| Chapter title |
Optogenetics: optical control of a photoactivatable rac in living cells.
|
|---|---|
| Chapter number | 15 |
| Book title |
Advanced Fluorescence Microscopy
|
| Published in |
Methods in molecular biology, October 2014
|
| DOI | 10.1007/978-1-4939-2080-8_15 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-2079-2, 978-1-4939-2080-8
|
| Authors |
Yin T, Wu YI, Taofei Yin, Yi I. Wu |
| Editors |
Peter J. Verveer |
| Abstract |
Recent developments in optogenetics have extended optical control of signaling to intracellular proteins, including Rac, a small G protein in the Rho family. A blue light-sensing LOV (light, oxygen, or voltage) domain derived from Avena sativa (oat) phototropin was fused to the N-terminus of a constitutively active mutant of Rac, via an α-helix (Jα) that is conserved among plant phototropins. The fused LOV domain occluded binding of downstream effectors to Rac in the dark. Exposure to blue light caused a conformational change of the LOV domain and unwinding of the Jα helix, relieving steric inhibition. The LOV domain incorporates a flavin as the photon-absorbing cofactor and can be activated by light in a reversible and repeatable fashion. In cultured cells, global illumination with blue light rapidly activated Rac and led to cell spreading and membrane ruffling. Localized and pulsed illumination generated a gradient of Rac activity and induced directional migration. In this chapter, we will describe the techniques in detail and present some examples of applications of using photoactivatable Rac (PA-Rac) in living cells. |
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Mendeley readers
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|---|---|---|
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| Student > Ph. D. Student | 4 | 22% |
| Student > Doctoral Student | 2 | 11% |
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| Student > Master | 2 | 11% |
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| Physics and Astronomy | 1 | 6% |
| Other | 2 | 11% |
| Unknown | 2 | 11% |