Ubiquitination screen using protein microarrays for comprehensive identification of Rsp5 substrates in yeast.
AuthorsGupta R,Kus B,Fladd C,Wasmuth J,Tonikian R,Sidhu S,Krogan NJ,Parkinson J,Rotin D
JournalMolecular systems biology
PubMed ID17551511
Ubiquitin-protein ligases (E3s) are responsible for target recognition and regulate stability, localization or function of their substrates. However, the substrates of most E3 enzymes remain unknown. Here, we describe the development of a novel proteomic in vitro ubiquitination screen using a protein microarray platform that can be utilized for the ... More
Authors
Journal
PubMed ID16263709
Surface plasmon field-enhanced fluorescence spectroscopy studies of the interaction between an antibody and its surface-coupled antigen.
AuthorsYu F, Yao D, Knoll W
JournalAnal Chem
PubMed ID12948127
Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) uses the greatly enhanced electromagnetic field of a surface plasmon mode for the excitation of surface-confined fluorophores. The ability to simultaneously monitor the interfacial refractive index changes and the fluorescence signals in real time offers a huge potential for applications of SPFS in surface ... More
Niemann-Pick C1 functions in regulating lysosomal amine content.
AuthorsKaufmann AM, Krise JP,
JournalJ Biol Chem
PubMed ID18591242
'Mutations in the late endosomal/lysosomal membrane protein Niemann-Pick C1 (NPC1) are known to cause a generalized block in retrograde vesicle-mediated transport, resulting in the hyper-accumulation of multiple lysosomal cargos. An important, yet often overlooked, category of lysosomal cargo includes the vast array of small molecular weight amine-containing molecules that are ... More
AuthorsAustin CD, Lawrence DA, Peden AA, Varfolomeev EE, Totpal K, De Mazière AM, Klumperman J, Arnott D, Pham V, Scheller RH, Ashkenazi A
JournalProc Natl Acad Sci U S A
PubMed ID16801533
'Endocytosis is crucial for various aspects of cell homeostasis. Here, we show that proapoptotic death receptors (DRs) trigger selective destruction of the clathrin-dependent endocytosis machinery. DR stimulation induced rapid, caspase-mediated cleavage of key clathrin-pathway components, halting cellular uptake of the classic cargo protein transferrin. DR-proximal initiator caspases cleaved the clathrin ... More
Tyramide signal amplification for analysis of kinase activity by intracellular flow cytometry.
'Intracellular flow cytometry permits quantitation of diverse molecular targets at the single-cell level. However, limitations in detection sensitivity inherently restrict the method, sometimes resulting in the inability to measure proteins of very low abundance or to differentiate cells expressing subtly different protein concentrations. To improve these measurements, an enzymatic amplification ... More
Restriction of receptor movement alters cellular response: physical force sensing by EphA2.
AuthorsSalaita K, Nair PM, Petit RS, Neve RM, Das D, Gray JW, Groves JT,
JournalScience
PubMed ID20223987
'Activation of the EphA2 receptor tyrosine kinase by ephrin-A1 ligands presented on apposed cell surfaces plays important roles in development and exhibits poorly understood functional alterations in cancer. We reconstituted this intermembrane signaling geometry between live EphA2-expressing human breast cancer cells and supported membranes displaying laterally mobile ephrin-A1. Receptor-ligand binding, ... More
Functional cross-interaction of the fragments produced by the cleavage of distinct adhesion G-protein-coupled receptors.
AuthorsSilva JP, Lelianova V, Hopkins C, Volynski KE, Ushkaryov Y,
JournalJ Biol Chem
PubMed ID19124473
'The unusual adhesion G-protein-coupled receptors (aGPCRs) contain large extracellular N-terminal domains, which resemble cell-adhesion receptors, and C-terminal heptahelical domains, which may couple to G-proteins. These receptors are cleaved post-translationally between these domains into two fragments (NTF and CTF). Using the aGPCR latrophilin 1, we previously demonstrated that the fragments behave ... More
Multiplex detection of surface molecules on colorectal cancers.
AuthorsEllmark P, Belov L, Huang P, Lee CS, Solomon MJ, Morgan DK, Christopherson RI
JournalProteomics
PubMed ID16485257
'A technique of fluorescence multiplexing is described for analysis of the plasma membrane proteome of colorectal cancer cells from surgically resected specimens, enabling detection and immunophenotyping when the cancer cells are in the minority. A single-cell suspension was prepared from a colorectal tumour, and the mixed population of cells was ... More
Accurate FRET measurements within single diffusing biomolecules using alternating-laser excitation.
AuthorsLee NK, Kapanidis AN, Wang Y, Michalet X, Mukhopadhyay J, Ebright RH, Weiss S
JournalBiophys J
PubMed ID15653725
'Fluorescence resonance energy transfer (FRET) between a donor (D) and an acceptor (A) at the single-molecule level currently provides qualitative information about distance, and quantitative information about kinetics of distance changes. Here, we used the sorting ability of confocal microscopy equipped with alternating-laser excitation (ALEX) to measure accurate FRET efficiencies ... More
Characterization of a single molecule DNA switch in free solution.
AuthorsWhite SS, Li H, Marsh RJ, Piper JD, Leonczek ND, Nicolaou N, Bain AJ, Ying L, Klenerman D
JournalJ Am Chem Soc
PubMed ID16939265
'We have studied a donor-acceptor fluorophore-labeled DNA switch where the acceptor is Alexa-647, a carbocyanine dye, in solution at the single molecule level to elucidate the fluorescence switching mechanism. The acceptor, which is in an initial high fluorescence trans state, undergoes a photoisomerization reaction resulting in two additional states during ... More
3D multicolor super-resolution imaging offers improved accuracy in neuron tracing.
