Chemogenetic Control of Nanobodies

Abstract

We introduce an engineered nanobody whose affinity to green fluorescent protein (GFP) can be switched on and off with small molecules. By controlling the cellular localization of GFP fusion proteins, the engineered nanobody allows interrogation of their roles in basic biological processes, an approach that should be applicable to numerous previously described GFP fusions. We also outline how the binding affinities of other nanobodies can be controlled by small molecules.

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Fig. 1: Generation of LAMAs from nanobodies and cpDHFR.
Fig. 2: Sequester and release of protein localization in live cells using LAMAs.

Data availability

Plasmids encoding for LAMAs have been deposited on Addgene with accession codes 130704 to 130718 and 136618 to 136635. All requests for the Nup62-mEGFP genome-edited cell line should be directed to J.E. Structures of GFPLAMAF98 and GFPLAMAG97 have been deposited to the PDB with deposition codes 6RUL and 6RUM, respectively. The source data for Figs. 1c–g,j,l and 2e,f,j,k are provided with the paper online. Additional datasets that support the finding of this study are available from the corresponding author upon request.

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Acknowledgements

This work was supported by the Max Planck Society, the École Polytechnique Fédérale de Lausanne and NCCR Chemical Biology. Research in Kräusslich’s group was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) (Projektnummer 240245660) SFB 1129 project 5 (H.-G.K). Research in Ellenberg’s group was supported by the Paul G. Allen Frontiers Group through an Allen Distinguished Investigators Grant to J.E., the National Institutes of Health Common Fund 4D Nucleome Program (grant no. U01 EB021223/U01 DA047728 to J.E.) and the EMBL (S.O., M.K. and J.E.). We thank I. Schlichting for X-ray data collection. Diffraction data were collected at the Swiss Light Source, beamline X10SA, of the Paul Scherrer Institute, Villigen, Switzerland. We thank L. Reymond, J. Broichhagen, B. Mathes and A. Bergner for providing reagents and M. Eguren for valuable discussions.

Author information

Affiliations

Authors

Contributions

H.F. and K.J. designed the study. H.F generated, characterized and applied all LAMAs. M.T. solved the crystal structures of GFPLAMAs. J.H. helped analyze the crystal structures. M.K. generated the NUP62-mEGFP cell line and S.O. performed the NUP62-mEGFP translocation experiments. B.K. helped with generation of stable cell lines with LAMAs. T.G.M. generated stable cells lines of p24LAMA and characterized them. H.-G.K., J.E. and K.J. supervised the work. H.F and K.J. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Kai Johnsson.

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Competing interests

The authors declare no competing interests.

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Peer review information Arunima Singh was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

41592_2020_746_MOESM3_ESM.mov

Reversibility of mito-GFPLAMAF98 with EGFP in live cells; HeLa Kyoto cells expressing mito-GFPLAMAF98 IRES EGFP. TMP (10 µM) in complete media was perfused over the cells at 3:38–17:19 min s. Complete media was perfused over the cells at 23:24–33:42 min s. TMP was again perfused over the cells at 39:49–49:51 min s. Complete media was again perfused over the cells at 00:55:56–1:08:05 h min s. Fluorescence of EGFP was imaged (green). Scale bar, 10 µm.

41592_2020_746_MOESM4_ESM.mov

Reversibility of mito-GFPLAMAG97 with EGFP in live cells. HeLa Kyoto cells expressing mito-GFPLAMAG97 IRES EGFP. TMP (10 µM) in complete media was perfused over the cells at 3:38–17:19 min s. Complete media was perfused over the cells at 48:01–58:03 min s. Fluorescence of EGFP was imaged (green). Scale bar, 10 µm.

41592_2020_746_MOESM5_ESM.mov

Reversibility of Lyn-GFPLAMAF98 with EGFP in live cells. HeLa Kyoto cells expressing Lyn-GFPLAMAF98 IRES EGFP. TMP (10 µM) in complete media was perfused over the cells at 3:38–15:30 min s. Complete media was perfused over the cells at 20:40–28:34 min s. Fluorescence of EGFP was imaged (green). Scale bar, 10 µm.

41592_2020_746_MOESM6_ESM.mov

Reversibility of nuc-GFPLAMAF98 with EGFP in live cells. HeLa Kyoto cells expressing nuc-GFPLAMAF98 IRES EGFP. TMP (10 µM) in complete media was perfused over the cells at 3:38–17:01 min s. Complete media was perfused over the cells at 22:11–31:36 min s. Fluorescence of EGFP was imaged (green). Scale bar, 10 µm.

41592_2020_746_MOESM7_ESM.mov

Mitosis in a genome-edited Mad2L1-EGFP cell line with mito-GFPLAMAF98 in the absence of TMP. Genome-edited Mad2L1-EGFP cells stably expressing SNAP-tagged mito-GFPLAMAF98. TMP (50 µM) was washed out at time point 0. Nucleus (blue), mito-GFPLAMAF98 (magenta) and transmission image (gray). Scale bar, 20 µm.

