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
Identification of an intracellular trafficking and assembly pathway for HIV-1 gag.
AuthorsPerlman M, Resh MD
JournalTraffic
PubMed ID16683918
'Retroviral Gag proteins are membrane-bound polyproteins that are necessary and sufficient for virus-like particle (VLP) formation. It is not known how Gag traffics through the cell or how the site of particle production is determined. Here we use two techniques, biarsenical/tetracysteine (TC) labeling and release from a cycloheximide block, to ... More
Real-time visualization of HIV-1 GAG trafficking in infected macrophages.
AuthorsGousset K, Ablan SD, Coren LV, Ono A, Soheilian F, Nagashima K, Ott DE, Freed EO,
JournalPLoS Pathog
PubMed ID18369466
'HIV-1 particle production is driven by the Gag precursor protein Pr55(Gag). Despite significant progress in defining both the viral and cellular determinants of HIV-1 assembly and release, the trafficking pathway used by Gag to reach its site of assembly in the infected cell remains to be elucidated. The Gag trafficking ... More
Site-specific, orthogonal labeling of proteins in intact cells with two small biarsenical fluorophores.
AuthorsZürn A, Klenk C, Zabel U, Reiner S, Lohse MJ, Hoffmann C,
JournalBioconjug Chem
PubMed ID20429545
'The fusion of fluorescent proteins to proteins of interest has greatly advanced fluorescence microscopy, but is often limited by their large size. Here, we report site-specific, orthogonal labeling of two cellular proteins in intact cells with two small fluorescent dyes: fluorescein arsenical hairpin binder, FlAsH, and its red analogue, ReAsH, ... 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
ReAsH: another viable option for in vivo protein labelling in Dictyostelium.
AuthorsHwang RD, Chen CC, Knecht DA,
JournalJ Microsc
PubMed ID19335452
'Biarsenical-tetracysteine fluorescent protein tagging has been effectively used in a variety of cell types. It has the advantage of requiring a much smaller peptide alteration to existing proteins than fusion to green fluorescent protein (GFP) or monomeric red fluorescent protein (mRFP). However, there are no reports of the tetracysteine tagging ... 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
Biarsenical-tetracysteine motif as a fluorescent tag for detection in capillary electrophoresis.
'Biarsenical dyes complexed to tetracysteine motifs have proven to be highly useful fluorescent dyes in labeling specific cellular proteins for microscopic imaging. Their many advantages include membrane permeability, relatively small size, stoichiometric labeling, high affinity, and an assortment of excitation/emission wavelengths. The goal of the current study was to determine ... 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
Bipartite tetracysteine display requires site flexibility for ReAsH coordination.
AuthorsGoodman JL, Fried DB, Schepartz A,
JournalChembiochem
PubMed ID19533719
Flexibility required: We designed intramolecular bipartite tetracysteine sites in loops of p53 and the beta-sheets of EmGFP. We found that ReAsH binding preferentially favors tetracysteine sites with flexible geometries such as loops; flexibility was assessed by comparing Calpha B-factor values. This information is important for directing successful bipartite tetracysteine site ... More
FlAsH-based live-cell fluorescent imaging of synthetic peptides expressed in Arabidopsis and tobacco.
AuthorsEstévez JM, Somerville C
JournalBiotechniques
PubMed ID17140113
Genes encoding synthetic hydroxyproline-rich peptides with repetitive (Ser-Pro) units linked to an extensin signal sequence and a tetracysteine (TC) sequence were expressed transiently in tobacco, and transiently and stably in Arabidopsis under control of a strong constitutive promoter Expression of these peptides could be visualized in live cells by confocal ... 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
Tracking of human Y receptors in living cells--a fluorescence approach.
AuthorsBöhme I, Mörl K, Bamming D, Meyer C, Beck-Sickinger AG
JournalPeptides
PubMed ID17207557
Non-invasive methods for studying biological processes in living cells have become very important, also in the field of GPCR biochemistry. Great advancements in the application of fluorescence techniques as well as in the development and improvement of novel fluorophores allow the visualization of dynamic processes. Using these technologies, problems concerning ... 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
Studying the dynamics of flagella in multicellular communities of Escherichia coli by using biarsenical dyes.
This paper describes a new approach for labeling intact flagella using the biarsenical dyes FlAsH and ReAsH and imaging their spatial and temporal dynamics on live Escherichia coli cells in swarming communities of bacteria by using epifluorescence microscopy. Using this approach, we observed that (i) bundles of flagella on swarmer ... 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
Hairpin structure of a biarsenical-tetracysteine motif determined by NMR spectroscopy.
The biarsenical-tetracysteine motif is a useful tag for genetic labeling of proteins with small molecules in living cells. The present study concerns the structure of a 12 amino acid peptide FLNCCPGCCMEP bound to the fluorophore ReAsH based on resorufin. (1)H NMR spectroscopy was used to determine the solution structure of ... More
Bridging fluorescence microscopy and electron microscopy.