AuthorsLakadamyali M, Babcock H, Bates M, Zhuang X, Lichtman J,
JournalPLoS One
PubMed ID22292051
'The connectivity among neurons holds the key to understanding brain function. Mapping neural connectivity in brain circuits requires imaging techniques with high spatial resolution to facilitate neuron tracing and high molecular specificity to mark different cellular and molecular populations. Here, we tested a three-dimensional (3D), multicolor super-resolution imaging method, stochastic ... More
Inter- and intramolecular fluorescence quenching of organic dyes by tryptophan.
AuthorsMarmé N, Knemeyer JP, Sauer M, Wolfrum J
JournalBioconjug Chem
PubMed ID14624626
'Steady-state and time-resolved fluorescence measurements were performed to elucidate the fluorescence quenching of oxazine, rhodamine, carbocyanine, and bora-diaza-indacene dyes by amino acids. Among the natural amino acids, tryptophan exhibits the most pronounced quenching efficiency. Especially, the red-absorbing dyes ATTO 655, ATTO 680, and the oxazine derivative MR 121 are strongly ... More
Single particle characterization of iron-induced pore-forming alpha-synuclein oligomers.
AuthorsKostka M, Högen T, Danzer KM, Levin J, Habeck M, Wirth A, Wagner R, Glabe CG, Finger S, Heinzelmann U, Garidel P, Duan W, Ross CA, Kretzschmar H, Giese A,
JournalJ Biol Chem
PubMed ID18258594
'Aggregation of alpha-synuclein is a key event in several neurodegenerative diseases, including Parkinson disease. Recent findings suggest that oligomers represent the principal toxic aggregate species. Using confocal single-molecule fluorescence techniques, such as scanning for intensely fluorescent targets (SIFT) and atomic force microscopy, we monitored alpha-synuclein oligomer formation at the single ... More
Genetic analysis of beta1 integrin "activation motifs" in mice.
AuthorsCzuchra A, Meyer H, Legate KR, Brakebusch C, Fässler R
JournalJ Cell Biol
PubMed ID16954348
'Akey feature of integrins is their ability to regulate the affinity for ligands, a process termed integrin activation. The final step in integrin activation is talin binding to the NPXY motif of the integrin beta cytoplasmic domains. Talin binding disrupts the salt bridge between the alpha/beta tails, leading to tail ... More
Preparation of photoswitchable labeled antibodies for STORM imaging.
AuthorsBates M, Jones SA, Zhuang X,
Journal
PubMed ID23734027
'Stochastic optical reconstruction microscopy (STORM) is a method for superresolution fluorescence imaging based on the high accuracy localization of individual fluorophores. It uses optically switchable fluorophores: molecules that can be switched between a nonfluorescent and a fluorescent state by exposure to light. Many synthetic fluorescent dye molecules show photoswitchable fluorescence ... More
Molecule by molecule direct and quantitative counting of antibody-protein complexes in solution.
'We have used two-color fluorescence coincidence detection to directly count individual protein-antibody complexes of protein G or herpes simplex virus labeled with one or more red- and blue-excited antibodies. This allowed quantitative measurement of the concentration of the protein-antibody complexes over 3 orders of magnitude down to the femtomolar level. ... More
Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes: fluorescence of the dyes and their bioconjugates.
AuthorsBerlier JE, Rothe A, Buller G, Bradford J, Gray DR, Filanoski BJ, Telford WG, Yue S, Liu J, Cheung CY, Chang W, Hirsch JD, Beechem JM, Haugland RP, Haugland RP
JournalJ Histochem Cytochem
PubMed ID14623938
'Amine-reactive N-hydroxysuccinimidyl esters of Alexa Fluor fluorescent dyes with principal absorption maxima at about 555 nm, 633 nm, 647 nm, 660 nm, 680 nm, 700 nm, and 750 nm were conjugated to antibodies and other selected proteins. These conjugates were compared with spectrally similar protein conjugates of the Cy3, Cy5, ... More
Three-color alternating-laser excitation of single molecules: monitoring multiple interactions and distances.
AuthorsLee NK, Kapanidis AN, Koh HR, Korlann Y, Ho SO, Kim Y, Gassman N, Kim SK, Weiss S
JournalBiophys J
PubMed ID17040983
'We introduce three-color alternating-laser excitation (3c-ALEX), a fluorescence resonance energy transfer (FRET) method that measures up to three intramolecular distances and complex interaction stoichiometries of single molecules in solution. This tool extends substantially the capabilities of two-color ALEX, which employs two alternating lasers to study molecular interactions (through probe stoichiometry ... More
CD8 T cell competition for dendritic cells in vivo is an early event in activation.
AuthorsWillis RA, Kappler JW, Marrack PC
JournalProc Natl Acad Sci U S A
PubMed ID16880405
'T cell responses against an antigen are often focused on a small fraction of potentially immunogenic determinants, a phenomenon known as immunodominance. Immunodominance can be established at several stages of antigen presentation, including antigen processing, binding of peptides to MHC, and competition between T cells for dendritic cells (DCs). The ... More
Prostate specific antigen biosensor based on long range surface plasmon-enhanced fluorescence spectroscopy and dextran hydrogel binding matrix.
AuthorsWang Y, Brunsen A, Jonas U, Dostálek J, Knoll W,
JournalAnal Chem
PubMed ID19894697
'A new biosensor based on surface plasmon-enhanced fluorescence spectroscopy (SPFS), which employs long-range surface plasmons (LRSP) and a photo-cross-linkable carboxymethyl dextran (PCDM) hydrogel binding matrix, is reported. LRSPs are surface plasmon modes that propagate along a thin metallic film with orders of magnitude lower damping compared to regular surface plasmons. ... More
Higher order Rab programming in phagolysosome biogenesis.
