Visualization of mitochondria in living cells with a genetically encoded yellow fluorescent protein originating from a yellow-emitting luminous bacterium

ABSTRACT Studies with live cells demonstrate that agonist and antagonist rapidly (within minutes) modulate the subnuclear dynamics of estrogen receptor α (ER) and steroid receptor coactivator 1 (SRC-1). A functional cyan fluorescent protein (CFP)-taggedlac repressor-ER chimera (CFP-LacER) was used in live cells to discretely immobilize ER on stably integratedlac operator arrays to study recruitment of yellow fluorescent protein (YFP)-steroid receptor coactivators (YFP–SRC-1 and YFP-CREB binding protein [CBP]). In the absence of ligand, YFP–SRC-1 is found dispersed throughout the nucleoplasm, with a surprisingly high accumulation on the CFP-LacER arrays. Agonist addition results in the rapid (within minutes) recruitment of nucleoplasmic YFP–SRC-1, while antagonist additions diminish YFP–SRC-1–CFP-LacER associations. Less ligand-independent colocalization is observed with CFP-LacER and YFP-CBP, but agonist-induced recruitment occurs within minutes. The agonist-induced recruitment of coactivators requires helix 12 and critical residues in the ER–SRC-1 interaction surface, but not the F, AF-1, or DNA binding domains. Fluorescence recovery after photobleaching indicates that YFP–SRC-1, YFP-CBP, and CFP-LacER complexes undergo rapid (within seconds) molecular exchange even in the presence of an agonist. Taken together, these data suggest a dynamic view of receptor-coregulator interactions that is now amenable to real-time study in living cells.

Download Full-text

Trafficking of Yellow-Fluorescent-Protein-Tagged µ1 Subunit of Clathrin Adaptor AP-1 Complex in Living Cells

Traffic ◽

10.1034/j.1600-0854.2001.25020506.x ◽

2001 ◽

Vol 2 (5) ◽

pp. 345-357 ◽

Cited By ~ 33

Author(s):

Fangtian Huang ◽

Alexandre Nesterov ◽

Royston E. Carter ◽

Alexander Sorkin

Keyword(s):

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Living Cells ◽

Clathrin Adaptor

Download Full-text

Oligomeric Sensor Kinase DcuS in the Membrane of Escherichia coli and in Proteoliposomes: Chemical Cross-linking and FRET Spectroscopy

Journal of Bacteriology ◽

10.1128/jb.00082-10 ◽

2010 ◽

Vol 192 (13) ◽

pp. 3474-3483 ◽

Cited By ~ 24

Author(s):

Patrick D. Scheu ◽

Yun-Feng Liao ◽

Julia Bauer ◽

Holger Kneuper ◽

Thomas Basché ◽

...

Keyword(s):

Escherichia Coli ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Living Cells ◽

Full Length ◽

Cross Linking ◽

Oligomeric State ◽

Bacterial Membranes ◽

Chemical Cross Linking

ABSTRACT DcuS is the membrane-integral sensor histidine kinase of the DcuSR two-component system in Escherichia coli that responds to extracellular C4-dicarboxylates. The oligomeric state of full-length DcuS was investigated in vitro and in living cells by chemical cross-linking and by f luorescence r esonance e nergy t ransfer (FRET) spectroscopy. The FRET results were quantified by an improved method using background-free spectra of living cells for determining FRET efficiency (E) and donor fraction {fD = (donor)/[(donor) + (acceptor)]}. Functional fusions of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) variants of green fluorescent protein to DcuS were used for in vivo FRET measurements. Based on noninteracting membrane proteins and perfectly interacting proteins (a CFP-YFP fusion), the results of FRET of cells coexpressing DcuS-CFP and DcuS-YFP were quantitatively evaluated. In living cells and after reconstitution of purified recombinant DcuS in proteoliposomes, DcuS was found as a dimer or higher oligomer, independent of the presence of an effector. Chemical cross-linking with disuccinimidyl suberate showed tetrameric, in addition to dimeric, DcuS in proteoliposomes and in membranes of bacteria, whereas purified DcuS in nondenaturing detergent was mainly monomeric. The presence and amount of tetrameric DcuS in vivo and in proteoliposomes was not dependent on the concentration of DcuS. Only membrane-embedded DcuS (present in the oligomeric state) is active in (auto)phosphorylation. Overall, the FRET and cross-linking data demonstrate the presence in living cells, in bacterial membranes, and in proteoliposomes of full-length DcuS protein in an oligomeric state, including a tetramer.

