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Nat Protoc. 2017 Sep;12(9):1871-1889. doi: 10.1038/nprot.2017.071. Epub 2017 Aug 17.

Single-cell mechanogenetics using monovalent magnetoplasmonic nanoparticles.

Kim JW1,2,3, Seo D4,5, Lee JU1,2,3, Southard KM4,6, Lim Y1,2,3, Kim D1,2,3, Gartner ZJ5,6, Jun YW1,2,4, Cheon J1,2,3.

Author information

1
Center for Nanomedicine, Institute for Basic Science (IBS), Seoul, Republic of Korea.
2
Yonsei-IBS Institute, Yonsei University, Seoul, Republic of Korea.
3
Department of Chemistry, Yonsei University, Seoul, Republic of Korea.
4
Department of Otolaryngology, University of California, San Francisco, San Francisco, California, USA.
5
Department of Emerging Materials Science, Daegu Gyeongbuk Institute of Science &Technology (DGIST), Daegu, Republic of Korea.
6
Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California, USA.

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.

PMID:
28817122
PMCID:
PMC5742362
DOI:
10.1038/nprot.2017.071
[Indexed for MEDLINE]
Free PMC Article

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