AuthorsRoberts EA, Chua J, Kyei GB, Deretic V
JournalJ Cell Biol
PubMed ID16982798
'Phagosomes offer kinetically and morphologically tractable organelles to dissect the control of phagolysosome biogenesis by Rab GTPases. Model phagosomes harboring latex beads undergo a coordinated Rab5-Rab7 exchange, which is akin to the process of endosomal Rab conversion, the control mechanisms of which are unknown. In the process of blocking phagosomal ... More
Practical guidelines for dual-color fluorescence cross-correlation spectroscopy.
AuthorsBacia K, Schwille P,
JournalNat Protoc
PubMed ID18007619
'Dual-color fluorescence cross-correlation spectroscopy (FCCS) allows for the determination of molecular mobility and concentrations and for the quantitative analysis of molecular interactions such as binding or cleavage at very low concentrations. This protocol discusses considerations for preparing a biological system for FCCS experiments and offers practical advice for performing FCCS ... More
Activation-induced deaminase cloning, localization, and protein extraction from young VH-mutant rabbit appendix.
'Studies in mouse, human, and chicken suggest that activation-induced deaminase (AID) is involved in three known processes leading to antibody diversification: somatic hypermutation, gene conversion, and class-switch recombination. Developing rabbit appendix provides a particularly good site for studying all three of these B cell maturation events. We report here successful ... More
Conformational heterogeneity in RNA polymerase observed by single-pair FRET microscopy.
AuthorsCoban O, Lamb DC, Zaychikov E, Heumann H, Nienhaus GU
JournalBiophys J
PubMed ID16581837
'Kinetic, structural, and single-molecule transcription measurements suggest that RNA polymerase can adopt many different conformations during elongation. We have measured the geometry of the DNA and RNA in ternary elongation complexes using single-pair fluorescence resonance energy transfer. Six different synthetic transcription elongation complexes were constructed from DNA containing an artificial ... More
Libraries against libraries for combinatorial selection of replicating antigen-antibody pairs.
AuthorsBowley DR, Jones TM, Burton DR, Lerner RA,
JournalProc Natl Acad Sci U S A
PubMed ID19139405
'Antibodies are among the most highly selective tight-binding ligands for proteins. Because the human genome project has deciphered the proteome, there is an opportunity to use combinatorial antibody libraries to select high-affinity antibodies to every protein encoded by the genome. However, this is a large task because the selection formats ... More
Optimizing a waveguide-based sandwich immunoassay for tumor biomarkers: evaluating fluorescent labels and functional surfaces.
AuthorsMukundan H, Xie H, Anderson AS, Grace WK, Shively JE, Swanson BI,
JournalBioconjug Chem
PubMed ID19173652
'The sensor team at the Los Alamos National Laboratory has developed a waveguide-based optical biosensor for the detection of biomarkers associated with disease. We have previously demonstrated the application of this technology to the sensitive detection of carcinoembryonic antigen in serum and nipple aspirate fluid from breast cancer patients. In ... More
Visualizing the site and dynamics of IgG salvage by the MHC class I-related receptor, FcRn.
AuthorsOber RJ, Martinez C, Vaccaro C, Zhou J, Ward ES
JournalJ Immunol
PubMed ID14764666
'The MHC class I-related receptor, FcRn, plays a central role in regulating the serum levels of IgG. FcRn is expressed in endothelial cells, suggesting that these cells may be involved in maintaining IgG levels. We have used live cell imaging of FcRn-green fluorescent protein transfected human endothelial cells to analyze ... More
A fluorescence resonance energy transfer-based approach for investigating late endosome-lysosome retrograde fusion events.
AuthorsKaufmann AM, Goldman SD, Krise JP,
JournalAnal Biochem
PubMed ID19109922
'Traditionally, lysosomes have been considered to be a terminal endocytic compartment. Recent studies suggest that lysosomes are quite dynamic, being able to fuse with other late endocytic compartments as well as with the plasma membrane. Here we describe a quantitative fluorescence energy transfer (FRET)-based method for assessing rates of retrograde ... More
Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging.
AuthorsDempsey GT, Vaughan JC, Chen KH, Bates M, Zhuang X,
JournalNat Methods
PubMed ID22056676
'One approach to super-resolution fluorescence imaging uses sequential activation and localization of individual fluorophores to achieve high spatial resolution. Essential to this technique is the choice of fluorescent probes; the properties of the probes, including photons per switching event, on-off duty cycle, photostability and number of switching cycles, largely dictate ... More
Simultaneous multiple immunoassays in a compact disc-shaped microfluidic device based on centrifugal force.
AuthorsHonda N, Lindberg U, Andersson P, Hoffmann S, Takei H
JournalClin Chem
PubMed ID16081503
'BACKGROUND: We explored the potential of a microfluidic device based on centrifugal force as an immunoassay platform by examining the imprecision of assays carried out with 200 nL of sample. METHODS: Biotinylated antibodies against alpha-fetoprotein (AFP), interleukin-6 (IL-6), and carcinoembryonic antigen [(CEA); 0.1 g/L each in 15 mmol/L phosphate-buffered saline ... More
Molecular evolution of antibody cross-reactivity for two subtypes of type A botulinum neurotoxin.
AuthorsGarcia-Rodriguez C, Levy R, Arndt JW, Forsyth CM, Razai A, Lou J, Geren I, Stevens RC, Marks JD
JournalNat Biotechnol
PubMed ID17173035
'Broadening antibody specificity without compromising affinity should facilitate detection and neutralization of toxin and viral subtypes. We used yeast display and a co-selection strategy to increase cross-reactivity of a single chain (sc) Fv antibody to botulinum neurotoxin type A (BoNT/A). Starting with a scFv that binds the BoNT/A1 subtype with ... More
Multicolor super-resolution fluorescence imaging via multi-parameter fluorophore detection.
