Advertisement

Nanoscale monitoring of mitochondria and lysosome interactions for drug screening and discovery

  • Qixin Chen
  • Xintian Shao
  • Zhiqi Tian
  • Yang Chen
  • Payel Mondal
  • Fei Liu
  • Fengshan Wang
  • Peixue LingEmail author
  • Weijiang HeEmail author
  • Kai ZhangEmail author
  • Zijian Guo
  • Jiajie DiaoEmail author
Research Article
  • 22 Downloads

Abstract

Technology advances in genomics, proteomics, and metabolomics largely expanded the pool of potential therapeutic targets. Compared with the in vitro setting, cell-based screening assays have been playing a key role in the processes of drug discovery and development. Besides the commonly used strategies based on colorimetric and cell viability, we reason that methods that capture the dynamic cellular events will facilitate optimal hit identification with high sensitivity and specificity. Herein, we propose a live-cell screening strategy using structured illumination microscopy (SIM) combined with an automated cell colocalization analysis software, Cellprofiler™, to screen and discover drugs for mitochondria and lysosomes interaction at a nanoscale resolution in living cells. This strategy quantitatively benchmarks the mitochondria-lysosome interactions such as mitochondria and lysosomes contact (MLC) and mitophagy. The automatic quantitative analysis also resolves fine changes of the mitochondria-lysosome interaction in response to genetic and pharmacological interventions. Super-resolution live-cell imaging on the basis of quantitative analysis opens up new avenues for drug screening and development by targeting dynamic organelle interactions at the nanoscale resolution, which could facilitate optimal hit identification and potentially shorten the cycle of drug discovery.

Keywords

drug screening mitochondria lysosome mitophagy structured illumination microscopy 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Notes

Acknowledgements

This research was supported by the National Basic Research Program of China (No. 2015CB856300), National Institutes of Health (NIH R35GM128837 to J.D.), Natural Science Foundation of Shandong Province (Nos. ZR2017PH072, ZR2017BH051, and ZR2015QL007), and Key Research and Development Plan of Shandong Province (No. 2018GSF121033). K. Z. was supported by the University of Illinois at Urbana-Champaign. The Light Microscopy Imaging Center (LMIC) is supported in part with funds from Indiana University Office of the Vice Provost for Research. The 3D-SIM microscope was provided by NIH grant NIH1S10OD024988-01.

Supplementary material

12274_2019_2331_MOESM1_ESM.pdf (2.6 mb)
Nanoscale monitoring of mitochondria and lysosome interactions for drug screening and discovery

