論文 - 詳細
| RRC ID | 18567 |
|---|---|
| 著者 | Kaida A, Miura M. |
| タイトル | Visualizing the effect of hypoxia on fluorescence kinetics in living HeLa cells using the fluorescent ubiquitination-based cell cycle indicator (Fucci). |
| ジャーナル | Exp Cell Res |
| Abstract |
Fluorescent proteins are widely used for the direct visualization of events such as gene expression and subcellular localization in mammalian cells. It is well established that oxygen is required for formation of functional chromophore; however, the effect of hypoxia on fluorescence emission has rarely been studied. For this purpose, under hypoxic conditions, we investigated the kinetics of red and green fluorescence in HeLa cells from two fluorescent proteins, monomeric Kusabira Orange 2 (mKO2) and monomeric Azami Green (mAG), respectively, using the fluorescent ubiquitination-based cell cycle indicator (Fucci). In this system, cells in G1 or other phases emit red or green fluorescence, respectively. We found that hypoxia abrogated both red and green fluorescence about ~10h after the treatment, although their protein levels were almost maintained. The treatment did not significantly affect fluorescence in cells constitutively expressing the same fluorescent proteins lacking the ubiquitin ligase-binding domains. The abrogation of fluorescence resulted from a combination of ubiquitination-dependent degradation of pre-existing functional proteins during specific cell cycle phases, and the expression of newly synthesized non-fluorescent proteins containing non-oxidized chromophore during hypoxic treatment. Indeed, non-fluorescent cells after hypoxic treatment gradually developed fluorescence after reoxygenation in the presence of cycloheximide; kinetics of recovery were much faster for mAG than for mKO2. Using the Fucci system, we could clearly visualize for the first time the effect of hypoxia on the fluorescence kinetics of proteins expressed in living mammalian cells. |
| 巻・号 | 318(3) |
| ページ | 288-97 |
| 公開日 | 2012-2-1 |
| DOI | 10.1016/j.yexcr.2011.10.016 |
| PII | S0014-4827(11)00423-X |
| PMID | 22079518 |
| MeSH | Cell Cycle* / genetics Cell Tracking / methods Flow Cytometry Fluorescence Green Fluorescent Proteins / genetics Green Fluorescent Proteins / metabolism* HeLa Cells Humans Hypoxia / diagnosis* Hypoxia / genetics Hypoxia / metabolism* Indicators and Reagents / pharmacokinetics Kinetics Models, Biological Oxygen Consumption / physiology Transfection Ubiquitination / physiology* |
| IF | 3.383 |
| 引用数 | 19 |
| WOS 分野 | ONCOLOGY CELL BIOLOGY |
| オルトメトリクス指標 |
オルトメトリクス指標項目
|
| 最多言及媒体 | X(Twitter) |
| 各媒体での言及数の合計 | 1 |
| 過去6か月間でのオルトメトリクス指標の変動値 | 0.0 |
| リソース情報 | |
| ヒト・動物細胞 | HeLa/Fucci(RCB2812) |