Document Detail


Visualizing the effect of hypoxia on fluorescence kinetics in living HeLa cells using the fluorescent ubiquitination-based cell cycle indicator (Fucci).
MedLine Citation:
PMID:  22079518     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
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.
Authors:
Atsushi Kaida; Masahiko Miura
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2011-11-3
Journal Detail:
Title:  Experimental cell research     Volume:  -     ISSN:  1090-2422     ISO Abbreviation:  -     Publication Date:  2011 Nov 
Date Detail:
Created Date:  2011-11-14     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0373226     Medline TA:  Exp Cell Res     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Copyright Information:
Copyright © 2011. Published by Elsevier Inc.
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