| Autor: |
Shang Yang, Oana M. Constantin, Divya Sachidanandan, Hannes Hofmann, Tobias C. Kunz, Vera Kozjak-Pavlovic, Thomas G. Oertner, Georg Nagel, Robert J. Kittel, Christine E. Gee, Shiqiang Gao |
| Jazyk: |
angličtina |
| Rok vydání: |
2021 |
| Předmět: |
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| Zdroj: |
BMC Biology, Vol 19, Iss 1, Pp 1-17 (2021) |
| Druh dokumentu: |
article |
| ISSN: |
1741-7007 |
| DOI: |
10.1186/s12915-021-01151-9 |
| Popis: |
Abstract Background Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger that transduces extracellular signals in virtually all eukaryotic cells. The soluble Beggiatoa photoactivatable adenylyl cyclase (bPAC) rapidly raises cAMP in blue light and has been used to study cAMP signaling pathways cell-autonomously. But low activity in the dark might raise resting cAMP in cells expressing bPAC, and most eukaryotic cyclases are membrane-targeted rather than soluble. Our aim was to engineer a plasma membrane-anchored PAC with no dark activity (i.e., no cAMP accumulation in the dark) that rapidly increases cAMP when illuminated. Results Using a streamlined method based on expression in Xenopus oocytes, we compared natural PACs and confirmed bPAC as the best starting point for protein engineering efforts. We identified several modifications that reduce bPAC dark activity. Mutating a phenylalanine to tyrosine at residue 198 substantially decreased dark cyclase activity, which increased 7000-fold when illuminated. Whereas Drosophila larvae expressing bPAC in mechanosensory neurons show nocifensive-like behavior even in the dark, larvae expressing improved soluble (e.g., bPAC(R278A)) and membrane-anchored PACs exhibited nocifensive responses only when illuminated. The plasma membrane-anchored PAC (PACmn) had an undetectable dark activity which increased >4000-fold in the light. PACmn does not raise resting cAMP nor, when expressed in hippocampal neurons, affect cAMP-dependent kinase (PKA) activity in the dark, but rapidly and reversibly increases cAMP and PKA activity in the soma and dendrites upon illumination. The peak responses to brief (2 s) light flashes exceed the responses to forskolin-induced activation of endogenous cyclases and return to baseline within seconds (cAMP) or ~10 min (PKA). Conclusions PACmn is a valuable optogenetic tool for precise cell-autonomous and transient stimulation of cAMP signaling pathways in diverse cell types. |
| Databáze: |
Directory of Open Access Journals |
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