Resolution of de novo HIV production and trafficking in immature dendritic cells.
AuthorsTurville SG, Aravantinou M, Stössel H, Romani N, Robbiani M,
JournalNat Methods
PubMed ID18059278
'The challenge in observing de novo virus production in human immunodeficiency virus (HIV)-infected dendritic cells (DCs) is the lack of resolution between cytosolic immature and endocytic mature HIV gag protein. To track HIV production, we developed an infectious HIV construct bearing a diothiol-resistant tetracysteine motif (dTCM) at the C terminus ... More
Dynamic fluorescent imaging of human immunodeficiency virus type 1 gag in live cells by biarsenical labeling.
AuthorsRudner L, Nydegger S, Coren LV, Nagashima K, Thali M, Ott DE,
JournalJ Virol
PubMed ID15767407
'Human immunodeficiency virus type 1 (HIV-1) Gag is the primary structural protein of the virus and is sufficient for particle formation. We utilized the recently developed biarsenical-labeling method to dynamically observe HIV-1 Gag within live cells by adding a tetracysteine tag (C-C-P-G-C-C) to the C terminus of Gag in both ... More
Fluorescence imaging of amyloid formation in living cells by a functional, tetracysteine-tagged alpha-synuclein.
'Alpha-synuclein is a major component of intraneuronal protein aggregates constituting a distinctive feature of Parkinson disease. To date, fluorescence imaging of dynamic processes leading to such amyloid deposits in living cells has not been feasible. To address this need, we generated a recombinant alpha-synuclein (alpha-synuclein-C4) bearing a tetracysteine target for ... More
Short tetracysteine tags to beta-tubulin demonstrate the significance of small labels for live cell imaging.
AuthorsAndresen M, Schmitz-Salue R, Jakobs S
JournalMol Biol Cell
PubMed ID15469986
'Genetically encoded tags are of fundamental importance for live cell imaging. We show that small tetracysteine (TetCys) tags can be highly advantageous for the functionality of the host protein compared with large fluorescent protein tags. One to three concatenated small TetCys tags as well as the large green fluorescent protein ... More
Specific covalent labeling of recombinant protein molecules inside live cells.
AuthorsGriffin BA, Adams SR, Tsien RY,
JournalScience
PubMed ID9657724
'Recombinant proteins containing four cysteines at the i, i + 1, i + 4, and i + 5 positions of an alpha helix were fluorescently labeled in living cells by extracellular administration of 4'',5''-bis(1,3, 2-dithioarsolan-2-yl)fluorescein. This designed small ligand is membrane-permeant and nonfluorescent until it binds with high affinity and ... More
Mammalian cell-based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity.
'Membrane-permeant biarsenical dyes such as FlAsH and ReAsH fluoresce upon binding to genetically encoded tetracysteine motifs expressed in living cells, yet spontaneous nonspecific background staining can prevent detection of weakly expressed or dilute proteins. If the affinity of the tetracysteine peptide could be increased, more stringent dithiol washes should increase ... More
Multicolor and electron microscopic imaging of connexin trafficking.
Authors Gaietta Guido; Deerinck Thomas J; Adams Stephen R; Bouwer James; Tour Oded; Laird Dale W; Sosinsky Gina E; Tsien Roger Y; Ellisman Mark H;
JournalScience
PubMed ID11964472
'Recombinant proteins containing tetracysteine tags can be successively labeled in living cells with different colors of biarsenical fluorophores so that older and younger protein molecules can be sharply distinguished by both fluorescence and electron microscopy. Here we used this approach to show that newly synthesized connexin43 was transported predominantly in ... More
Preparation of the membrane-permeant biarsenicals FlAsH-EDT2 and ReAsH-EDT2 for fluorescent labeling of tetracysteine-tagged proteins.
AuthorsAdams SR, Tsien RY,
JournalNat Protoc
PubMed ID18772880
The membrane-permeant fluorogenic biarsenicals FlAsH-EDT(2) and ReAsH-EDT(2) can be prepared in good yields by a straightforward two-step procedure from the inexpensive precursor dyes fluorescein and resorufin, respectively. Handling of toxic reagents such as arsenic trichloride is minimized so the synthesis can be carried out in a typical chemistry laboratory, usually ... More
Fluorescent labeling of recombinant proteins in living cells with FlAsH.