AuthorsBates M, Dempsey GT, Chen KH, Zhuang X,
JournalChemphyschem
PubMed ID22213647
'Understanding the complexity of the cellular environment will benefit from the ability to unambiguously resolve multiple cellular components, simultaneously and with nanometer-scale spatial resolution. Multicolor super-resolution fluorescence microscopy techniques have been developed to achieve this goal, yet challenges remain in terms of the number of targets that can be simultaneously ... More
Superresolution imaging of chemical synapses in the brain.
AuthorsDani A, Huang B, Bergan J, Dulac C, Zhuang X,
JournalNeuron
PubMed ID21144999
'Determination of the molecular architecture of synapses requires nanoscopic image resolution and specific molecular recognition, a task that has so far defied many conventional imaging approaches. Here, we present a superresolution fluorescence imaging method to visualize the molecular architecture of synapses in the brain. Using multicolor, three-dimensional stochastic optical reconstruction ... More
SR-BI-mediated high density lipoprotein (HDL) endocytosis leads to HDL resecretion facilitating cholesterol efflux.
AuthorsPagler TA, Rhode S, Neuhofer A, Laggner H, Strobl W, Hinterndorfer C, Volf I, Pavelka M, Eckhardt ER, van der Westhuyzen DR, Schütz GJ, Stangl H
JournalJ Biol Chem
PubMed ID16488891
'The high density lipoprotein (HDL) receptor, scavenger receptor class B, type I (SR-BI), mediates selective cholesteryl ester uptake from lipoproteins into liver and steroidogenic tissues but also cholesterol efflux from macrophages to HDL. Recently, we demonstrated the uptake of HDL particles in SR-BI overexpressing Chinese hamster ovarian cells (ldlA7-SRBI) using ... More
Two-color fluorescence analysis of individual virions determines the distribution of the copy number of proteins in herpes simplex virus particles.
AuthorsClarke RW, Monnier N, Li H, Zhou D, Browne H, Klenerman D,
JournalBiophys J
PubMed ID17513380
'We present a single virion method to determine absolute distributions of copy number in the protein composition of viruses and apply it to herpes simplex virus type 1. Using two-color coincidence fluorescence spectroscopy, we determine the virion-to-virion variability in copy numbers of fluorescently labeled tegument and envelope proteins relative to ... More
Possible sources of dye-related signal correlation bias in two-color DNA microarray assays.
AuthorsCox WG, Beaudet MP, Agnew JY, Ruth JL
JournalAnal Biochem
PubMed ID15265729
'DNA microarray analyses commonly use two spectrally distinct fluorescent labels to simultaneously compare different mRNA pools. Signal correlation bias currently limits accepted resolution to twofold changes in gene expression. This bias was investigated by (i) examining fluorescence and absorption spectra and changes in relative fluorescence of DNAs labeled with the ... More
Electron transfer reaction in a single protein molecule observed by total internal reflection fluorescence microscopy.
AuthorsFurukawa Y, Ban T, Hamada D, Ishimori K, Goto Y, Morishima I
JournalJ Am Chem Soc
PubMed ID15713086
'To observe an electron transfer (ET) process in a single protein molecule, we constructed a model system, Alexa-HCytb5, in which cytochrome b5 (Cytb5) is modified with a fluorescent probe, Alexa Fluor 647 dye. In this model system, intramolecular transfer of an electron from the Alexa dye to heme in Cytb5 ... More
Single-molecule level analysis of the subunit composition of the T cell receptor on live T cells.
AuthorsJames JR, White SS, Clarke RW, Johansen AM, Dunne PD, Sleep DL, Fitzgerald WJ, Davis SJ, Klenerman D,
JournalProc Natl Acad Sci U S A
PubMed ID17971442
'The T cell receptor (TCR) expressed on most T cells is a protein complex consisting of TCRalphabeta heterodimers that bind antigen and cluster of differentiation (CD) 3epsilondelta, epsilongamma, and zetazeta dimers that initiate signaling. A long-standing controversy concerns whether there is one, or more than one, alphabeta heterodimer per complex. ... More
Ultrabright photoactivatable fluorophores created by reductive caging.
AuthorsVaughan JC, Jia S, Zhuang X,
JournalNat Methods
PubMed ID23103881
'Sub-diffraction-limit imaging can be achieved by sequential localization of photoactivatable fluorophores, for which the image resolution depends on the number of photons detected per localization. We report a strategy for fluorophore caging that creates photoactivatable probes with high photon yields. Upon photoactivation, these probes can provide 10(4)-10(6) photons per localization ... More
Statistical deconvolution for superresolution fluorescence microscopy.
AuthorsMukamel EA, Babcock H, Zhuang X,
JournalBiophys J
PubMed ID22677393
'Superresolution microscopy techniques based on the sequential activation of fluorophores can achieve image resolution of ~10 nm but require a sparse distribution of simultaneously activated fluorophores in the field of view. Image analysis procedures for this approach typically discard data from crowded molecules with overlapping images, wasting valuable image information that ... More
Single-molecule tracking of mRNA exiting from RNA polymerase II.
AuthorsAndrecka J, Lewis R, Brückner F, Lehmann E, Cramer P, Michaelis J,
JournalProc Natl Acad Sci U S A
PubMed ID18162559
'Single-pair fluorescence resonance energy transfer was used to track RNA exiting from RNA polymerase II (Pol II) in elongation complexes. Measuring the distance between the RNA 5'' end and three known locations within the elongation complex allows us determine its position by means of triangulation. RNA leaves the polymerase active ... More
Segregation of PIP2 and PIP3 into distinct nanoscale regions within the plasma membrane.
