 (2017). [PMID: 29074583]
  29. Sagne, C. et al. Identification and characterization of a lysosomal transporter for small neutral amino acids. Proc. Natl Acad. Sci. USA 98, 7206–7211 (2001). [PMID: 11390972]
  30. Liu, B., Du, H., Rutkowski, R., Gartner, A. & Wang, X. LAAT-1 is the lysosomal lysine/arginine transporter that maintains amino acid homeostasis. Science 337, 351–554 (2012). [PMID: 22822152]
  31. Jézégou, A. et al. Heptahelical protein PQLC2 is a lysosomal cationic amino acid exporter underlying the action of cysteamine in cystinosis therapy. Proc. Natl Acad. Sci. USA 12, E3434–E3443 (2012).
  32. Jewell, J. L. et al. Lysosomal amino acid transporter SLC38A9 signals arginine sufficiency to mTORC1. Science 347, 188–195 (2015). [DOI: 10.1126/science.1257132]
  33. Marceau, F. et al. Cation trapping by cellular acidic compartments: beyond the concept of lysosomotropic drugs. Toxicol. Appl. Pharmacol. 259, 1–12 (2012). [PMID: 22198553]
  34. Stuart, T. et al. Comprehensive integration of single-cell data. Cell 177, 1888–1902 (2019). [PMID: 31178118]
  35. Butler, A., Hoffman, P., Smibert, P., Papalexi, E. & Satija, R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat. Biotechnol. 36, 411–420 (2018). [PMID: 29608179]
  36. Sanchez-Illana, A. et al. Evaluation of batch effect elimination using quality control replicates in LC–MS metabolite profiling. Anal. Chim. Acta 1019, 38–48 (2018). [PMID: 29625683]
  37. Kiselev, V. Y., Andrews, T. S. & Hemberg, M. Challenges in unsupervised clustering of single-cell RNA-seq data. Nat. Rev. Genet. 20, 273–282 (2019). [PMID: 30617341]
  38. Huotari, J. & Helenius, A. Endosome maturation. EMBO J. 30, 3481–3500 (2011). [PMID: 21878991]
  39. Li, J. et al. Lysozyme-assisted photothermal eradication of methicillin-resistant Staphylococcus aureus infection and accelerated tissue repair with natural melanosome nanostructures. ACS Nano 13, 11153–11167 (2019). [PMID: 31425647]
  40. Wang, J. et al. Integrated proteomic and metabolomic analysis to study the effects of spaceflight on Candida albicans. BMC Genomics 21, 57 (2020). [PMID: 31952470]
  41. Luo, X. et al. High-performance chemical isotope labeling liquid chromatography–mass spectrometry for profiling the metabolomic reprogramming elicited by ammonium limitation in yeast. J. Proteome Res. 15, 1602–1612 (2016). [PMID: 26947805]
  42. Luzio, J. P., Pryor, P. R. & Bright, N. A. Lysosomes: fusion and function. Nat. Rev. Mol. Cell Biol. 8, 622–632 (2007). [PMID: 17637737]
  43. Klionsky, D. J. et al. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12, 1–222 (2016).
  44. Platt, F. M., Boland, B. & van der Spoel, A. C. The cell biology of disease: lysosomal storage disorders: the cellular impact of lysosomal dysfunction. J. Cell Biol. 199, 723–734 (2012). [PMID: 23185029]
  45. Trapnell, C. et al. The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells. Nat. Biotechnol. 32, 381–386 (2014). [PMID: 24658644]
  46. Qiu, X. et al. Reversed graph embedding resolves complex single-cell trajectories. Nat. Methods 14, 979–982 (2017). [PMID: 28825705]
  47. Gomez-Sintes, R., Ledesma, M. D. & Boya, P. Lysosomal cell death mechanisms in aging. Ageing Res. Rev. 32, 150–168 (2016). [PMID: 26947122]
  48. Carmona-Gutierrez, D., Hughes, A. L., Madeo, F. & Ruckenstuhl, C. The crucial impact of lysosomes in aging and longevity. Ageing Res. Rev. 32, 2–12 (2016). [PMID: 27125853]
  49. Yang, H., Wang, H., Ren, J., Chen, Q. & Chen, Z. J. cGAS is essential for cellular senescence. Proc. Natl Acad. Sci. USA 114, E4612–E4620 (2017). [PMID: 28533362]
  50. Paik, M. J. et al. Polyamine patterns in the cerebrospinal fluid of patients with Parkinson’s disease and multiple system atrophy. Clin. Chim. Acta 411, 1532–1535 (2010). [PMID: 20515677]
  51. Trushina, E., Dutta, T., Persson, X.-M. T., Mielke, M. M. & Petersen, R. C. Identification of altered metabolic pathways in plasma and CSF in mild cognitive impairment and Alzheimer’s disease using metabolomics. PLoS ONE 8, e63644 (2013). [PMID: 23700429]
  52. Yu, Q. et al. Lipidome alterations in human prefrontal cortex during development, aging, and cognitive disorders. Mol. Psychiatry 25, 2952–2969 (2018).
  53. Leidal, A. M., Levine, B. & Debnath, J. Autophagy and the cell biology of age-related disease. Nat. Cell Biol. 20, 1338–1348 (2018). [PMID: 30482941]
  54. Davidson, S. M. & Vander Heiden, M. G. Critical functions of the lysosome in cancer biology. Annu. Rev. Pharmacol. Toxicol. 57, 481–507 (2017). [PMID: 27732799]
  55. Tang, T. et al. The role of lysosomes in cancer development and progression. Cell Biosci. 10, 131 (2020). [PMID: 33292489]
  56. Lozy, F. & Karantza, V. Autophagy and cancer cell metabolism. Semin. Cell Dev. Biol. 23, 395–401 (2012). [PMID: 22281437]
  57. Lin, Y. C. et al. Inhibition of high basal level of autophagy induces apoptosis in human bladder cancer cells. J. Urol. 195, 1126–1135 (2016). [PMID: 26519656]
  58. Huang, L. et al. Spray-capillary-based capillary electrophoresis mass spectrometry for metabolite analysis in single cells. Anal. Chem. 93, 4479–4487 (2021).
  59. Chen, Y. et al. Ultrafast microelectrophoresis: behind direct mass spectrometry measurements of proteins and metabolites in living cell/cells. Anal. Chem. 91, 10441–10447 (2019). [PMID: 31195797]
  60. Cerny, J. et al. The small chemical vacuolin-1 inhibits Ca-dependent lysosomal exocytosis but not cell resealing. EMBO Rep. 5, 883–888 (2004). [PMID: 15332114]
  61. Xu, H. & Ren, D. Lysosomal physiology. Annu. Rev. Physiol. 77, 57–80 (2015). [PMID: 25668017]
  62. Folick, A. et al. Lysosomal signaling molecules regulate longevity in Caenorhabditis elegans. Science 347, 83–87 (2015). [PMID: 25554789]
  63. Leeman, D. S. et al. Lysosome activation clears aggregates and enhances quiescent neural stem cell activation during aging. Science 359, 1277–1283 (2018). [PMID: 29590078]
  64. Liu, G. Y. & Sabatini, D. M. mTOR at the nexus of nutrition, growth, ageing and disease. Nat. Rev. Mol. Cell Biol. 21, 183–203 (2020). [PMID: 31937935]
  65. Ramachandran, P. V. et al. Lysosomal signaling promotes longevity by adjusting mitochondrial activity. Dev. Cell 48, 685–696 (2019). [PMID: 30713071]
  66. Salminen, A. & Kaarniranta, K. Regulation of the aging process by autophagy. Trends Mol. Med. 15, 217–224 (2009). [PMID: 19380253]
  67. Durkin, M. E., Qian, X., Popescu, N. C. & Lowy, D. R. Isolation of mouse embryo fibroblasts. Bio. Protoc. 3, e908 (2013). [PMID: 27376106]
  68. Seluanov, A., Vaidya, A. & Gorbunova, V. Establishing primary adult fibroblast cultures from rodents. J. Vis. Exp. 44, 2033 (2010).
  69. Blazenovic, I. et al. Increasing compound identification rates in untargeted lipidomics research with liquid chromatography drift time-ion mobility mass spectrometry. Anal. Chem. 90, 10758–10764 (2018). [PMID: 30096227]
  70. Yang, B., Patterson, N. H., Tsui, T., Caprioli, R. M. & Norris, J. L. Single-cell mass spectrometry reveals changes in lipid and metabolite expression in RAW 264.7 cells upon lipopolysaccharide stimulation. J. Am. Soc. Mass Spectrom. 29, 1012–1020 (2018). [PMID: 29536413]
  71. Warrack, B. M. et al. Normalization strategies for metabonomic analysis of urine samples. J. Chromatogr. B 877, 547–552 (2009). [DOI: 10.1016/j.jchromb.2009.01.007]
  72. Chen, Y. et al. Combination of injection volume calibration by creatinine and MS signals’ normalization to overcome urine variability in LC–MS-based metabolomics studies. Anal. Chem. 85, 7659–7665 (2013). [PMID: 23855648]
  73. Alfassi, Z. B. On the normalization of a mass spectrum for comparison of two spectra. J. Am. Soc. Mass Spectrom. 15, 385–387 (2004). [PMID: 14998540]

