Live-cell measurements of kinase activity in single cells using translocation reporters
| Autor: | Derek N. Macklin, Jacob J. Hughey, Sajia Akhter, Markus W. Covert, Takamasa Kudo, Sergi Regot, Stevan Jeknić |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Cytoplasm Recombinant Fusion Proteins Nuclear Localization Signals General Biochemistry Genetics and Molecular Biology 03 medical and health sciences Genes Reporter Image Processing Computer-Assisted Humans Kinase activity Phosphorylation Nuclear export signal Cell Nucleus Kinase Chemistry HEK 293 cells Phosphotransferases Cell biology Molecular Imaging Luminescent Proteins 030104 developmental biology HEK293 Cells Nuclear transport Single-Cell Analysis Nuclear localization sequence |
| Zdroj: | Nature protocols. 13(1) |
| ISSN: | 1750-2799 |
| Popis: | Although kinases are important regulators of many cellular processes, measuring their activity in live cells remains challenging. We have developed kinase translocation reporters (KTRs), which enable multiplexed measurements of the dynamics of kinase activity at a single-cell level. These KTRs are composed of an engineered construct in which a kinase substrate is fused to a bipartite nuclear localization signal (bNLS) and nuclear export signal (NES), as well as to a fluorescent protein for microscopy-based detection of its localization. The negative charge introduced by phosphorylation of the substrate is used to directly modulate nuclear import and export, thereby regulating the reporter's distribution between the cytoplasm and nucleus. The relative cytoplasmic versus nuclear fluorescence of the KTR construct (the C/N ratio) is used as a proxy for the kinase activity in living, single cells. Multiple KTRs can be studied in the same cell by fusing them to different fluorescent proteins. Here, we present a protocol to execute and analyze live-cell microscopy experiments using KTRs. We describe strategies for development of new KTRs and procedures for lentiviral expression of KTRs in a cell line of choice. Cells are then plated in a 96-well plate, from which multichannel fluorescent images are acquired with automated time-lapse microscopy. We provide detailed guidance for a computational analysis and parameterization pipeline. The entire procedure, from virus production to data analysis, can be completed in ∼10 d. |
| Databáze: | OpenAIRE |
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