41592_2020_746_MOESM8_ESM.mov

Mitosis in a genome-edited Mad2L1-EGFP cell line with mito-GFPLAMAF98 in the presence of TMP. Genome-edited Mad2L1-EGFP cells stably expressing SNAP-tagged mito-GFPLAMAF98 and kept in TMP (50 µM) during imaging. Nucleus (blue), mito-GFPLAMAF98 (magenta) and transmission image (gray). Scale bar, 20 µm.

41592_2020_746_MOESM9_ESM.mov

Mitosis in a genome-edited Mad2L1-EGFP cell line with mito-SNAP-tag in the absence of TMP. Genome-edited Mad2L1-EGFP cells stably expressing SNAP-tag on the outer membrane of the mitochondria. TMP (50 µM) was washed out at time point 0. Nucleus (blue), mito-SNAP-tag (magenta) and transmission image (gray). Scale bar, 20 µm.

41592_2020_746_MOESM10_ESM.mov

Mitosis in a genome-edited Mad2L1-EGFP cell line with mito-SNAP-tag in the presence of TMP. Genome-edited Mad2L1-EGFP cells stably expressing SNAP-tag on the outer membrane of the mitochondria and kept in TMP (50 µM) during imaging. Nucleus (blue), mitoSNAP-tag (magenta) and transmission image (gray). Scale bar, 20 µm.

Supplementary Information

Supplementary Figs. 1–19 and Tables 1 and 2.

Reporting Summary

Supplementary Video 1

Reversibility of mito-GFPLAMAF98 with EGFP in live cells; HeLa Kyoto cells expressing mito-GFPLAMAF98 IRES EGFP. TMP (10 µM) in complete media was perfused over the cells at 3:38–17:19 min s. Complete media was perfused over the cells at 23:24–33:42 min s. TMP was again perfused over the cells at 39:49–49:51 min s. Complete media was again perfused over the cells at 00:55:56–1:08:05 h min s. Fluorescence of EGFP was imaged (green). Scale bar, 10 µm.

Supplementary Video 2

Reversibility of mito-GFPLAMAG97 with EGFP in live cells. HeLa Kyoto cells expressing mito-GFPLAMAG97 IRES EGFP. TMP (10 µM) in complete media was perfused over the cells at 3:38–17:19 min s. Complete media was perfused over the cells at 48:01–58:03 min s. Fluorescence of EGFP was imaged (green). Scale bar, 10 µm.

Supplementary Video 3

Reversibility of Lyn-GFPLAMAF98 with EGFP in live cells. HeLa Kyoto cells expressing Lyn-GFPLAMAF98 IRES EGFP. TMP (10 µM) in complete media was perfused over the cells at 3:38–15:30 min s. Complete media was perfused over the cells at 20:40–28:34 min s. Fluorescence of EGFP was imaged (green). Scale bar, 10 µm.

Supplementary Video 4

Reversibility of nuc-GFPLAMAF98 with EGFP in live cells. HeLa Kyoto cells expressing nuc-GFPLAMAF98 IRES EGFP. TMP (10 µM) in complete media was perfused over the cells at 3:38–17:01 min s. Complete media was perfused over the cells at 22:11–31:36 min s. Fluorescence of EGFP was imaged (green). Scale bar, 10 µm.

Supplementary Video 5

Mitosis in a genome-edited Mad2L1-EGFP cell line with mito-GFPLAMAF98 in the absence of TMP. Genome-edited Mad2L1-EGFP cells stably expressing SNAP-tagged mito-GFPLAMAF98. TMP (50 µM) was washed out at time point 0. Nucleus (blue), mito-GFPLAMAF98 (magenta) and transmission image (gray). Scale bar, 20 µm.

Supplementary Video 6

Mitosis in a genome-edited Mad2L1-EGFP cell line with mito-GFPLAMAF98 in the presence of TMP. Genome-edited Mad2L1-EGFP cells stably expressing SNAP-tagged mito-GFPLAMAF98 and kept in TMP (50 µM) during imaging. Nucleus (blue), mito-GFPLAMAF98 (magenta) and transmission image (gray). Scale bar, 20 µm.

Supplementary Video 7

Mitosis in a genome-edited Mad2L1-EGFP cell line with mito-SNAP-tag in the absence of TMP. Genome-edited Mad2L1-EGFP cells stably expressing SNAP-tag on the outer membrane of the mitochondria. TMP (50 µM) was washed out at time point 0. Nucleus (blue), mito-SNAP-tag (magenta) and transmission image (gray). Scale bar, 20 µm.

Supplementary Video 8

Mitosis in a genome-edited Mad2L1-EGFP cell line with mito-SNAP-tag in the presence of TMP. Genome-edited Mad2L1-EGFP cells stably expressing SNAP-tag on the outer membrane of the mitochondria and kept in TMP (50 µM) during imaging. Nucleus (blue), mitoSNAP-tag (magenta) and transmission image (gray). Scale bar, 20 µm.

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Farrants, H., Tarnawski, M., Müller, T.G. et al. Chemogenetic Control of Nanobodies. Nat Methods 17, 279–282 (2020). https://doi.org/10.1038/s41592-020-0746-7

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