AuthorsGriffin BA, Adams SR, Jones J, Tsien RY,
JournalMethods Enzymol
PubMed ID11045009
This chapter describes methods for site-specific labeling of proteins in living cells based on the well-known affinity of arsenoxides (R-As= 0) for a pair of closely spaced cysteines. To prevent labeling of such endogenous cellular sites (and the associated toxicity), a fluorescein containing two arsenoxides (FlAsH) was designed that ... More
Visualization of mRNA translation in living cells.
The role of mRNA localization is presumably to effect cell asymmetry by synthesizing proteins in specific cellular compartments. However, protein synthesis has never been directly demonstrated at the sites of mRNA localization. To address this, we developed a live cell method for imaging translation of beta-actin mRNA. Constructs coding for ... More
New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications.
AuthorsAdams SR, Campbell RE, Gross LA, Martin BR, Walkup GK, Yao Y, Llopis J, Tsien RY,
JournalJ Am Chem Soc
PubMed ID12022841
We recently introduced a method (Griffin, B. A.; Adams, S. R.; Tsien, R. Y. Science 1998, 281, 269-272 and Griffin, B. A.; Adams, S. R.; Jones, J.; Tsien, R. Y. Methods Enzymol. 2000, 327, 565-578) for site-specific fluorescent labeling of recombinant proteins in living cells. The sequence Cys-Cys-Xaa-Xaa-Cys-Cys, where Xaa ... More
A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells.
Fluorescence resonance energy transfer (FRET) from cyan to yellow fluorescent proteins (CFP/YFP) is a well-established method to monitor protein-protein interactions or conformational changes of individual proteins. But protein functions can be perturbed by fusion of large tags such as CFP and YFP. Here we use G protein-coupled receptor (GPCR) activation ... More
Protein labeling with FlAsH and ReAsH.
AuthorsMachleidt T, Robers M, Hanson GT
JournalMethods Mol Biol
PubMed ID16988405
The ability to image biochemical and phenotypical changes in living cells has become crucial for the investigation and understanding of the molecular mechanisms that govern all physiological cellular functions in health and disease. Genetically encoded reporters derived from fluorescent proteins (FPs) have proved to be extremely useful for localization and ... More
Different mitochondrial intermembrane space proteins are released during apoptosis in a manner that is coordinately initiated but can vary in duration.
AuthorsMuñoz-Pinedo C, Guío-Carrión A, Goldstein JC, Fitzgerald P, Newmeyer DD, Green DR
JournalProc Natl Acad Sci U S A
PubMed ID16864784
The release of mitochondrial intermembrane space proteins to the cytosol is a key event during apoptosis. We used in situ fluorescent labeling of proteins tagged with a short tetracysteine-containing sequence to follow the release of Smac, Omi, adenylate kinase-2, cytochrome c, and apoptosis-inducing factor (AIF) during apoptosis and compared the ... More
Phospholamban pentamer quaternary conformation determined by in-gel fluorescence anisotropy.
AuthorsRobia SL, Flohr NC, Thomas DD
JournalBiochemistry
PubMed ID15766259
We measured in-gel fluorescence anisotropy of phospholamban (PLB) labeled with the biarsenical fluorophore FlAsH at three different sites on the cytoplasmic domain. The 6 kDa monomer bands of FlAsH-tetracysPLB showed high anisotropy (r = 0.29), reflecting null homotransfer and low mobility (S = 0.85) on the nanosecond time scale of ... More
Golgi twins in late mitosis revealed by genetically encoded tags for live cell imaging and correlated electron microscopy.
AuthorsGaietta GM, Giepmans BN, Deerinck TJ, Smith WB, Ngan L, Llopis J, Adams SR, Tsien RY, Ellisman MH
JournalProc Natl Acad Sci U S A
PubMed ID17101980
Combinations of molecular tags visible in light and electron microscopes become particularly advantageous in the analysis of dynamic cellular components like the Golgi apparatus. This organelle disassembles at the onset of mitosis and, after a sequence of poorly understood events, reassembles after cytokinesis. The precise location of Golgi membranes and ... More