Single-cell mechanogenetics using monovalent magnetoplasmonic nanoparticles

Ji Wook Kim, Daeha Seo, Jung Uk Lee, Kaden M. Southard, Yongjun Lim, Daehyun Kim, Zev J. Gartner, Young Wook Jun, Jinwoo Cheon

Research output: Contribution to journalArticle

10 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)1871-1889
Number of pages19
JournalNature Protocols
Volume12
Issue number9
DOIs
Publication statusPublished - 2017 Sep 1

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)

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    Kim, J. W., Seo, D., Lee, J. U., Southard, K. M., Lim, Y., Kim, D., Gartner, Z. J., Jun, Y. W., & Cheon, J. (2017). Single-cell mechanogenetics using monovalent magnetoplasmonic nanoparticles. Nature Protocols, 12(9), 1871-1889. https://doi.org/10.1038/nprot.2017.071