AuthorsWang J, Richards DA,
JournalBiol Open
PubMed ID23213479
'PIP(2) and PIP(3) are implicated in a wide variety of cellular signaling pathways at the plasma membrane. We have used STORM imaging to localize clusters of PIP(2) and PIP(3) to distinct nanoscale regions within the plasma membrane of PC12 cells. With anti-phospholipid antibodies directly conjugated with AlexaFluor 647, we found ... More
In-gel fluorescence probing of RNA-RNA interactions.
AuthorsDayie KT, Gumbs OH, Eldho NV, Seetharaman M, Thompson M
JournalAnal Biochem
PubMed ID17275775
Multicolor imaging: the important question of co-localization.
AuthorsSmallcombe A
JournalBiotechniques
PubMed ID11414212
Whole-genome genotyping with the single-base extension assay.
We describe an efficient, accurate and robust whole-genome genotyping (WGG) assay based on a two-color, single-base extension (SBE), single-nucleotide polymorphism (SNP)-scoring step. We report genotyping results for biallelic International HapMap quality control (QC) SNPs using a single probe per locus. We show scalability, throughput and accuracy of the system by ... More
Multicolor super-resolution imaging with photo-switchable fluorescent probes.
AuthorsBates M, Huang B, Dempsey GT, Zhuang X,
JournalScience
PubMed ID17702910
Recent advances in far-field optical nanoscopy have enabled fluorescence imaging with a spatial resolution of 20 to 50 nanometers. Multicolor super-resolution imaging, however, remains a challenging task. Here, we introduce a family of photo-switchable fluorescent probes and demonstrate multicolor stochastic optical reconstruction microscopy (STORM). Each probe consists of a photo-switchable ... More
Evidence for resonance optical trapping of individual fluorophore-labeled antibodies using single molecule fluorescence spectroscopy.
AuthorsLi H, Zhou D, Browne H, Klenerman D
JournalJ Am Chem Soc
PubMed ID16637638
We report single molecule fluorescence studies of the diffusion of individual multiple fluorophore-labeled antibodies in solution, which show that a trapping potential of about 3.6 k(B)T can be obtained at laser powers below 1 mW with resonant excitation. Individual antibodies can be trapped for up to 140 ms, and bound ... More
Phosphine quenching of cyanine dyes as a versatile tool for fluorescence microscopy.
AuthorsVaughan JC, Dempsey GT, Sun E, Zhuang X,
JournalJ Am Chem Soc
PubMed ID23311875
We report that the cyanine dye Cy5 and several of its structural relatives are reversibly quenched by the phosphine tris(2-carboxyethyl)phosphine (TCEP). Using Cy5 as a model, we show that the quenching reaction occurs by 1,4-addition of the phosphine to the polymethine bridge of Cy5 to form a covalent adduct. Illumination ... More
Fluorescence quantification for surface plasmon excitation.
AuthorsFeller BE, Kellis JT, Cascão-Pereira LG, Knoll W, Robertson CR, Frank CW,
JournalLangmuir
PubMed ID18844383
Surface plasmon resonance and surface plasmon fluorescence spectroscopy in combination have the potential to distinguish multicomponent surface processes. However, surface intensity variations from resonance angle shifts lead to a nonlinear response in the fluorescence intensity. We report a method to account for surface intensity variations using the experimentally measured relationship ... More
Monitoring of real-time streptavidin-biotin binding kinetics using droplet microfluidics.
AuthorsSrisa-Art M, Dyson EC, deMello AJ, Edel JB,
JournalAnal Chem
PubMed ID18712935
Rapid kinetic measurements are important in understanding chemical interactions especially for biological molecules. Herein, we present a droplet-based microfluidic platform to study streptavidin-biotin binding kinetics with millisecond time resolution. With integration of a confocal fluorescence detection system, individual droplets can be monitored and characterized online to extract kinetic information. Using ... More
Engineering the Fc region of immunoglobulin G to modulate in vivo antibody levels.
AuthorsVaccaro C, Zhou J, Ober RJ, Ward ES
JournalNat Biotechnol
PubMed ID16186811
We have engineered the Fc region of a human immunoglobulin G (IgG) to generate a mutated antibody that modulates the concentrations of endogenous IgGs in vivo. This has been achieved by targeting the activity of the Fc receptor, FcRn, which serves through its IgG salvage function to maintain and regulate ... More
Multicolor single molecule tracking of stochastically active synthetic dyes.
AuthorsBenke A, Olivier N, Gunzenhäuser J, Manley S,
JournalNano Lett
PubMed ID22519662
Single particle tracking can reveal dynamic information at the scale of single molecules in living cells but thus far has been limited either in the range of potential protein targets or in the quality and number of tracks attainable. We demonstrate a new approach to single molecule tracking by using ... More
Ultrasensitive coincidence fluorescence detection of single DNA molecules.
AuthorsLi H, Ying L, Green JJ, Balasubramanian S, Klenerman D
JournalAnal Chem
PubMed ID12705600
We have detected individual DNA molecules labeled with two different fluorophores in solution by using two-color excitation and detection of coincidence fluorescence bursts. The confocal volumes of the two excitation lasers were carefully matched so that the volume overlap was 30% of the total confocal volume illuminated. This method greatly ... More
Characterizing specific phage-protein interactions by fluorescence correlation spectroscopy.
AuthorsBahns JT, Liu CM, Chen L
JournalProtein Sci
PubMed ID15388856
The interactions of several affinity reagent displayed T7 and M13 phage particles with their corresponding target molecules were examined using Fluorescence Correlation Spectroscopy (FCS). Diffusion times, relative fractions of each component in the recognition reactions at the equilibrium state, and ultimately the dissociation constants were deduced from analyzing the fluorescence ... More
Super-resolution fluorescence imaging of telomeres reveals TRF2-dependent T-loop formation.