References

  1. [1]
    Bialer, M.; White, H. S. Key factors in the discovery and development of new antiepileptic drugs. Nat. Rev. Drug Discov. 2010, 9, 68–82.CrossRefGoogle Scholar
  2. [2]
    Chen, Q. X.; Shao, X. T.; Ling, P. X.; Liu, F.; Han, G. Y.; Wang, F. S. Recent advances in polysaccharides for osteoarthritis therapy. Eur. J. Med. Chem. 2017, 139, 926–935.CrossRefGoogle Scholar
  3. [3]
    Prasad, V.; Mailankody, S. Research and development spending to bring a single cancer drug to market and revenues after approval. JAMA Intern. Med. 2017, 177, 1569–1575.CrossRefGoogle Scholar
  4. [4]
    Schulze, K.; Imbeaud, S.; Letouzé, E.; Alexandrov, L. B.; Calderaro, J.; Rebouissou, S.; Couchy, G.; Meiller, C.; Shinde, J.; Soysouvanh, F. et al. Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets. Nat. Genet. 2015, 47, 505–511.CrossRefGoogle Scholar
  5. [5]
    Xu, D. C.; Jin, T. J.; Zhu, H.; Chen, H. B.; Ofengeim, D.; Zou, C. Y.; Mifflin, L.; Pan, L. F.; Amin, P.; Li, W. J. et al. TBK1 suppresses RIPK1-driven apoptosis and inflammation during development and in aging. Cell 2018, 174, 1477–1491.E19.CrossRefGoogle Scholar
  6. [6]
    Han, M. H.; Hwang, S. I.; Roy, D. B.; Lundgren, D. H.; Price, J. V.; Ousman, S. S.; Fernald, G. H.; Gerlitz, B.; Robinson, W. H.; Baranzini, S. E. et al. Proteomic analysis of active multiple sclerosis lesions reveals therapeutic targets. Nature 2008, 451, 1076–1081.CrossRefGoogle Scholar
  7. [7]
    Wishart, D. S. Emerging applications of metabolomics in drug discovery and precision medicine. Nat. Rev. Drug Discov. 2016, 15, 473–484.CrossRefGoogle Scholar
  8. [8]
    Vega-Avila, E.; Pugsley, M. K. An overview of colorimetric assay methods used to assess survival or proliferation of mammalian cells. Proc. West. Pharmacol. Soc. 2011, 54, 10–14.Google Scholar
  9. [9]
    Chen, Q. X.; Mei, X. F.; Han, G. Y.; Ling, P. X.; Guo, B.; Guo, Y. W.; Shao, H. R.; Wang, G.; Cui, Z.; Bai, Y. X. et al. Xanthan gum protects rabbit articular chondrocytes against sodium nitroprusside-induced apoptosis in vitro. Carbohydr. Polym. 2015, 131, 363–369.CrossRefGoogle Scholar
  10. [10]
    Chen, Q. X.; Shao, X. T.; Ling, P. X.; Liu, F.; Shao, H. R.; Ma, A. B.; Wu, J. X.; Zhang, W.; Liu, F. Y.; Han, G. Y. et al. Low molecular weight xanthan gum suppresses oxidative stress-induced apoptosis in rabbit chondrocytes. Carbohydr. Polym. 2017, 169, 255–263.CrossRefGoogle Scholar
  11. [11]
    Frankfurt, O. S.; Krishan, A. Enzyme-linked immunosorbent assay (ELISA) for the specific detection of apoptotic cells and its application to rapid drug screening. J. Immunol. Methods 2001, 253, 133–144.CrossRefGoogle Scholar
  12. [12]
    Krutzik, P. O.; Nolan, G. P. Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling. Nat. Methods 2006, 3, 361–368.CrossRefGoogle Scholar
  13. [13]
    Krutzik, P. O.; Crane, J. M.; Clutter, M. R.; Nolan, G. P. High-content single-cell drug screening with phosphospecific flow cytometry. Nat. Chem. Biol. 2008, 4, 132–142.CrossRefGoogle Scholar
  14. [14]
    Rudin, M.; Weissleder, R. Molecular imaging in drug discovery and development. Nat. Rev. Drug Discov. 2003, 2, 123–131.CrossRefGoogle Scholar
  15. [15]
    Neefjes, J.; Dantuma, N. P. Fluorescent probes for proteolysis: Tools for drug discovery. Nat. Rev. Drug Discov. 2004, 3, 58–69.CrossRefGoogle Scholar
  16. [16]