MeSH Term

Amino Acid Transport Systems
Amino Acid Transport Systems, Basic
Cellular Senescence
Fibroblasts
Green Fluorescent Proteins
HEK293 Cells
Humans
Hydrophobic and Hydrophilic Interactions
Lidocaine
Lysosomes
Metabolomics
Patch-Clamp Techniques
Reproducibility of Results
Signal-To-Noise Ratio
Spectrometry, Mass, Electrospray Ionization
Urinary Bladder Neoplasms

Chemicals

Amino Acid Transport Systems
Amino Acid Transport Systems, Basic
SLC66A1 protein, human
enhanced green fluorescent protein
Green Fluorescent Proteins
Lidocaine
Journal Article Research Support, Non-U.S. Gov't
Article has an altmetric score of 79

Word Cloud

Created with Highcharts 10.0.0lysosomallysosomesmetaboliccellularvariousprocessesphysiologicalspectrometrySLMSprofilingenlargedsingleLysosomescriticalmetabolismheterogeneouslyinvolvedabilitymeasureheterogeneityessentialunderstandingrolesthereforebuiltsingle-lysosomemassplatformintegratingpatch-clamprecordinginducednano-electrosprayionizationnanoESI/massMSenablesconcurrentelectrophysiologicalindividualaccuracyreliabilitytechniquevalidatedsupportingpreviousfindingstransportabilitycationicaminoacidstransportersPQLC2trappinglysosomotropichydrophobicweakbasedrugslidocainederivedmetabolitescelltypesclassifiedfivemajorsubpopulationsbasedchemicalbiologicaldivergenceSenescencecarcinomaalteredprofilestype-specificmannerThuscanopenavenuesinvestigatingheterogeneouschangespathologicalMetabolomic

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