AuthorsDoksani Y, Wu JY, de Lange T, Zhuang X,
Journal
PubMed ID24120135
We have applied a super-resolution fluorescence imaging method, stochastic optical reconstruction microscopy (STORM), to visualize the structure of functional telomeres and telomeres rendered dysfunctional through removal of shelterin proteins. The STORM images showed that functional telomeres frequently exhibit a t-loop configuration. Conditional deletion of individual components of shelterin showed that ... More
Fluorescent carrier ampholytes assay for portable, label-free detection of chemical toxins in tap water.
AuthorsBercovici M, Kaigala GV, Backhouse CJ, Santiago JG,
JournalAnal Chem
PubMed ID20141152
We present a novel method for fluorescence-based indirect detection of analytes and demonstrate its use for label-free detection of chemical toxins in a hand-held device. We fluorescently label a mixture of low-concentration carrier ampholytes and introduce it into an isotachophoresis (ITP) separation. The carrier ampholytes provide a large number of ... More
Measuring, in solution, multiple-fluorophore labeling by combining fluorescence correlation spectroscopy and photobleaching.
AuthorsDelon A, Wang I, Lambert E, Mache S, Mache R, Derouard J, Motto-Ros V, Galland R,
JournalJ Phys Chem B
PubMed ID20143802
Determining the number of fluorescent entities that are coupled to a given molecule (DNA, protein, etc.) is a key point of numerous biological studies, especially those based on a single molecule approach. Reliable methods are important, in this context, not only to characterize the labeling process but also to quantify ... More
Import and fate of fluorescent analogs of oxidized phospholipids in vascular smooth muscle cells.
AuthorsMoumtzi A, Trenker M, Flicker K, Zenzmaier E, Saf R, Hermetter A,
JournalJ Lipid Res
PubMed ID17135656
Lipid oxidation is now thought to be an initiating and sustaining event in atherogenesis. Oxidatively fragmented phospholipids, namely 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), present in minimally modified LDL and atherosclerotic lesions, have been reported to elicit a wide range of pathophysiological responses in the cells of the vascular wall. Nevertheless, ... More
Synthesis and evaluation of near-infrared (NIR) dye-herceptin conjugates as photoacoustic computed tomography (PCT) probes for HER2 expression in breast cancer.
We are evaluating PCT imaging in conjunction with NIR dye labeled Herceptin antibody for noninvasive assessment of HER2 expression in tumors. Herceptin was labeled with Alexa Fluor-750 amine reactive dye for characterization of photoacoustic and fluorescence signals. Measurements were performed in solution and after incubation in cultured cell lines that ... More
Transport and detection of unlabeled nucleotide targets by microtubules functionalized with molecular beacons.
AuthorsRaab M, Hancock WO,
JournalBiotechnol Bioeng
PubMed ID17879297
Shrinking biosensors down to microscale dimensions enables increases in sensitivity and the ability to analyze minute samples such as the contents of individual cells. The goal of the present study is to create mobile microscale biosensors by attaching molecular beacons to microtubules and using kinesin molecular motors to transport these ... More
Systematic identification of antiprion drugs by high-throughput screening based on scanning for intensely fluorescent targets.
AuthorsBertsch U, Winklhofer KF, Hirschberger T, Bieschke J, Weber P, Hartl FU, Tavan P, Tatzelt J, Kretzschmar HA, Giese A,
JournalJ Virol
PubMed ID15919931
Conformational changes and aggregation of specific proteins are hallmarks of a number of diseases, like Alzheimer's disease, Parkinson's disease, and prion diseases. In the case of prion diseases, the prion protein (PrP), a neuronal glycoprotein, undergoes a conformational change from the normal, mainly alpha-helical conformation to a disease-associated, mainly beta-sheeted ... More
Novel pentablock copolymers for selective gene delivery to cancer cells.
AuthorsZhang B, Kanapathipillai M, Bisso P, Mallapragada S,
JournalPharm Res
PubMed ID19142716
PURPOSE: In this study, the novel poly(diethylaminoethylmethacrylate) (PDEAEM)/Pluronic F127 pentablock copolymers were found to be able to mediate high-efficiency transfection of human epithelial ovarian carcinoma (SKOV3) cell line while showing significantly lower efficacy in human epithelial retinal (ARPE-19) cell line and Swiss Mouse Fibroblast (3T3) cell line. METHODS: The intracellular ... More
Sequential treatment of drug-resistant tumors with targeted minicells containing siRNA or a cytotoxic drug.
The dose-limiting toxicity of chemotherapeutics, heterogeneity and drug resistance of cancer cells, and difficulties of targeted delivery to tumors all pose daunting challenges to effective cancer therapy. We report that small interfering RNA (siRNA) duplexes readily penetrate intact bacterially derived minicells previously shown to cause tumor stabilization and regression when ... More
Toward efficient drug screening by homogeneous assays based on the development of new fluorescent vasopressin and oxytocin receptor ligands.
A series of fluorescent ligands designed for vasopressin and oxytocin G protein-coupled receptors was synthesized and characterized to develop fluorescence polarization or homogeneous time-resolved fluorescence (HTRF) binding assays. These ligands, labeled with europium pyridine-bis-bipyridine cryptate or with Alexa 488,546,647 selectively bound to the vasopressin V1a and oxytocin receptors with high ... More
Photobleaching pathways in single-molecule FRET experiments.
AuthorsKong X, Nir E, Hamadani K, Weiss S,
JournalJ Am Chem Soc
PubMed ID17375921
To acquire accurate structural and dynamical information on complex biomolecular machines using single-molecule fluorescence resonance energy transfer (sm-FRET), a large flux of donor and acceptor photons is needed. To achieve such fluxes, one may use higher laser excitation intensity; however, this induces increased rates of photobleaching. Anti-oxidant additives have been ... More
Design of recombinant antibody microarrays for complex proteome analysis: choice of sample labeling-tag and solid support.