    Willets, K. A. Super-resolution imaging of SERS hot spots. Chem. Soc. Rev. 2014, 43, 3854–3864.CrossRefGoogle Scholar
  17. [17]
    Jia, S.; Vaughan, J. C.; Zhuang, X. W. Isotropic three-dimensional superresolution imaging with a self-bending point spread function. Nat. Photonics 2014, 8, 302–306.CrossRefGoogle Scholar
  18. [18]
    Boutros, M.; Heigwer, F.; Laufer, C. Microscopy-based high-content screening. Cell 2015, 163, 1314–1325.CrossRefGoogle Scholar
  19. [19]
    Usaj, M. M.; Styles, E. B.; Verster, A. J.; Friesen, H.; Boone, C.; Andrews, B. J. High-content screening for quantitative cell biology. Trends Cell Biol. 2016, 26, 598–611.CrossRefGoogle Scholar
  20. [20]
    Liu, Z.; Lavis, L. D.; Betzig, E. Imaging live-cell dynamics and structure at the single-molecule level. Mol. Cell 2015, 58, 644–659.CrossRefGoogle Scholar
  21. [21]
    Chen, Q. X.; Jin, C. Z.; Shao, X. T.; Guan, R. L.; Tian, Z. Q.; Wang, C. R.; Liu, F.; Ling, P. X.; Guan, J. L.; Ji, L. N. et al. Super-resolution tracking of mitochondrial dynamics with an iridium(III) luminophore. Small 2018, 14, 1802166.CrossRefGoogle Scholar
  22. [22]
    Hanne, J.; Falk, H. J.; Görlitz, F.; Hoyer, P.; Engelhardt, J.; Sahl, S. J.; Hell, S. W. STED nanoscopy with fluorescent quantum dots. Nat. Commun. 2015, 6, 7127.CrossRefGoogle Scholar
  23. [23]
    Tian, X. H.; Liu, T. Y.; Fang, B.; Wang, A. D.; Zhang, M. Z.; Hussain, S.; Luo, L.; Zhang, R. L.; Zhang, Q.; Wu, J. Y. et al. Neun-specific fluorescent probe revealing neuronal nuclei protein and nuclear acids association in living neurons under STED nanoscopy. ACS Appl. Mater. Interfaces 2018, 10, 31959–31964.CrossRefGoogle Scholar
  24. [24]
    Huang, X. S.; Fan, J. C.; Li, L. J.; Liu, H. S.; Wu, R. L.; Wu, Y.; Wei, L. S.; Mao, H.; Lal, A.; Xi, P. et al. Fast, long-term, super-resolution imaging with Hessian structured illumination microscopy. Nat. Biotechnol. 2018, 36, 451–459.CrossRefGoogle Scholar
  25. [25]
    Sigal, Y. M.; Zhou, R. B.; Zhuang, X. W. Visualizing and discovering cellular structures with super-resolution microscopy. Science 2018, 361, 880–887.CrossRefGoogle Scholar
  26. [26]
    Huang, B.; Wang, W. Q.; Bates, M.; Zhuang, X. W. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 2008, 319, 810–813.CrossRefGoogle Scholar
  27. [27]
    Ha, T.; Tinnefeld, P. Photophysics of fluorescent probes for single-molecule biophysics and super-resolution imaging. Annu. Rev. Phys. Chem. 2012, 63, 595–617.CrossRefGoogle Scholar
  28. [28]
    Fernández-Suárez, M.; Ting, A. Y. Fluorescent probes for super-resolution imaging in living cells. Nat. Rev. Mol. Cell Biol. 2008, 9, 929–943.CrossRefGoogle Scholar
  29. [29]
    Onnis, A.; Cianfanelli, V.; Cassioli, C.; Samardzic, D.; Pelicci, P. G.; Cecconi, F.; Baldari, C. T. The pro-oxidant adaptor p66SHC promotes B cell mitophagy by disrupting mitochondrial integrity and recruiting LC3-II. Autophagy 2018, 14, 2117–2138.CrossRefGoogle Scholar
  30. [30]
    Wong, Y. C.; Ysselstein, D.; Krainc, D. Mitochondria–lysosome contacts regulate mitochondrial fission via RAB7 GTP hydrolysis. Nature 2018, 554, 382–386.CrossRefGoogle Scholar
  31. [31]
    Burbulla, L. F.; Song, P. P.; Mazzulli, J. R.; Zampese, E.; Wong, Y. C.; Jeon, S.; Santos, D. P.; Blanz, J.; Obermaier, C. D.; Strojny, C. et al. Dopamine oxidation mediates mitochondrial and lysosomal dysfunction in Parkinson’s disease. Science 2017, 357, 1255–1261.CrossRefGoogle Scholar
  32. [32]
