Journal Home > Volume 13 , Issue 2
Nano Research 2020, 13(2): 485-495 https://doi.org/10.1007/s12274-020-2633-z
Research Article | Issue | Published: 18 January 2020

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

Show Author's Information Jing Liu1,  Tim Hebbrecht2,  Toon Brans1,  Eef Parthoens3,4,5,  Saskia Lippens3,4,5,  Chengnan Li6,  Herlinde De Keersmaecker1,8,  Winnok H. De Vos7,  Stefaan C. De Smedt1,8,  Rabah Boukherroub6,  Jan Gettemans2,  Ranhua Xiong1,  Kevin Braeckmans1,8( )
Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmacy, Ghent University, Ghent B-9000, Belgium
Department of Biomolecular medicine, Faculty of Medicine and Health Sciences, Ghent University, Ghent B-9000, Belgium
VIB-UGent Center for Inflammation Research, VIB, Ghent B-9000, Belgium
VIB Bioimaging Core Ghent, VIB, Ghent B-9000, Belgium
Department of Biomedical Molecular Biology, Ghent University, Ghent B-9000, Belgium
Univ. Lille, CNRS, Centrale Lille, ISEN, Univ. Valenciennes, UMR 8520-IEMN, Lille F-59000, France
Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, 2020 Antwerp, Belgium
Centre for Advanced Light Microscopy, Ghent University, Ghent B-9000, Belgium
Keywords:
intracellular delivery, laser-induced photoporation, vapor nanobubble, long-term microscopy imaging, nanobody, living cell labeling
Topics:
Cell membranes Cell proliferation Fluorescence microscopyProteins
Cite this article:
Liu J, Hebbrecht T, Brans T, et al. Long-term live-cell microscopy with labeled nanobodies delivered by laser-induced photoporation. Nano Research, 2020, 13(2): 485-495. https://doi.org/10.1007/s12274-020-2633-z
Download citation
EndNote(RIS)
BibTeX

707

Views

22

Downloads

Citations

13

Crossref

N/A

WoS

13

Scopus

1

CSCD

Abstract Full text Electronic supplementary material About this article

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.


menu
Abstract
Full text
Outline
Electronic supplementary material
About this article

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

Show Author's information Jing Liu1, Tim Hebbrecht2, Toon Brans1, Eef Parthoens3,4,5, Saskia Lippens3,4,5, Chengnan Li6, Herlinde De Keersmaecker1,8, Winnok H. De Vos7, Stefaan C. De Smedt1,8, Rabah Boukherroub6, Jan Gettemans2, Ranhua Xiong1, Kevin Braeckmans1,8( )
Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmacy, Ghent University, Ghent B-9000, Belgium
Department of Biomolecular medicine, Faculty of Medicine and Health Sciences, Ghent University, Ghent B-9000, Belgium
VIB-UGent Center for Inflammation Research, VIB, Ghent B-9000, Belgium
VIB Bioimaging Core Ghent, VIB, Ghent B-9000, Belgium
Department of Biomedical Molecular Biology, Ghent University, Ghent B-9000, Belgium
Univ. Lille, CNRS, Centrale Lille, ISEN, Univ. Valenciennes, UMR 8520-IEMN, Lille F-59000, France
Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, 2020 Antwerp, Belgium
Centre for Advanced Light Microscopy, Ghent University, Ghent B-9000, Belgium

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, vapor nanobubble, long-term microscopy imaging, nanobody, living cell labeling

References(54)

