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.
|Number of pages||19|
|Publication status||Published - 2017 Sept 1|
Bibliographical noteFunding Information:
acknowleDGMents 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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All Science Journal Classification (ASJC) codes
- Biochemistry, Genetics and Molecular Biology(all)