    Tian, Z. Q.; Gong, J. H.; Crowe, M.; Lei, M.; Li, D. C.; Ji, B. H.; Diao, J. J. Biochemical studies of membrane fusion at the single-particle level. Prog. Lipid Res. 2019, 73, 92–100.CrossRefGoogle Scholar
  33. [33]
    Liu, K.; Lee, J.; Ou, J. H. J. Autophagy and mitophagy in hepatocarcinogenesis. Mol. Cell. Oncol. 2018, 5, e1405142.CrossRefGoogle Scholar
  34. [34]
    Lamprecht, M. R.; Sabatini, D. M.; Carpenter, A. E. CellProfiler™: Free, versatile software for automated biological image analysis. Biotechniques 2007, 42, 71–75.CrossRefGoogle Scholar
  35. [35]
    Kamentsky, L.; Jones, T. R.; Fraser, A.; Bray, M. A.; Logan, D. J.; Madden, K. L.; Ljosa, V.; Rueden, C.; Eliceiri, K. W.; Carpenter, A. E. Improved structure, function and compatibility for CellProfiler: Modular high-throughput image analysis software. Bioinformatics 2011, 27, 1179–1180.CrossRefGoogle Scholar
  36. [36]
    Kobayashi, S.; Liang, Q. R. Autophagy and mitophagy in diabetic cardiomyopathy. Biochim. Biophys. Acta 2015, 1852, 252–261.CrossRefGoogle Scholar
  37. [37]
    Ryan, B. J.; Hoek, S.; Fon, E. A.; Wade-Martins, R. Mitochondrial dysfunction and mitophagy in Parkinson’s: From familial to sporadic disease. Trends Biochem. Sci. 2015, 40, 200–210.CrossRefGoogle Scholar
  38. [38]
    Burchell, V. S.; Nelson, D. E.; Sanchez-Martinez, A.; Delgado-Camprubi, M.; Ivatt, R. M.; Pogson, J. H.; Randle, S. J.; Wray, S.; Lewis, P. A.; Houlden, H. et al. The Parkinson’s disease–linked proteins Fbxo7 and Parkin interact to mediate mitophagy. Nat. Neurosci. 2013, 16, 1257–1265.CrossRefGoogle Scholar
  39. [39]
    Youle, R. J.; Narendra, D. P. Mechanisms of mitophagy. Nat. Rev. Mol. Cell Biol. 2011, 12, 9–14.CrossRefGoogle Scholar
  40. [40]
    Li, H. Y.; Ham, A.; Ma, T. C.; Kuo, S. H.; Kanter, E.; Kim, D.; Ko, H. S.; Quan, Y.; Sardi, S. P.; Li, A. Q. Mitochondrial dysfunction and mitophagy defect triggered by heterozygous GBA mutations. Autophagy 2019, 15, 113–130.CrossRefGoogle Scholar
  41. [41]
    Williams, J. A.; Ni, H. M.; Ding, W. X. Mitochondrial dynamics, mitophagy and mitochondrial spheroids in drug-induced liver injury. In Mitochondria in Liver Disease. Han, D.; Kaplowitz, N., Eds.; Apple Academic Press Inc.: Florida, 2015; pp 237.Google Scholar
  42. [42]
    Maldonado, E. N.; Gooz, M.; DeHart, D. N.; Lemasters, J. J. VDAC opening drugs to induce mitochondrial dysfunction and cell death. Biophys. J. 2015, 108, 369a.CrossRefGoogle Scholar
  43. [43]
    Barutcu, S. A.; Girnius, N.; Vernia, S.; Davis, R. J. Role of the MAPK/cJun NH2-terminal kinase signaling pathway in starvation-induced autophagy. Autophagy 2018, 14, 1586–1595.CrossRefGoogle Scholar
  44. [44]
    Wallot-Hieke, N.; Verma, N.; Schlütermann, D.; Berleth, N.; Deitersen, J.; Böhler, P.; Stuhldreier, F.; Wu, W. X.; Seggewiß, S.; Peter, C. et al. Systematic analysis of ATG13 domain requirements for autophagy induction. Autophagy 2018, 14, 743–763.CrossRefGoogle Scholar
  45. [45]
    Liu, F.; Guan, J. L. FIP200, an essential component of mammalian autophagy is indispensible for fetal hematopoiesis. Autophagy 2011, 7, 229–230.CrossRefGoogle Scholar
  46. [46]
    Lazarou, M.; Sliter, D. A.; Kane, L. A.; Sarraf, S. A.; Wang, C. X.; Burman, J. L.; Sideris, D. P.; Fogel, A. I.; Youle, R. J. The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy. Nature 2015, 524, 309–314.CrossRefGoogle Scholar
  47. [47]
