| RRC ID |
82131
|
| 著者 |
Ohta Y, Furuta T, Nagai T, Horikawa K.
|
| タイトル |
Red fluorescent cAMP indicator with increased affinity and expanded dynamic range.
|
| ジャーナル |
Sci Rep
|
| Abstract |
cAMP is one of the most important second messengers in biological processes. Cellular dynamics of cAMP have been investigated using a series of fluorescent indicators; however, their sensitivity was sub-optimal for detecting cAMP dynamics at a low concentration range, due to a low ligand affinity and/or poor dynamic range. Seeking an indicator with improved detection sensitivity, we performed insertion screening of circularly permuted mApple, a red fluorescent protein, into the cAMP-binding motif of PKA regulatory subunit Iα and developed an improved cAMP indicator named R-FlincA (Red Fluorescent indicator for cAMP). Its increased affinity (Kd = 0.3 μM) and expanded dynamic range (860% at pH 7.2) allowed the detection of subtle changes in the cellular cAMP dynamics at sub-μM concentrations, which could not be easily observed with existing indicators. Increased detection sensitivity also strengthened the advantages of using R-FlincA as a red fluorescent indicator, as it permits a series of applications, including multi-channel/function imaging of multiple second messengers and combinatorial imaging with photo-manipulation. These results strongly suggest that R-FlincA is a promising tool that accelerates cAMP research by revealing unobserved cAMP dynamics at a low concentration range.
|
| 巻・号 |
8(1)
|
| ページ |
1866
|
| 公開日 |
2018-1-30
|
| DOI |
10.1038/s41598-018-20251-1
|
| PII |
10.1038/s41598-018-20251-1
|
| PMID |
29382930
|
| PMC |
PMC5789972
|
| MeSH |
Calcium / metabolism
Cells, Cultured
Cyclic AMP / metabolism*
Dictyostelium / metabolism*
Fluorescent Dyes / chemistry*
Humans
Insulin-Secreting Cells / metabolism*
Luminescent Proteins / metabolism*
Molecular Imaging / methods*
Red Fluorescent Protein
Spectrometry, Fluorescence
|
| IF |
3.998
|
| リソース情報 |
| ヒト・動物細胞 |
293T(RCB2202) |