[1]
Skylaki, S.; Hilsenbeck, O.; Schroeder, T. Challenges in long-term imaging and quantification of single-cell dynamics. Nat. Biotechnol. 2016, 34, 1137-1144.
DOI Google Scholar
[2]
Lee Snapp, E. Fluorescent proteins: A cell biologist's user guide. Trends Cell Biol. 2009, 19, 649-655.
DOI Google Scholar
[3]
Fernández-Suárez, M.; Ting, A. Y. Fluorescent probes for super-resolution imaging in living cells. Nat. Rev. Mol. Cell Biol. 2008, 9, 929-943.
DOI Google Scholar
[4]
Zheng, Q. S.; Juette, M. F.; Jockusch, S.; Wasserman, M. R.; Zhou, Z.; Altman, R. B.; Blanchard, S. C. Ultra-stable organic fluorophores for single-molecule research. Chem. Soc. Rev. 2014, 43, 1044-1056.
DOI Google Scholar
[5]
Jaiswal, J. K.; Mattoussi, H.; Mauro, J. M.; Simon, S. M. Long-term multiple color imaging of live cells using quantum dot bioconjugates. Nat. Biotechnol. 2003, 21, 47-51.
DOI Google Scholar
[6]
Canton, I.; Massignani, M.; Patikarnmonthon, N.; Chierico, L.; Robertson, J.; Renshaw, S. A.; Warren, N. J.; Madsen, J. P.; Armes, S. P.; Lewis, A. L. et al. Fully synthetic polymer vesicles for intracellular delivery of antibodies in live cells. FASEB J. 2013, 27, 98-108.
DOI Google Scholar
[7]
Lukinavičius, G.; Reymond, L.; D'Este, E.; Masharina, A.; Göttfert, F.; Ta, H.; Güther, A.; Fournier, M.; Rizzo, S.; Waldmann, H. et al. Fluorogenic probes for live-cell imaging of the cytoskeleton. Nat. Methods 2014, 11, 731-733.
DOI Google Scholar
[8]
Lukinavičius, G.; Reymond, L.; Umezawa, K.; Sallin, O.; D’Este, E.; Göttfert, F.; Ta, H.; Hell, S. W.; Urano, Y.; Johnsson, K. Fluorogenic probes for multicolor imaging in living cells. J. Am. Chem. Soc. 2016, 138, 9365-9368.
DOI Google Scholar
[9]
Dalby, B.; Cates, S.; Harris, A.; Ohki, E. C.; Tilkins, M. L.; Price, P. J.; Ciccarone, V. C. Advanced transfection with Lipofectamine 2000 reagent: Primary neurons, siRNA, and high-throughput applications. Methods 2004, 33, 95-103.
DOI Google Scholar
[10]
Longo, P. A.; Kavran, J. M.; Kim, M. S.; Leahy, D. J. Transient mammalian cell transfection with polyethylenimine (PEI). Methods Enzymol. 2013, 529, 227-240.
DOI Google Scholar
[11]
Teng, K. W.; Ishitsuka, Y.; Ren, P.; Youn, Y.; Deng, X.; Ge, P. H.; Lee, S. H.; Belmont, A. S.; Selvin, P. R. Labeling proteins inside living cells using external fluorophores for microscopy. eLife 2016, 5, e20378.
DOI Google Scholar
[12]
Sharei A.; Zoldan J.; Adamo A.; Sim W. Y.; Cho N.; Jackson E.; Mao S., Schneider S., Han M. J., Lytton-Jean, A. et al. A vector-free microfluidic platform for intracellular delivery. Proceedings of the National Academy of Sciences. 2013, 110, 2082-2087.
DOI Google Scholar
[13]
Kollmannsperger, A.; Sharei, A.; Raulf, A.; Heilemann, M.; Langer, R.; Jensen, K. F.; Wieneke, R.; Tampé, R. Live-cell protein labelling with nanometre precision by cell squeezing. Nat. Commun. 2016, 7, 10372.
DOI Google Scholar
[14]
Xu, J. M.; Teslaa, T.; Wu, T. H.; Chiou, P. Y.; Teitell, M. A.; Weiss, S. Nanoblade delivery and incorporation of quantum dot conjugates into tubulin networks in live cells. Nano Lett. 2012, 12, 5669-5672.
DOI Google Scholar
[15]
Xiong, R. H.; Raemdonck, K.; Peynshaert, K.; Lentacker, I.; de Cock, I.; Demeester, J.; de Smedt, S. C.; Skirtach, A. G.; Braeckmans, K. Comparison of gold nanoparticle mediated photoporation: Vapor nanobubbles outperform direct heating for delivering macromolecules in live cells. ACS Nano 2014, 8, 6288-6296.
DOI Google Scholar
[16]
