Dynamic copper(I) imaging in mammalian cells with a genetically encoded fluorescent copper(I) sensor.
Copper, a key cofactor for many life processes, is toxic at elevated levels, and its availability is strictly controlled inside cells. Therefore, it is a challenge to visualize copper availability in the tight copper-binding environment of the cell. We report a genetically encoded fluorescent copper(I) sensor based on the copper(I)-binding-induced conformational change of a copper-responsive transcriptional regulator, Amt1. The resulting reporter, Amt1-FRET, is ratiometric, highly sensitive (K(d) = 2.5 x 10(-18) M), and selective toward copper(I). Its measured high affinity to copper(I) confirms the extremely low copper availability in yeast since Amt1 senses the upper limit of cellular copper levels in yeast and activates copper detoxification genes. Amt1-FRET operates in the dynamic range of the cellular copper buffer in mammalian cells and can report dynamic fluctuations of the cellular copper availability within minutes of perturbation. Thus, Amt1-FRET visualizes the tightly controlled copper availability in mammalian cells.
Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.
Seraphine V
SV
Wegner
Hasan
H
Arslan
Murat
M
Sunbul
Jun
J
Yin
Chuan
C
He
D000818:Animals; D016466:CHO Cells; D003300:Copper / Q000032:analysis* / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D014157:Transcription Factors; C069807:AMT1 protein, Candida glabrate; D003300:Copper