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
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
Correlated three-dimensional light and electron microscopy reveals transformation of mitochondria during apoptosis.
AuthorsSun MG, Williams J, Munoz-Pinedo C, Perkins GA, Brown JM, Ellisman MH, Green DR, Frey TG
JournalNat Cell Biol
PubMed ID17721514
'In addition to their role in cellular bioenergetics, mitochondria also initiate common forms of programmed cell death (apoptosis) through the release of proteins such as cytochrome c from the intermembrane and intracristal spaces. The release of these proteins is studied in populations of cells by western blotting mitochondrial and cytoplasmic ... More
Fluorescence anisotropy assay for proteolysis of specifically labeled fusion proteins.
AuthorsBlommel PG, Fox BG
JournalAnal Biochem
PubMed ID15582561
'A cloning method and plasmid vectors that permit fluorescence-anisotropy-based measurement of proteolysis are reported. The recombinant protein substrates produced by this method contain a tetracysteine motif that can be site-specifically labeled with bis-arsenical fluorophore [Science 281 (1998) 269]. Six protein substrates with an N-terminal fusion of the tetracysteine motif and ... 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
Accumulation of FlAsH/Lumio Green in active mitochondria can be reversed by beta-mercaptoethanol for specific staining of tetracysteine-tagged proteins.
AuthorsLanghorst MF, Genisyuerek S, Stuermer CA
JournalHistochem Cell Biol
PubMed ID16395611
'Recent advances in the field of small molecule labels for live cell imaging promise to overcome some of the limitations set by the size of fluorescent proteins. We tested the tetracysteine-biarsenical labeling system in live cell fluorescence microscopy of reggie-1/flotillin-2 in HeLa and N2a cells. In both cell types, the ... 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
Dual-tagging system for the affinity purification of mammalian protein complexes.
AuthorsGiannone RJ, McDonald WH, Hurst GB, Huang Y, Wu J, Liu Y, Wang Y,
JournalBiotechniques
PubMed ID17907572
'Although affinity purification coupled with mass spectrometry (MS) provides a powerful tool to study protein-protein interactions, this strategy has encountered numerous difficulties when adapted to mammalian cells. Here we describe a Gateway-compatible dual-tag affinity purification system that integrates regulatable expression, tetracysteine motifs, and various combinations ofaffinity tags to facilitate the ... More
The endogenous CXXC motif governs the cadmium sensitivity of the renal Na+/glucose co-transporter.
AuthorsXia X, Wang G, Peng Y, Tu MG, Jen J, Fang H
JournalJ Am Soc Nephrol
PubMed ID15829715
'Cadmium (Cd2+) poisoning causes severe renal disorders manifested by defects in reabsorptive transport of various compounds. It is reported here that the renal brush-border membrane Na+/glucose co-transporter-1 (SGLT1) is a molecular target for Cd2+ toxicity. In micromolar concentrations, Cd2+ acted as a noncompetitive, partial inhibitor of methyl-D-glucopyranoside uptake in vesicles ... More
Dynamics of the upper 50-kDa domain of myosin V examined with fluorescence resonance energy transfer.
AuthorsSun M, Oakes JL, Ananthanarayanan SK, Hawley KH, Tsien RY, Adams SR, Yengo CM
JournalJ Biol Chem
PubMed ID16377637
'The upper 50-kDa region of myosin may be critical for coupling between the nucleotide- and actin-binding regions. We introduced a tetracysteine motif in the upper 50-kDa domain (residues 292-297) of myosin V containing a single IQ domain (MV 1IQ), allowing us to label this site with the fluorescein biarscenical hairpin-binding ... More
The potential of nucleic acid repair in functional genomics.
AuthorsRice MC, Czymmek K, Kmiec EB
JournalNat Biotechnol
PubMed ID11283588
'Chimeric RNA/DNA oligonucleotides have been used successfully to correct point and frameshift mutations in cells as well as in animal and plant models. This approach is one of several nucleic acid repair technologies that will help elucidate the function of newly discovered genes. Understanding the mechanisms by which these different ... More
Inhibition of protein aggregation in vitro and in vivo by a natural osmoprotectant.
AuthorsIgnatova Z, Gierasch LM
JournalProc Natl Acad Sci U S A
PubMed ID16899544
'Small organic molecules termed osmolytes are harnessed by a variety of cell types in a wide range of organisms to counter unfavorable physiological conditions that challenge protein stability and function. Using a well characterized reporter system that we developed to allow in vivo observations, we have explored how the osmolyte ... 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
Monitoring protein stability and aggregation in vivo by real-time fluorescent labeling.