AuthorsWingren C, Ingvarsson J, Dexlin L, Szul D, Borrebaeck CA,
JournalProteomics
PubMed ID17787036
Antibody-based microarray is a novel technology with great potential within high-throughput proteomics. The process of designing high-performing antibody (protein) microarrays has, however, turned out to be a challenging process. In this study, we have developed further our human recombinant single-chain variable-fragment (scFv) antibody microarray methodology by addressing two crucial technological ... More
Fluorescence spectroscopic profiling of compound libraries.
AuthorsSimeonov A, Jadhav A, Thomas CJ, Wang Y, Huang R, Southall NT, Shinn P, Smith J, Austin CP, Auld DS, Inglese J,
JournalJ Med Chem
PubMed ID18363325
Chromo/fluorophoric properties often accompany the heterocyclic scaffolds and impurities that comprise libraries used for high-throughput screening (HTS). These properties affect assay outputs obtained with optical detection, thus complicating analysis and leading to false positives and negatives. Here, we report the fluorescence profile of more than 70,000 samples across spectral regions ... More
Assessing the sensitivity of commercially available fluorophores to the intracellular environment.
AuthorsChen AK, Cheng Z, Behlke MA, Tsourkas A,
JournalAnal Chem
PubMed ID18700780
The use of fluorescence has become commonplace in the biological sciences, with many studies utilizing probes based on commercially available fluorophores to provide insight into cell function and behavior. As these imaging applications become more advanced, it becomes increasingly important to acquire accurate quantitative measurements of the fluorescence signal. Absolute ... More
Programmable in situ amplification for multiplexed imaging of mRNA expression.
AuthorsChoi HM, Chang JY, Trinh le A, Padilla JE, Fraser SE, Pierce NA,
JournalNat Biotechnol
PubMed ID21037591
In situ hybridization methods enable the mapping of mRNA expression within intact biological samples. With current approaches, it is challenging to simultaneously map multiple target mRNAs within whole-mount vertebrate embryos, representing a significant limitation in attempting to study interacting regulatory elements in systems most relevant to human development and disease. ... More
Evidence to support the cellular mechanism involved in serum IgG homeostasis in humans.
AuthorsWard ES, Zhou J, Ghetie V, Ober RJ
JournalInt Immunol
PubMed ID12578848
IgG is the most abundant serum antibody and is an essential component of the humoral immune response. It is known that the 'neonatal' Fc receptor (FcRn) plays a role in maintaining constant serum IgG levels by acting as a protective receptor which binds and salvages IgG from degradation. However, the ... More
Effects of atmospheric ozone on microarray data quality.
AuthorsFare TL, Coffey EM, Dai H, He YD, Kessler DA, Kilian KA, Koch JE, LeProust E, Marton MJ, Meyer MR, Stoughton RB, Tokiwa GY, Wang Y
JournalAnal Chem
PubMed ID14632079
A data anomaly was observed that affected the uniformity and reproducibility of fluorescent signal across DNA microarrays. Results from experimental sets designed to identify potential causes (from microarray production to array scanning) indicated that the anomaly was linked to a batch process; further work allowed us to localize the effect ... More
Direct imaging of lateral movements of AMPA receptors inside synapses.
AuthorsTardin C, Cognet L, Bats C, Lounis B, Choquet D
JournalEMBO J
PubMed ID12970178
Trafficking of AMPA receptors in and out of synapses is crucial for synaptic plasticity. Previous studies have focused on the role of endo/exocytosis processes or that of lateral diffusion of extra-synaptic receptors. We have now directly imaged AMPAR movements inside and outside synapses of live neurons using single-molecule fluorescence microscopy. ... More
Nine-analyte detection using an array-based biosensor.
AuthorsTaitt CR, Anderson GP, Lingerfelt BM, Feldstein MJ, Ligler FS
JournalAnal Chem
PubMed ID12498211
A fluorescence-based multianalyte immunosensor has been developed for simultaneous analysis of multiple samples. While the standard 6 x 6 format of the array sensor has been used to analyze six samples for six different analytes, this same format has the potential to allow a single sample to be tested for ... More
Flow cytometric analysis of DNA binding and cleavage by cell surface-displayed homing endonucleases.
LAGLIDADG homing endonucleases (LHEs) cleave 18-24 bp DNA sequences and are promising enzymes for applications requiring sequence-specific DNA cleavage amongst genome-sized DNA backgrounds. Here, we report a method for cell surface display of LHEs, which facilitates analysis of their DNA binding and cleavage properties by flow cytometry. Cells expressing surface ... More
Improved fluoroimmunoassays using the dye Alexa Fluor 647 with the RAPTOR, a fiber optic biosensor.
AuthorsAnderson GP, Nerurkar NL
JournalJ Immunol Methods
PubMed ID12445725
The performance of the fluorescent dye Alexa Fluor 647 (AF647) was explored as an alternative to Cy5 for immunoassays on the RAPTOR, a fiber optic biosensor. The RAPTOR performs sandwich fluoroimmunoassays on the surface of small polystyrene optical waveguides for analyte detection. Fluorescence and immunoassay data were examined at various ... More
Dissecting RNA-interference pathway with small molecules.
AuthorsChiu YL, Dinesh CU, Chu CY, Ali A, Brown KM, Cao H, Rana TM
JournalChem Biol
PubMed ID15975509
RNA interference (RNAi) is a process whereby short-interfering RNAs (siRNA) silence gene expression in a sequence-specific manner. We have screened a chemical library of substituted dihydropteridinones and identified a nontoxic, cell permeable, and reversible inhibitor of the RNAi pathway in human cells. Biochemical and fluorescence resonance-energy transfer experiments demonstrated that ... More
Cell surface detection of membrane protein interaction with homogeneous time-resolved fluorescence resonance energy transfer technology.