    Li, F. X.; Xu, D. C.; Wang, Y. L.; Zhou, Z. X.; Liu, J. P.; Hu, S. C.; Gong, Y. K.; Yuan, J. Y.; Pan, L. F. Structural insights into the ubiquitin recognition by OPTN (optineurin) and its regulation by TBK1-mediated phosphorylation. Autophagy 2018, 14, 66–79.CrossRefGoogle Scholar
  48. [48]
    Youle, R. J.; Van Der Bliek, A. M. Mitochondrial fission, fusion, and stress. Science 2012, 337, 1062–1065.CrossRefGoogle Scholar
  49. [49]
    Novak, I. Mitophagy: A complex mechanism of mitochondrial removal. Antioxid. Redox Signal. 2012, 17, 794–802.CrossRefGoogle Scholar
  50. [50]
    Vakifahmetoglu-Norberg, H.; Xia, H. G.; Yuan, J. Y. Pharmacologic agents targeting autophagy. J. Clin. Invest. 2015, 125, 5–13.CrossRefGoogle Scholar
  51. [51]
    Xu, Y. Q.; Yuan, J. Y.; Lipinski, M. M. Live imaging and single-cell analysis reveal differential dynamics of autophagy and apoptosis. Autophagy 2013, 9, 1418–1430.CrossRefGoogle Scholar
  52. [52]
    Cristofani, R.; Marelli, M. M.; Cicardi, M. E.; Fontana, F.; Marzagalli, M.; Limonta, P.; Poletti, A.; Moretti, R. M. Dual role of autophagy on docetaxelsensitivity in prostate cancer cells. Cell Death Dis. 2018, 9, 889.CrossRefGoogle Scholar
  53. [53]
    Mauthe, M.; Orhon, I.; Rocchi, C.; Zhou, X. D.; Luhr, M.; Hijlkema, K. J.; Coppes, R. P.; Engedal, N.; Mari, M.; Reggiori, F. Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion. Autophagy 2018, 14, 1435–1455.CrossRefGoogle Scholar
  54. [54]
    Quintana-Cabrera, R.; Quirin, C.; Glytsou, C.; Corrado, M.; Urbani, A.; Pellattiero, A.; Calvo, E.; Vázquez, J.; Enríquez, J. A.; Gerle, C. et al. The cristae modulator Optic atrophy 1 requires mitochondrial ATP synthase oligomers to safeguard mitochondrial function. Nat. Commun. 2018, 9, 3399.CrossRefGoogle Scholar
  55. [55]
    Chen, Q. X.; Shao, X. T.; Hao, M. G.; Tian, Z. Q.; Wang, C. R.; Liu, F.; Zhang, K.; Wang, F. S.; Ling, P. X.; Guan, J. L. et al. Quantitative analysis of interactive behavior of mitochondria and lysosomes using structured illumination microscopy. 2018, bioRxiv:  https://doi.org/10.1101/445841.CrossRefGoogle Scholar
  56. [56]
    Zemanová, L.; Schenk, A.; Valler, M. J.; Nienhaus, G. U.; Heilker, R. Confocal optics microscopy for biochemical and cellular high-throughput screening. Drug Discov. Today 2003, 8, 1085–1093.CrossRefGoogle Scholar
  57. [57]
    Simm, J.; Klambauer, G.; Arany, A.; Steijaert, M.; Wegner, J. K.; Gustin, E.; Chupakhin, V.; Chong, Y. T.; Vialard, J.; Buijnsters, P. et al. Repurposing high-throughput image assays enables biological activity prediction for drug discovery. Cell Chem. Biol. 2018, 25, 611–618.e3.CrossRefGoogle Scholar

Copyright information

© Tsinghua University Press and Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.School of Pharmaceutical SciencesShandong UniversityJinanChina
  2. 2.Shandong Academy of Pharmaceutical Science, Key Laboratory of Biopharmaceuticals, Engineering Laboratory of Polysaccharide DrugsNational-Local Joint Engineering Laboratory of Polysaccharide DrugsJinanChina
  3. 3.Department of Cancer BiologyUniversity of Cincinnati College of MedicineCincinnatiUSA
  4. 4.Department of BiochemistryUniversity of Illinois at Urbana-ChampaignUrbanaUSA
  5. 5.State Key Laboratory of Coordination Chemistry, Coordination Chemistry Institute, School of Chemistry and Chemical EngineeringNanjing UniversityNanjingChina

Personalised recommendations