AuthorsIgnatova Z, Gierasch LM,
JournalProc Natl Acad Sci U S A
PubMed ID14701904
'In vivo fluorescent labeling of an expressed protein has enabled the observation of its stability and aggregation directly in bacterial cells. Mammalian cellular retinoic acid-binding protein I (CRABP I) was mutated to incorporate in a surface-exposed omega loop the sequence Cys-Cys-Gly-Pro-Cys-Cys, which binds specifically to a biarsenical fluorescein dye (FlAsH). ... 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
The amino-conserved domain of human cytomegalovirus UL80a proteins is required for key interactions during early stages of capsid formation and virus production.
AuthorsLoveland AN, Nguyen NL, Brignole EJ, Gibson W
JournalJ Virol
PubMed ID17079329
'Assembly of many spherical virus capsids is guided by an internal scaffolding protein or group of proteins that are often cleaved and eliminated in connection with maturation and incorporation of the genome. In cytomegalovirus there are at least two proteins that contribute to this scaffolding function; one is the maturational ... 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
Ligand interaction scan: a general method for engineering ligand-sensitive protein alleles.
AuthorsErster O, Eisenstein M, Liscovitch M
JournalNat Methods
PubMed ID17450147
The ligand interaction scan (LIScan) method is a general procedure for engineering small molecule ligand-regulated forms of a protein that is complementary to other 'reverse' genetic and chemical-genetic methods for drug-target validation. It involves insertional mutagenesis by a chemical-genetic 'switch', comprising a genetically encoded peptide module that binds with high ... 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
Bisarsenical labeling of HIV-1 for real-time fluorescence microscopy.
AuthorsArhel NJ, Charneau P,
JournalMethods Mol Biol
PubMed ID19020824
Imaging studies have benefited from the development of a novel technique for non-destructive labeling of proteins within living cells, based on the use of a reagent called FlAsH-EDT2, a bisarsenical derivative of fluorescein capable of binding with high affinity and specificity to a tetracysteine motif in the protein of interest. ... 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
Quantification of real-time Salmonella effector type III secretion kinetics reveals differential secretion rates for SopE2 and SptP.
AuthorsVan Engelenburg SB, Palmer AE,
JournalChem Biol
PubMed ID18559272
Gram-negative pathogenic bacteria such as Salmonella utilize the type III secretion system to inject bacterial effector proteins into a host cell. Upon entry, these effectors bind mammalian cell proteins, hijack cellular signaling pathways, and redirect cellular function, thus enabling bacterial infection. In this study we use the FlAsH/tetracysteine labeling system ... 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
Genetically engineered, biarsenically labeled influenza virus allows visualization of viral NS1 protein in living cells.
AuthorsLi Y, Lu X, Li J, Bérubé N, Giest KL, Liu Q, Anderson DH, Zhou Y,
JournalJ Virol
PubMed ID20463066
Real-time fluorescence imaging of viral proteins in living cells provides a valuable means to study virus-host interactions. The challenge of generating replication-competent fluorescent influenza A virus is that the segmented genome does not allow fusion of a fluorescent protein gene to any viral gene. Here, we introduced the tetracysteine (TC) ... 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
Structure and conformational changes in the C-terminal domain of the beta2-adrenoceptor: insights from fluorescence resonance energy transfer studies.
The C terminus of the beta(2)-adrenoceptor (AR) interacts with G protein-coupled receptor kinases and arrestins in an agonist-dependent manner, suggesting that conformational changes induced by ligands in the transmembrane domains are transmitted to the C terminus. We used fluorescence resonance energy transfer (FRET) to examine ligand-induced structural changes in the ... More
Specific biarsenical labeling of cell surface proteins allows fluorescent- and biotin-tagging of amyloid precursor protein and prion proteins.
AuthorsTaguchi Y, Shi ZD, Ruddy B, Dorward DW, Greene L, Baron GS,
JournalMol Biol Cell
PubMed ID18987338
Fluorescent tagging is a powerful tool for imaging proteins in living cells. However, the steric effects imposed by fluorescent tags impair the behavior of many proteins. Here, we report a novel technique, Instant with DTT, EDT, And Low temperature (IDEAL)-labeling, for rapid and specific FlAsH-labeling of tetracysteine-tagged cell surface proteins ... 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
Detection of tetracysteine-tagged proteins using a biarsenical fluorescein derivative through dry microplate array gel electrophoresis.
AuthorsFeldman G, Bogoev R, Shevirov J, Sartiel A, Margalit I,
JournalElectrophoresis
PubMed ID15300761
The design of an extended-run 96-well sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system and the development of protein detection technology based upon fluorescein derivatives that bind to peptide epitope tags, allows the creation of a truly high-throughput analysis of protein expression, where less than 20 min are needed to separate ... 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
Secretion of type III effectors into host cells in real time.
AuthorsEnninga J, Mounier J, Sansonetti P, Tran Van Nhieu G
JournalNat Methods
PubMed ID16299482
Type III secretion (T3S) systems are key features of many gram-negative bacteria that translocate T3S effector proteins directly into eukaryotic cells. There, T3S effectors exert many effects, such as cellular invasion or modulation of host immune responses. Studying spatiotemporal orchestrated secretion of various effectors has been difficult without disrupting their ... More
Homogeneous stalled ribosome nascent chain complexes produced in vivo or in vitro.
