| Chapter title |
Optogenetic Control of Ras/Erk Signaling Using the Phy–PIF System
|
|---|---|
| Chapter number | 1 |
| Book title |
Kinase Signaling Networks
|
| Published in |
Methods in molecular biology, July 2017
|
| DOI | 10.1007/978-1-4939-7154-1_1 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-7152-7, 978-1-4939-7154-1
|
| Authors |
Alexander G. Goglia, Maxwell Z. Wilson, Daniel B. DiGiorno, Jared E. Toettcher |
| Abstract |
The Ras/Erk signaling pathway plays a central role in diverse cellular processes ranging from development to immune cell activation to neural plasticity to cancer. In recent years, this pathway has been widely studied using live-cell fluorescent biosensors, revealing complex Erk dynamics that arise in many cellular contexts. Yet despite these high-resolution tools for measurement, the field has lacked analogous tools for control over Ras/Erk signaling in live cells. Here, we provide detailed methods for one such tool based on the optical control of Ras activity, which we call "Opto-SOS." Expression of the Opto-SOS constructs can be coupled with a live-cell reporter of Erk activity to reveal highly quantitative input-to-output maps of the pathway. Detailed herein are protocols for expressing the Opto-SOS system in cultured cells, purifying the small molecule cofactor necessary for optical stimulation, imaging Erk responses using live-cell microscopy, and processing the imaging data to quantify Ras/Erk signaling dynamics. |
Mendeley readers
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 35 | 100% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Student > Ph. D. Student | 9 | 26% |
| Researcher | 5 | 14% |
| Student > Bachelor | 4 | 11% |
| Professor | 3 | 9% |
| Student > Master | 2 | 6% |
| Other | 5 | 14% |
| Unknown | 7 | 20% |
| Readers by discipline | Count | As % |
|---|---|---|
| Biochemistry, Genetics and Molecular Biology | 15 | 43% |
| Agricultural and Biological Sciences | 3 | 9% |
| Neuroscience | 3 | 9% |
| Engineering | 2 | 6% |
| Chemistry | 1 | 3% |
| Other | 2 | 6% |
| Unknown | 9 | 26% |