Xiong, R. H.; Joris, F.; Liang, S. Y.; de Rycke, R.; Lippens, S.; Demeester, J.; Skirtach, A.; Raemdonck, K.; Himmelreich, U.; de Smedt, S. C. et al. Cytosolic delivery of nanolabels prevents their asymmetric inheritance and enables extended quantitative in vivo cell imaging. Nano Lett. 2016, 16, 5975-5986.
DOI Google Scholar
[17]
Liu, J.; Xiong, R. H.; Brans, T.; Lippens, S.; Parthoens, E.; Zanacchi, F. C.; Magrassi, R.; Singh, S. K.; Kurungot, S.; Szunerits, S. et al. Repeated photoporation with graphene quantum dots enables homogeneous labeling of live cells with extrinsic markers for fluorescence microscopy. Light Sci. Appl. 2018, 7, 47.
DOI Google Scholar
[18]
Rothbauer, U.; Zolghadr, K.; Tillib, S.; Nowak, D.; Schermelleh, L.; Gahl, A.; Backmann, N.; Conrath, K.; Muyldermans, S.; Cardoso, M. C. et al. Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat. Methods 2006, 3, 887-889.
DOI Google Scholar
[19]
Chakravarty, R.; Goel, S.; Cai, W. B. Nanobody: The “magic bullet” for molecular imaging? Theranostics 2014, 4, 386-389.
DOI Google Scholar
[20]
Ries, J.; Kaplan, C.; Platonova, E.; Eghlidi, H.; Ewers, H. A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Nat. Methods 2012, 9, 582-584.
DOI Google Scholar
[21]
Yamane, D.; Wu, Y. C.; Wu, T. H.; Toshiyoshi, H.; Teitell, M. A.; Chiou, P. Y. Electrical impedance monitoring of photothermal porated mammalian cells. J. Lab. Autom. 2014, 19, 50-59.
DOI Google Scholar
[22]
Kubala, M. H.; Kovtun, O.; Alexandrov, K.; Collins, B. M. Structural and thermodynamic analysis of the GFP: GFP-nanobody complex. Protein Sci. 2010, 19, 2389-2401.
DOI Google Scholar
[23]
Bolte, S.; Cordelières, F. P. A guided tour into subcellular colocalization analysis in light microscopy. J. Microsc. 2006, 224, 213-232.
DOI Google Scholar
[24]
Dunn, K. W.; Kamocka, M. M.; McDonald, J. H. A practical guide to evaluating colocalization in biological microscopy. Am. J. Physiol. Cell Physiol. 2011, 300, C723-C742.
DOI Google Scholar
[25]
Li, Q.; Lau, A.; Morris, T. J.; Guo, L.; Fordyce, C. B.; Stanley, E. F. A syntaxin 1, Gαo, and N-type calcium channel complex at a presynaptic nerve terminal: Analysis by quantitative immunocolocalization. J. Neurosc. 2004, 24, 4070-4081.
DOI Google Scholar
[26]
Chaudhry, A.; Shi, R.; Luciani, D. S. A pipeline for multidimensional confocal analysis of mitochondrial morphology, function and dynamics in pancreatic β-cells. Am. J. Physiol. Endocrinol. Metab., in press, .
DOI Google Scholar
[27]
Hashimoto, Y.; Skacel, M.; Adams, J. C. Roles of fascin in human carcinoma motility and signaling: Prospects for a novel biomarker? Int. J. Biochem. Cell Biol. 2005, 37, 1787-1804.
DOI Google Scholar
[28]
Jayo, A.; Parsons, M. Fascin: A key regulator of cytoskeletal dynamics. Int. J. Biochem. Cell Biol. 2010, 42, 1614-1617.
DOI Google Scholar
[29]
Adams, J. C. Formation of stable microspikes containing actin and the 55 kDa actin bundling protein, fascin, is a consequence of cell adhesion to thrombospondin-1: Implications for the anti-adhesive activities of thrombospondin-1. J. Cell Sci. 1995, 108, 1977-1990.
DOI Google Scholar
[30]
Mattila, P. K.; Lappalainen, P. Filopodia: Molecular architecture and cellular functions. Nat. Rev. Mol. Cell Biol. 2008, 9, 446-454.
DOI Google Scholar
[31]
Adams, J. C. Fascin protrusions in cell interactions. Trends Cardiovasc. Med. 2004, 14, 221-226.
DOI Google Scholar
[32]
Cory, G. Scratch-wound assay. In Cell Migration. Wells, C. M.; Parsons, M., Eds.; Humana Press: New York, 2011; pp 25-30.
DOI
[33]
Allen, T. D.; Cronshaw, J. M.; Bagley, S.; Kiseleva, E.; Goldberg, M. W. The nuclear pore complex: Mediator of translocation between nucleus and cytoplasm. J. Cell Sci. 2000, 113, 1651-1659.
