Journal
NATURE PROTOCOLS
Volume 12, Issue 9, Pages 1871-1889Publisher
NATURE PORTFOLIO
DOI: 10.1038/nprot.2017.071
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Funding
- IBS [IBS-R026-D1]
- Korea Healthcare Technology RD Project [HI08C2149]
- National Research Foundation of Korea (NRF) - Korean government (MSIP) [2017R1C1B2010945]
- National Institute of General Medical Science (NIGMS) [1R01GM112081-01]
- National Institute of Health (NIH)
- UCSF Program for Breakthrough Biomedical Research-Sandler Foundation
- National Heart, Lung, and Blood Institute and NIH [1R21HL123329-01]
- NIH [DP2 HD080351-01]
- NIGMS UCSF [P50 GM081879]
- National Research Foundation of Korea [2017R1C1B2010945] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
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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 (mu MT) 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 mu MT-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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