AuthorsGiepmans BN,
JournalHistochem Cell Biol
PubMed ID18575880
Development of new fluorescent probes and fluorescence microscopes has led to new ways to study cell biology. With the emergence of specialized microscopy units at most universities and research centers, the use of these techniques is well within reach for a broad research community. A major breakthrough in fluorescence microscopy ... More
Surveying polypeptide and protein domain conformation and association with FlAsH and ReAsH.
AuthorsLuedtke NW, Dexter RJ, Fried DB, Schepartz A,
JournalNat Chem Biol
PubMed ID17982447
Recombinant polypeptides and protein domains containing two cysteine pairs located distal in primary sequence but proximal in the native folded or assembled state are labeled selectively in vitro and in mammalian cells using the profluorescent biarsenical reagents FlAsH-EDT2 and ReAsH-EDT2. This strategy, termed bipartite tetracysteine display, enables the detection of ... More
Biarsenical labeling of vesicular stomatitis virus encoding tetracysteine-tagged m protein allows dynamic imaging of m protein and virus uncoating in infected cells.
AuthorsDas SC, Panda D, Nayak D, Pattnaik AK,
JournalJ Virol
PubMed ID19153240
A recombinant vesicular stomatitis virus (VSV-PeGFP-M-MmRFP) encoding enhanced green fluorescent protein fused in frame with P (PeGFP) in place of P and a fusion matrix protein (monomeric red fluorescent protein fused in frame at the carboxy terminus of M [MmRFP]) at the G-L gene junction, in addition to wild-type (wt) ... 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
Fluorescence applications in molecular neurobiology.
AuthorsTaraska JW, Zagotta WN,
JournalNeuron
PubMed ID20434995
Macromolecules drive the complex behavior of neurons. For example, channels and transporters control the movements of ions across membranes, SNAREs direct the fusion of vesicles at the synapse, and motors move cargo throughout the cell. Understanding the structure, assembly, and conformational movements of these and other neuronal proteins is essential ... 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
The class I HLA repertoire of pancreatic islets comprises the nonclassical class Ib antigen HLA-G.
AuthorsCirulli V, Zalatan J, McMaster M, Prinsen R, Salomon DR, Ricordi C, Torbett BE, Meda P, Crisa L
JournalDiabetes
PubMed ID16644675
Selective expression of the human class Ib HLA molecule HLA-G in immunologically protected sites and its function in the inhibition of NK and T-cell effector functions support an important role of this molecule in immunoregulation. Here, we demonstrate that HLA-G is constitutively expressed in the endocrine compartment of the human ... 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
Cytochrome c is released in a single step during apoptosis.
AuthorsGoldstein JC, Muñoz-Pinedo C, Ricci JE, Adams SR, Kelekar A, Schuler M, Tsien RY, Green DR
JournalCell Death Differ
PubMed ID15933725
Release of cytochrome c from mitochondria is a central event in apoptotic signaling. In this study, we utilized a cytochrome c fusion that binds fluorescent biarsenical ligands (cytochrome c-4CYS (cyt. c-4CYS)) as well as cytochrome c-green fluorescent protein (cyt. c-GFP) to measure its release from mitochondria in different cell types ... More
Cellular trafficking of phospholamban and formation of functional sarcoplasmic reticulum during myocyte differentiation.
AuthorsStenoien DL, Knyushko TV, Londono MP, Opresko LK, Mayer MU, Brady ST, Squier TC, Bigelow DJ
JournalAm J Physiol Cell Physiol
PubMed ID17287364
Phospholamban (PLB) associates with the Ca(2+)-ATPase in sarcoplasmic reticulum (SR) membranes to permit the modulation of contraction in response to beta-adrenergic signaling. To understand how coordinated changes in the abundance and intracellular trafficking of PLB and the Ca(2+)-ATPase contribute to the maturation of functional muscle, we measured changes in abundance, ... 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
Mutation of a conserved threonine in the third transmembrane helix of alpha- and beta-connexins creates a dominant-negative closed gap junction channel.
AuthorsBeahm DL, Oshima A, Gaietta GM, Hand GM, Smock AE, Zucker SN, Toloue MM, Chandrasekhar A, Nicholson BJ, Sosinsky GE
JournalJ Biol Chem
PubMed ID16407179
Single site mutations in connexins have provided insights about the influence specific amino acids have on gap junction synthesis, assembly, trafficking, and functionality. We have discovered a single point mutation that eliminates functionality without interfering with gap junction formation. The mutation occurs at a threonine residue located near the cytoplasmic ... More
PERSIA for Direct Fluorescence Measurements of Transcription, Translation, and Enzyme Activity in Cell-Free Systems.
Authors
JournalACS Synth Biol
PubMed ID30920800
Fluorescent labeling of tetracysteine-tagged proteins in intact cells.
Authors
JournalNat Protoc
PubMed ID20885379
Phase-separated condensates of metabolic complexes in living cells: Purinosome and glucosome.