DOI Google Scholar
[34]
Martens, T. F.; Remaut, K.; Demeester, J.; de Smedt, S. C.; Braeckmans, K. Intracellular delivery of nanomaterials: How to catch endosomal escape in the act. Nano Today 2014, 9, 344-364.
DOI Google Scholar
[35]
Gilleron, J.; Querbes, W.; Zeigerer, A.; Borodovsky, A.; Marsico, G.; Schubert, U.; Manygoats, K.; Seifert, S.; Andree, C.; Stöter, M. et al. Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape. Nat. Biotechnol. 2013, 31, 638-646.
DOI Google Scholar
[36]
Berger, S. M.; Pesold, B.; Reber, S.; Schönig, K.; Berger, A. J.; Weidenfeld, I.; Miao, J.; Berger, M. R.; Gruss, O. J.; Bartsch, D. et al. Quantitative analysis of conditional gene inactivation using rationally designed, tetracycline-controlled miRNAs. Nucleic Acids Res. 2010, 38, e168.
DOI Google Scholar
[37]
Klein, A.; Hank, S.; Raulf, A.; Joest, E. F.; Tissen, F.; Heilemann, M.; Wieneke, R.; Tampé, R. Live-cell labeling of endogenous proteins with nanometer precision by transduced nanobodies. Chem. Sci. 2018, 9, 7835-7842.
DOI Google Scholar
[38]
Herce, H. D.; Schumacher, D.; Schneider, A. F. L.; Ludwig, A. K.; Mann, F. A.; Fillies, M.; Kasper, M. A.; Reinke, S.; Krause, E.; Leonhardt, H. et al. Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells. Nat. Chem. 2017, 9, 762-771.
DOI Google Scholar
[39]
Sagar; Pröls, F.; Wiegreffe, C.; Scaal, M. Communication between distant epithelial cells by filopodia-like protrusions during embryonic development. Development 2015, 142, 665-671.
DOI Google Scholar
[40]
Metwally, K.; Mensah, S.; Baffou, G. Fluence threshold for photothermal bubble generation using plasmonic nanoparticles. J. Phys. Chem. C 2015, 119, 28586-28596.
DOI Google Scholar
[41]
Gilje, S.; Dubin, S.; Badakhshan, A.; Farrar, J.; Danczyk, S. A.; Kaner, R. B. Photothermal deoxygenation of graphene oxide for patterning and distributed ignition applications. Adv. Mater. 2010, 22, 419-423.
DOI Google Scholar
[42]
Abdelsayed, V.; Moussa, S.; Hassan, H. M.; Aluri, H. S.; Collinson, M. M.; El-Shall, M. S. Photothermal deoxygenation of graphite oxide with laser excitation in solution and graphene-aided increase in water temperature. J. Phys. Chem. Lett. 2010, 1, 2804-2809.
DOI Google Scholar
[43]
Xiong, R. H.; Drullion, C.; Verstraelen, P.; Demeester, J.; Skirtach, A. G.; Abbadie, C.; de Vos, W. H.; de Smedt, S. C.; Braeckmans, K. Fast spatial-selective delivery into live cells. J. Control. Release 2017, 266, 198-204.
DOI Google Scholar
[44]
Xiong, R. H.; Verstraelen, P.; Demeester, J.; Skirtach, A. G.; Timmermans, J. P.; de Smedt, S. C.; de Vos, W. H.; Braeckmans, K. Selective labeling of individual neurons in dense cultured networks with nanoparticle-enhanced photoporation. Front. Cell. Neurosc. 2018, 12, 80.
DOI Google Scholar
[45]
Wayteck, L.; Xiong, R. H.; Braeckmans, K.; de Smedt, S. C.; Raemdonck, K. Comparing photoporation and nucleofection for delivery of small interfering RNA to cytotoxic T cells. J. Control. Release 2017, 267, 154-162.
DOI Google Scholar
[46]
van Audenhove, I.; Boucherie, C.; Pieters, L.; Zwaenepoel, O.; Vanloo, B.; Martens, E.; Verbrugge, C.; Hassanzadeh-Ghassabeh, G.; Vandekerckhove, J.; Cornelissen, M. et al. Stratifying fascin and cortactin function in invadopodium formation using inhibitory nanobodies and targeted subcellular delocalization. FASEB J. 2014, 28, 1805-1818.
DOI Google Scholar
[47]