Download Full-text

Simultaneous Visualization of the Yellow and Green Forms of the Green Fluorescent Protein in Living Cells

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215549804600911 ◽

1998 ◽

Vol 46 (9) ◽

pp. 1073-1076 ◽

Cited By ~ 18

Author(s):

Chris T. Baumann ◽

Carol S. Lim ◽

Gordon L. Hager

Keyword(s):

Green Fluorescent Protein ◽

Laser Scanning ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Simultaneous Detection ◽

Argon Laser ◽

Living Cells ◽

Left Shoulder ◽

Enhanced Yellow Fluorescent Protein ◽

Green Fluorescent

In this study we sought to develop a method for the co-localization of proteins in living cells utilizing the enhanced green fluorescent protein (EGFP) and a redshifted EGFP variant, EYFP (enhanced yellow fluorescent protein). EYFP was expressed as an unsubstituted molecule while EGFP was fused to NF1 (EGFP-NF1), a transcription factor found exclusively in the nucleus. The Leica TCS SP laser scanning confocal microscope was used. This microscope allows the user to monitor the emitted light at defined wavelengths owing to the presence of a monochrometer in the emission light path. pEGFP-NF1 and pEYFP were co-expressed in the same cell and excited with the 476–nm and 488–nm argon laser lines. To separate the EYFP and EGFP fluorescence, EGFP-NF1 emission was recorded between 496 and 505 nm. These wavelengths are on the left shoulder of the EGFP emission peak and exclude most of the EYFP fluorescence. The EYFP emission was followed between 670 and 754 nm, utilizing the tail of EYFP emission that extends well beyond that for EGFP. Under these conditions we obtained excellent discrimination between EYFP fluorescence and EGFP-NF1 emission. These observations demonstrate that EYFP- and EGFP-substituted chimeras can be used for simultaneous detection in living cells.

Download Full-text

Dynamics and calcium sensitivity of the Ca2+/myristoyl switch protein hippocalcin in living cells

The Journal of Cell Biology ◽

10.1083/jcb.200306042 ◽

2003 ◽

Vol 163 (4) ◽

pp. 715-721 ◽

Cited By ~ 60

Author(s):

Dermott W. O'Callaghan ◽

Alexei V. Tepikin ◽

Robert D. Burgoyne

Keyword(s):

Fluorescent Protein ◽

Dynamic Range ◽

Yellow Fluorescent Protein ◽

Calcium Sensitivity ◽

Living Cells ◽

Enhanced Yellow Fluorescent Protein ◽

Neuronal Calcium Sensor Protein ◽

Narrow Dynamic Range ◽

Ef Hands ◽

Myristoyl Switch

Hippocalcin is a neuronal calcium sensor protein that possesses a Ca2+/myristoyl switch allowing it to translocate to membranes. Translocation of hippocalcin in response to increased cytosolic [Ca2+] was examined in HeLa cells expressing hippocalcin–enhanced yellow fluorescent protein (EYFP) to determine the dynamics and Ca2+ affinity of the Ca2+/myristoyl switch in living cells. Ca2+-free hippocalcin was freely diffusible, as shown by photobleaching and use of a photoactivable GFP construct. The translocation was dependent on binding of Ca2+ by EF-hands 2 and 3. Using photolysis of NP-EGTA, the maximal kinetics of translocation was determined (t1/2 = 0.9 s), and this was consistent with a diffusion driven process. Low intensity photolysis of NP-EGTA produced a slow [Ca2+] ramp and revealed that translocation of hippocalcin–EYFP initiated at around 180 nM and was half maximal at 290 nM. Histamine induced a reversible translocation of hippocalcin–EYFP. The data show that hippocalcin is a sensitive Ca2+ sensor capable of responding to increases in intracellular Ca2+ concentration over the narrow dynamic range of 200–800 nM free Ca2+.

Download Full-text