期刊
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
卷 487, 期 3, 页码 594-599出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2017.04.097
关键词
Cyclic AMP; Protein kinase activity; Fluorescence resonance energy transfer; Live cell imaging; Yeast
资金
- FAR grants
- Program Sys-BioNet, Italian Roadmap Research Infrastructure grant
- University of Milano-Bicocca
In Saccharomyces cerevisiae the second messenger cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) play a central role in metabolism regulation, stress resistance and cell cycle progression. To monitor cAMP levels and PKA activity in vivo in single S. cerevisiae cells, we expressed an Epac-based FRET probe and a FRET-based A-kinase activity reporter, which were proven to be useful live-cell bio-sensors for CAMP levels and PKA activity in mammalian cells. Regarding detection of cAMP in single yeast cells, we show that in wild type strains the CFP/YFP fluorescence ratio increased immediately after glucose addition to derepressed cells, while no changes were observed when glucose was added to a strain that is not able to produce CAMP. In addition, we had evidence for damped oscillations in cAMP levels at least in SP1 strain. Regarding detection of PKA activity, we show that in wild type strains the FRET increased after glucose addition to derepressed cells, while no changes were observed when glucose was added to either a strain that is not able to produce CAMP or to a strain with absent PKA activity. Taken together these probes are useful to follow activation of the cAMP/PICA pathway in single yeast cells and for long times (up to one hour). (C) 2017 Elsevier Inc. All rights reserved.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据