Bertier, L.; Hebbrecht, T.; Mettepenningen, E.; de Wit, N.; Zwaenepoel, O.; Verhelle, A.; Gettemans, J. Nanobodies targeting cortactin proline rich, helical and actin binding regions downregulate invadopodium formation and matrix degradation in SCC-61 cancer cells. Biomed. Pharmacother. 2018, 102, 230-241.
DOI Google Scholar
[48]
van den Abbeele, A.; de Clercq, S.; de Ganck, A.; de Corte, V.; van Loo, B.; Soror, S. H.; Srinivasan, V.; Steyaert, J.; Vandekerckhove, J.; Gettemans, J. A llama-derived gelsolin single-domain antibody blocks gelsolin-G-actin interaction. Cell. Mol. Life Sci. 2010, 67, 1519-1535.
DOI Google Scholar
[49]
van Overbeke, W.; Verhelle, A.; Everaert, I.; Zwaenepoel, O.; Vandekerckhove, J.; Cuvelier, C.; Derave, W.; Gettemans, J. Chaperone nanobodies protect gelsolin against MT1-MMP degradation and alleviate amyloid burden in the gelsolin amyloidosis mouse model. Mol. Ther. 2014, 22, 1768-1778.
DOI Google Scholar
[50]
Verhelle, A.; Nair, N.; Everaert, I.; van Overbeke, W.; Supply, L.; Zwaenepoel, O.; Peleman, C.; van Dorpe, J.; Lahoutte, T.; Devoogdt, N. et al. AAV9 delivered bispecific nanobody attenuates amyloid burden in the gelsolin amyloidosis mouse model. Hum. Mol. Genet. 2017, 26, 1353-1364.
DOI Google Scholar
[51]
Delanote, V.; Vanloo, B.; Catillon, M.; Friederich, E.; Vandekerckhove, J.; Gettemans, J. An alpaca single-domain antibody blocks filopodia formation by obstructing L-plastin-mediated F-actin bundling. FASEB J. 2010, 24, 105-118.
DOI Google Scholar
[52]
van Audenhove, I.; Denert, M.; Boucherie, C.; Pieters, L.; Cornelissen, M.; Gettemans, J. Fascin rigidity and L-plastin flexibility cooperate in cancer cell invadopodia and filopodia. J. Biol. Chem. 2016, 291, 9148-9160.
DOI Google Scholar
[53]
van Audenhove, I.; van Impe, K.; Ruano-Gallego, D.; de Clercq, S.; de Muynck, K.; Vanloo, B.; Verstraete, H.; Fernández, L. Á.; Gettemans, J. Mapping cytoskeletal protein function in cells by means of nanobodies. Cytoskeleton 2013, 70, 604-622.
DOI Google Scholar
[54]
Schindelin, J.; Arganda-Carreras, I.; Frise, E.; Kaynig, V.; Longair, M.; Pietzsch, T.; Preibisch, S.; Rueden, C.; Saalfeld, S.; Schmid, B. et al. Fiji: An open-source platform for biological-image analysis. Nat. Methods 2012, 9, 676-682.
DOI Google Scholar
Video
12274_2020_2633_MOESM1_ESM.mp4
12274_2020_2633_MOESM2_ESM.mp4
12274_2020_2633_MOESM3_ESM.mp4
12274_2020_2633_MOESM4_ESM.mp4
12274_2020_2633_MOESM5_ESM.mp4
File
12274_2020_2633_MOESM6_ESM.pdf (1.8 MB)
Publication history
Copyright
Acknowledgements

Publication history

Received: 22 October 2019
Revised: 23 December 2019
Accepted: 01 January 2020
Published: 18 January 2020
Issue date: February 2020

Copyright

© Tsinghua University Press and Springer-Verlag GmbH Germany, part of Springer Nature 2020

Acknowledgements

K. B. acknowledges financial support from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (No. 648124) and from the Ghent University Special Research Fund (No. 01B04912) with gratitude. J. L. gratefully acknowledges the financial support from the China Scholarship Council (CSC) (No. 201506750012) and the Ghent University Special Research Fund (No. 01SC1416). T. H. and J. G. acknowledges financial support from the Fonds Wetenschappelijk Onderzoek (No. G.0559.16N) and Ghent University (BOF-GOA) (No. BOF13/GOA010). We would like to thank the Centre for advanced light microscopy at Ghent University (Belgium) for the support during the data acquisition and analysis of the colocalization experiments. We would like to thank Mr. Dominique Deresmes for helping to perform the AFM imaging.

Return