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Single-cell mechanogenetics using monovalent magnetoplasmonic nanoparticles

Nature Protocols volume 12, pages 1871–1889 (2017)Cite this article

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Abstract

Spatiotemporal interrogation of signal transduction at the single-cell level is necessary to answer a host of important biological questions. This protocol describes a nanotechnology-based single-cell and single-molecule perturbation tool, termed mechanogenetics, that enables precise spatial and mechanical control over genetically encoded cell-surface receptors in live cells. The key components of this tool are a magnetoplasmonic nanoparticle (MPN) actuator that delivers defined spatial and mechanical cues to receptors through target-specific one-to-one engagement and a micromagnetic tweezers (μMT) that remotely controls the magnitude of force exerted on a single MPN. In our approach, a SNAP-tagged cell-surface receptor of interest is conjugated with a single-stranded DNA oligonucleotide, which hybridizes to its complementary oligonucleotide on the MPN. This protocol consists of four major stages: (i) chemical synthesis of MPNs, (ii) conjugation with DNA and purification of monovalent MPNs, (iii) modular targeting of MPNs to cell-surface receptors, and (iv) control of spatial and mechanical properties of targeted mechanosensitive receptors in live cells by adjusting the μMT-to-MPN distance. Using benzylguanine (BG)-functionalized MPNs and model cell lines expressing either SNAP-tagged Notch or vascular endothelial cadherin (VE-cadherin), we provide stepwise instructions for mechanogenetic control of receptor clustering and for mechanical receptor activation. The ability of this method to differentially control spatial and mechanical inputs to targeted receptors makes it particularly useful for interrogating the differential contributions of each individual cue to cell signaling. The entire procedure takes up to 1 week.

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Figure 1: Schematic illustration of magnetoplasmonic nanoparticles (MPNs).
Figure 2: Schematic workflow of the protocol (left) and key design principles (right).
Figure 3: Growth kinetics of MPNs.
Figure 4: Controlled synthesis of MPNs.
Figure 5: Monovalent DNA conjugation of MPNs.
Figure 6: Dark-field microscope and micro-magnetic tweezer (μMT) setup.
Figure 7: Distance-dependent force generation of MPNs.
Figure 8
Figure 9: Mechanogenetic control.

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Acknowledgements

We thank S. Blacklow (Harvard University) for the generous gift of Notch plasmids. This work was supported by IBS-R026-D1 from IBS (Y.J. and J.C.), HI08C2149 from Korea Healthcare Technology R&D Project (J.C.), 2017R1C1B2010945 from a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP, D.S.), 1R01GM112081-01 from the National Institute of General Medical Science (NIGMS) and the National Institute of Health (NIH) (Y.J.), the UCSF Program for Breakthrough Biomedical Research–Sandler Foundation (Y.J.), 1R21HL123329-01 from the National Heart, Lung, and Blood Institute and NIH (Y.J.), DP2 HD080351-01 from the NIH common fund (Z.J.G.) and P50 GM081879 from the NIGMS UCSF Center for Synthetic and Systems Biology (Z.J.G.).

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Author notes

  1. Ji-wook Kim, Daeha Seo, Jung-uk Lee and Young-wook Jun: These authors contributed equally to this work.

Authors and Affiliations

  1. Center for Nanomedicine, Institute for Basic Science (IBS), Seoul, Republic of Korea

    Ji-wook Kim, Jung-uk Lee, Yongjun Lim, Daehyun Kim, Young-wook Jun & Jinwoo Cheon

  2. Yonsei-IBS Institute, Yonsei University, Seoul, Republic of Korea

    Ji-wook Kim, Jung-uk Lee, Yongjun Lim, Daehyun Kim, Young-wook Jun & Jinwoo Cheon

  3. Department of Chemistry, Yonsei University, Seoul, Republic of Korea

    Ji-wook Kim, Jung-uk Lee, Yongjun Lim, Daehyun Kim & Jinwoo Cheon

  4. Department of Otolaryngology, University of California, San Francisco, San Francisco, California, USA

    Daeha Seo, Kaden M Southard & Young-wook Jun

  5. Department of Emerging Materials Science, Daegu Gyeongbuk Institute of Science & Technology (DGIST), Daegu, Republic of Korea

    Daeha Seo & Zev J Gartner

  6. Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California, USA

    Kaden M Southard & Zev J Gartner

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Contributions

J.K., D.S., Y.J. and J.C. conceived and designed the project; D.S., J.K., J.L., Y.L., D.K., Y.J. and J.C. contributed to nanoparticle synthesis; D.S., K.M.S., Z.J.G. and Y.J. designed the biological experiments; J.K., D.S., J.L., K.M.S., Y.L. and D.K. performed experiments and analyzed the data; and J.K., D.S., J.L., Y.J. and J.C. wrote the manuscript. All authors discussed and commented on the manuscript.

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Correspondence to Young-wook Jun or Jinwoo Cheon.

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Integrated supplementary information

Supplementary Figure 1 Functional group-dependent chemical stability of M-SiO2(Au2nm)n.

Reaction of Au2nm seeds with 20 nm APTMS or AEAPTMS functionalized M-SiO2 yielded 161±11 and 165±10 Au seeds per M-SiO2, respectively. The chemical stability of the respective conjugates was examined by incubating them in 1 mM Tris-HCl buffer (pH 8.0) for 48 hr. Significant portion of Au seeds were detached from M-SiO2(Au2nm)n (Au seeds per M-SiO2 after 48hr incubation: 98.1±17) produced by the APTMS conjugation chemistry, whereas negligible changes were observed for the AEAPTMS case (n = 161±15). Scale bar, 10 nm.

Supplementary Figure 2 Synthesis of MPNs having a 30 nm iron oxide magnetic core.

(a) TEM images of 30 nm iron oxide magnetic nanoparticles, (b) silica-coated magnetic nanoparticles (silica thickness, 4 nm), and (c) MPNs (diameter, 55 ± 6.7 nm). 30 nm iron oxide nanoparticles are used in step 14. Scale bar, 50 nm.

Supplementary Figure 3 Time dependent changes in absorbance of the gold growth solution.

(a) UV-Vis absorption spectra and (b) changes in absorbance at 290 nm of the gold growth solution as a function of time.

Supplementary information

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Supplementary Figures 1–3 (PDF 392 kb)

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Kim, Jw., Seo, D., Lee, Ju. et al. Single-cell mechanogenetics using monovalent magnetoplasmonic nanoparticles. Nat Protoc 12, 1871–1889 (2017). https://doi.org/10.1038/nprot.2017.071

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