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Liu J, Hebbrecht T, Brans T, et al. Long-term live-cell microscopy with labeled nanobodies delivered by laser-induced photoporation. Nano Res. 2020;13(2):485-495doi: 10.1007/s12274-020-2633-z.
Liu, J., Hebbrecht, T., Brans, T., Parthoens, E., Lippens, S., Li, C., De Keersmaecker, H., De Vos, W. H., De Smedt, S. C., Boukherroub, R., Gettemans, J., Xiong, R., & Braeckmans, K. (2020). Long-term live-cell microscopy with labeled nanobodies delivered by laser-induced photoporation. Nano research, 13(2), 485-495. https://doi.org/10.1007/s12274-020-2633-z
Liu, Jing, et al. "Long-term live-cell microscopy with labeled nanobodies delivered by laser-induced photoporation." Nano research vol. 13,2 (2020): 485-495. doi: https://doi.org/10.1007/s12274-020-2633-z
Liu J, Hebbrecht T, Brans T, Parthoens E, Lippens S, Li C, De Keersmaecker H, De Vos WH, De Smedt SC, Boukherroub R, Gettemans J, Xiong R, Braeckmans K. Long-term live-cell microscopy with labeled nanobodies delivered by laser-induced photoporation. Nano Res. 2020 Feb;13(2):485-495. doi: 10.1007/s12274-020-2633-z. Epub 2020 Jan 18. PMID: 33154805; PMCID: PMC7116313.
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Nano Res. 2020 Feb;13(2):485-495. doi: 10.1007/s12274-020-2633-z. Epub 2020 Jan 18.

Long-term live-cell microscopy with labeled nanobodies delivered by laser-induced photoporation.

Nano research

Jing Liu, Tim Hebbrecht, Toon Brans, Eef Parthoens, Saskia Lippens, Chengnan Li, Herlinde De Keersmaecker, Winnok H De Vos, Stefaan C De Smedt, Rabah Boukherroub, Jan Gettemans, Ranhua Xiong, Kevin Braeckmans

Affiliations

  1. Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmacy, Ghent University, Ghent B-9000, Belgium.
  2. Department of Biomolecular medicine, Faculty of Medicine and Health Sciences, Ghent University, Ghent B-9000, Belgium.
  3. VIB-UGent Center for Inflammation Research, VIB, Ghent B-9000, Belgium.
  4. VIB Bioimaging Core Ghent, VIB, Ghent B-9000, Belgium.
  5. Department of Biomedical Molecular Biology, Ghent University, Ghent B-9000, Belgium.
  6. Univ. Lille, CNRS, Centrale Lille, ISEN, Univ. Valenciennes, UMR 8520-IEMN, Lille F-59000, France.
  7. Centre for Advanced Light Microscopy, Ghent University, Ghent B-9000, Belgium.
  8. Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, 2020 Antwerp, Belgium.

PMID: 33154805 PMCID: PMC7116313 DOI: 10.1007/s12274-020-2633-z

Abstract

Fluorescence microscopy is the method of choice for studying intracellular dynamics. However, its success depends on the availability of specific and stable markers. A prominent example of markers that are rapidly gaining interest are nanobodies (Nbs, ~ 15 kDa), which can be functionalized with bright and photostable organic fluorophores. Due to their relatively small size and high specificity, Nbs offer great potential for high-quality long-term subcellular imaging, but suffer from the fact that they cannot spontaneously cross the plasma membrane of live cells. We have recently discovered that laser-induced photoporation is well suited to deliver extrinsic labels to living cells without compromising their viability. Being a laser-based technology, it is readily compatible with light microscopy and the typical cell recipients used for that. Spurred by these promising initial results, we demonstrate here for the first time successful long-term imaging of specific subcellular structures with labeled nanobodies in living cells. We illustrate this using Nbs that target GFP/YFP-protein constructs accessible in the cytoplasm, actin-bundling protein Fascin, and the histone H2A/H2B heterodimers. With an efficiency of more than 80% labeled cells and minimal toxicity (~ 2%), photoporation proved to be an excellent intracellular delivery method for Nbs. Time-lapse microscopy revealed that cell division rate and migration remained unaffected, confirming excellent cell viability and functionality. We conclude that laser-induced photoporation labeled Nbs can be easily delivered into living cells, laying the foundation for further development of a broad range of Nbs with intracellular targets as a toolbox for long-term live-cell microscopy.

