An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation
| Autor: | Hsien-Ming Lee, Yu-Ju Chen, Hua-De Gao, Yu-Hsu Chang, Pounraj Thanasekaran, Tzu-Ho Chen |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
Photolysis
General Immunology and Microbiology Chemistry General Chemical Engineering General Neuroscience Nanoparticle Bioengineering Photon upconversion General Biochemistry Genetics and Molecular Biology Cell biology Intracellular signal transduction Fluorometer Fluorescence microscope Nanoparticles Signal transduction Phototoxicity Protein kinase A Signal Transduction |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| DOI: | 10.3791/55769-v |
| Popis: | Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided. |
| Databáze: | OpenAIRE |
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