AuthorsEvans MS, Ugrinov KG, Frese MA, Clark PL
JournalNat Methods
PubMed ID16179922
Cotranslational protein maturation is often studied in cell-free translation mixtures, using stalled ribosome-nascent chain complexes produced by translating truncated mRNA. This approach has two limitations: (i) it can be technically challenging, and (ii) it only works in vitro, where the concentrations of cellular components differ from concentrations in vivo. We ... 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
Quantitative four-dimensional tracking of cytoplasmic and nuclear HIV-1 complexes.
AuthorsArhel N, Genovesio A, Kim KA, Miko S, Perret E, Olivo-Marin JC, Shorte S, Charneau P
JournalNat Methods
PubMed ID16990814
Emerging real-time techniques for imaging viral infections provide powerful tools for understanding the dynamics of virus-host cell interactions. Here we labeled human immunodeficiency virus-1 (HIV-1) integrase with a small tetracysteine tag, which preserved the virus' infectivity while allowing it to be labeled with the bis-arsenical fluorescein derivative FlAsH. This labeling ... 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
Revealing two-state protein-protein interactions of calmodulin by single-molecule spectroscopy.
AuthorsLiu R, Hu D, Tan X, Lu HP
JournalJ Am Chem Soc
PubMed ID16881631
We report a single-molecule fluorescence resonance energy transfer (FRET) and polarization study of conformational dynamics of calmodulin (CaM) interacting with a target peptide, C28W of a 28 amino acid oligomer. The C28W peptide represents the essential binding sequence domain of the Ca-ATPase protein interacting with CaM, which is important in ... More
From the test tube to the cell: exploring the folding and aggregation of a beta-clam protein.
A crucial challenge in present biomedical research is the elucidation of how fundamental processes like protein folding and aggregation occur in the complex environment of the cell. Many new physico-chemical factors like crowding and confinement must be considered, and immense technical hurdles must be overcome in order to explore these ... 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
Oscillations of cyclic AMP in hormone-stimulated insulin-secreting beta-cells.
AuthorsDyachok O, Isakov Y, Sågetorp J, Tengholm A
JournalNature
PubMed ID16421574
Cyclic AMP is a ubiquitous second messenger that transduces signals from a variety of cell surface receptors to regulate diverse cellular functions, including secretion, metabolism and gene transcription. In pancreatic beta-cells, cAMP potentiates Ca2+-dependent exocytosis and mediates the stimulation of insulin release exerted by the hormones glucagon and glucagon-like peptide-1 ... 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
Extended polyglutamine tracts cause aggregation and structural perturbation of an adjacent beta barrel protein.
AuthorsIgnatova Z, Gierasch LM
JournalJ Biol Chem
PubMed ID16524881
Formation of fibrillar intranuclear inclusions and related neuropathologies of the CAG-repeat disorders are linked to the expansion of a polyglutamine tract. Despite considerable effort, the etiology of these devastating diseases remains unclear. Although polypeptides with glutamine tracts recapitulate many of the observed characteristics of the gene products with CAG repeats, ... 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
Flippase-mediated phospholipid asymmetry promotes fast Cdc42 recycling in dynamic maintenance of cell polarity.
Authors
JournalNat Cell Biol
PubMed ID22344035
Live Visualization of Hemagglutinin Dynamics During Infection by Using a Novel Reporter Influenza A Virus.
Authors
JournalViruses
PubMed ID32604762
Homocysteine directly interacts and activates the angiotensin II type I receptor to aggravate vascular injury.
Authors
JournalNat Commun
PubMed ID29296021
The cargo adapter protein CLINT1 is phosphorylated by the Numb-associated kinase BIKE and mediates dengue virus infection.
Authors
JournalJ Biol Chem
PubMed ID35452674
Incomplete influenza A virus genomes occur frequently but are readily complemented during localized viral spread.
Authors
JournalNat Commun
PubMed ID31387995
Engineered kinesin motor proteins amenable to small-molecule inhibition.
Authors
JournalNat Commun
PubMed ID27045608
Conformational Profiling of the AT1 Angiotensin II Receptor Reflects Biased Agonism, G Protein Coupling, and Cellular Context.
Authors
JournalJ Biol Chem
PubMed ID28213525
An ionic lock and a hydrophobic zipper mediate the coupling between an insect pheromone receptor BmOR3 and downstream effectors.
Authors
JournalJ Biol Chem
PubMed ID34480896
A sensitive assay reveals structural requirements for α-synuclein fibril growth.
Authors
JournalJ Biol Chem
PubMed ID28373279
Fluorescent labeling of tetracysteine-tagged proteins in intact cells.