Keywords: intracellular delivery; laser-induced photoporation; living cell labeling; long-term microscopy imaging; nanobody; vapor nanobubble

References

  1. J Am Chem Soc. 2016 Aug 3;138(30):9365-8 - PubMed
  2. Mol Ther. 2014 Oct;22(10):1768-78 - PubMed
  3. Nat Chem. 2017 Aug;9(8):762-771 - PubMed
  4. Nat Commun. 2016 Jan 29;7:10372 - PubMed
  5. J Control Release. 2017 Nov 28;266:198-204 - PubMed
  6. Theranostics. 2014 Jan 29;4(4):386-98 - PubMed
  7. Nano Lett. 2012 Nov 14;12(11):5669-72 - PubMed
  8. J Control Release. 2017 Dec 10;267:154-162 - PubMed
  9. J Microsc. 2006 Dec;224(Pt 3):213-32 - PubMed
  10. Am J Physiol Endocrinol Metab. 2020 Feb 1;318(2):E87-E101 - PubMed
  11. Cytoskeleton (Hoboken). 2013 Oct;70(10):604-22 - PubMed
  12. Methods. 2004 Jun;33(2):95-103 - PubMed
  13. Nano Lett. 2016 Oct 12;16(10):5975-5986 - PubMed
  14. Nat Rev Mol Cell Biol. 2008 Dec;9(12):929-43 - PubMed
  15. Nucleic Acids Res. 2010 Sep;38(17):e168 - PubMed
  16. Cell Mol Life Sci. 2010 May;67(9):1519-35 - PubMed
  17. Nat Methods. 2014 Jul;11(7):731-3 - PubMed
  18. Nat Biotechnol. 2016 Nov 8;34(11):1137-1144 - PubMed
  19. Int J Biochem Cell Biol. 2010 Oct;42(10):1614-7 - PubMed
  20. Hum Mol Genet. 2017 Apr 1;26(7):1353-1364 - PubMed
  21. Chem Soc Rev. 2014 Feb 21;43(4):1044-56 - PubMed
  22. Front Cell Neurosci. 2018 Mar 29;12:80 - PubMed
  23. Nat Methods. 2012 Jun;9(6):582-4 - PubMed
  24. Protein Sci. 2010 Dec;19(12):2389-401 - PubMed
  25. J Cell Sci. 1995 May;108 ( Pt 5):1977-90 - PubMed
  26. Nat Biotechnol. 2013 Jul;31(7):638-46 - PubMed
  27. Nat Methods. 2006 Nov;3(11):887-9 - PubMed
  28. Nat Methods. 2012 Jun 28;9(7):676-82 - PubMed
  29. Int J Biochem Cell Biol. 2005 Sep;37(9):1787-804 - PubMed
  30. Trends Cell Biol. 2009 Nov;19(11):649-55 - PubMed
  31. FASEB J. 2013 Jan;27(1):98-108 - PubMed
  32. Nat Biotechnol. 2003 Jan;21(1):47-51 - PubMed
  33. Biomed Pharmacother. 2018 Jun;102:230-241 - PubMed
  34. J Neurosci. 2004 Apr 21;24(16):4070-81 - PubMed
  35. J Cell Sci. 2000 May;113 ( Pt 10):1651-9 - PubMed
  36. Methods Enzymol. 2013;529:227-40 - PubMed
  37. J Biol Chem. 2016 Apr 22;291(17):9148-60 - PubMed
  38. Trends Cardiovasc Med. 2004 Aug;14(6):221-6 - PubMed
  39. J Lab Autom. 2014 Feb;19(1):50-9 - PubMed
  40. Adv Mater. 2010 Jan 19;22(3):419-23 - PubMed
  41. Am J Physiol Cell Physiol. 2011 Apr;300(4):C723-42 - PubMed
  42. Chem Sci. 2018 Aug 20;9(40):7835-7842 - PubMed
  43. Elife. 2016 Dec 09;5: - PubMed
  44. Proc Natl Acad Sci U S A. 2013 Feb 5;110(6):2082-7 - PubMed
  45. FASEB J. 2010 Jan;24(1):105-18 - PubMed
  46. ACS Nano. 2014 Jun 24;8(6):6288-96 - PubMed
  47. Development. 2015 Feb 15;142(4):665-71 - PubMed
  48. FASEB J. 2014 Apr;28(4):1805-18 - PubMed
  49. Nat Rev Mol Cell Biol. 2008 Jun;9(6):446-54 - PubMed
  50. Light Sci Appl. 2018 Aug 8;7:47 - PubMed

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