AuthorsMaurel D, Kniazeff J, Mathis G, Trinquet E, Pin JP, Ansanay H
JournalAnal Biochem
PubMed ID15158484
Direct or indirect interactions between membrane proteins at the cell surface play a central role in numerous cell processes, including possible synergistic effects between different types of receptors. Here we describe a method and tools to analyze membrane protein-protein interaction at the surface of living cells. This technology is based ... More
CpG Island microarray probe sequences derived from a physical library are representative of CpG Islands annotated on the human genome.
AuthorsHeisler LE, Torti D, Boutros PC, Watson J, Chan C, Winegarden N, Takahashi M, Yau P, Huang TH, Farnham PJ, Jurisica I, Woodgett JR, Bremner R, Penn LZ, Der SD
JournalNucleic Acids Res
PubMed ID15911630
An effective tool for the global analysis of both DNA methylation status and protein-chromatin interactions is a microarray constructed with sequences containing regulatory elements. One type of array suited for this purpose takes advantage of the strong association between CpG Islands (CGIs) and gene regulatory regions. We have obtained 20,736 ... More
Correlation spectroscopy of minor fluorescent species: signal purification and distribution analysis.
AuthorsLaurence TA, Kwon Y, Yin E, Hollars CW, Camarero JA, Barsky D
JournalBiophys J
PubMed ID17189306
We are performing experiments that use fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) to monitor the movement of an individual donor-labeled sliding clamp protein molecule along acceptor-labeled DNA. In addition to the FRET signal sought from the sliding clamp-DNA complexes, the detection channel for FRET contains undesirable ... More
A hyperspectral fluorescence system for 3D in vivo optical imaging.
AuthorsZavattini G, Vecchi S, Mitchell G, Weisser U, Leahy RM, Pichler BJ, Smith DJ, Cherry SR
JournalPhys Med Biol
PubMed ID16585843
In vivo optical instruments designed for small animal imaging generally measure the integrated light intensity across a broad band of wavelengths, or make measurements at a small number of selected wavelengths, and primarily use any spectral information to characterize and remove autofluorescence. We have developed a flexible hyperspectral imaging instrument ... More
Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules.
AuthorsKapanidis AN, Lee NK, Laurence TA, Doose S, Margeat E, Weiss S
JournalProc Natl Acad Sci U S A
PubMed ID15175430
We use alternating-laser excitation to achieve fluorescence-aided molecule sorting (FAMS) and enable simultaneous analysis of biomolecular structure and interactions at the level of single molecules. This was performed by labeling biomolecules with fluorophores that serve as donor-acceptor pairs for Förster resonance energy transfer, and by using alternating-laser excitation to excite ... More
Monovalent ligation of the B cell receptor induces receptor activation but fails to promote antigen presentation.
AuthorsKim YM, Pan JY, Korbel GA, Peperzak V, Boes M, Ploegh HL
JournalProc Natl Acad Sci U S A
PubMed ID16492756
We explored the role of antigen valency in B cell receptor (BCR) activation and rearrangement of intracellular MHC class II compartments as factors that contribute to the efficacy of antigen presentation. Using primary B cells that express a hen egg lysozyme (HEL)-specific BCR, we found that oligomeric HEL more efficiently ... More
Spectroscopic study and evaluation of red-absorbing fluorescent dyes.
AuthorsBuschmann V, Weston KD, Sauer M
JournalBioconjug Chem
PubMed ID12526709
The spectroscopic characteristics (absorption, emission, and fluorescence lifetime) of 13 commercially available red-absorbing fluorescent dyes were studied under a variety of conditions. The dyes included in this study are Alexa647, ATTO655, ATTO680, Bodipy630/650, Cy5, Cy5.5, DiD, DY-630, DY-635, DY-640, DY-650, DY-655, and EVOblue30. The thorough characterization of this class of ... More
Fluorescence and solution NMR study of the active site of a 160-kDa group II intron ribozyme.
AuthorsGumbs OH, Padgett RA, Dayie KT
JournalRNA
PubMed ID16894219
We have reconstructed the group II intron from Pylaiella littoralis (PL) into a hydrolytic ribozyme, comprising domains 1-3 (D123) connected in cis plus domain 5 (D5) supplied in trans that efficiently cleaves spliced exon substrates. Using a novel gel-based fluorescence assay and nuclear magnetic resonance (NMR) spectroscopy, we monitored the ... More
Cholesterol depletion suppresses the translational diffusion of class II major histocompatibility complex proteins in the plasma membrane.
AuthorsVrljic M, Nishimura SY, Moerner WE, McConnell HM
JournalBiophys J
PubMed ID15516525
Glycosylphosphatidylinositol (GPI)-linked and native major histocompatibility complex class II I-E(k) were used as probes to determine the effect of varying cholesterol concentration on the mobility of proteins in the plasma membrane. These proteins were imaged in Chinese hamster ovary cells using single-molecule fluorescence microscopy. Observed diffusion coefficients of both native ... More
Modulation of antigen presentation by autoreactive B cell clones specific for GAD65 from a type I diabetic patient.
AuthorsBanga JP, Moore JK, Duhindan N, Madec AM, van Endert PM, Orgiazzi J, Endl J
JournalClin Exp Immunol
PubMed ID14678267
We used a GAD65-specific human B-T cell line cognate system in vitro to investigate the modulation of GAD65 presentation by autoantibody, assessed in a proliferation assay. Generally, if the T cell determinant overlaps or resides within the antibody epitope, effects of presentation are blunted while if they are distant can ... More
Arf gene loss enhances oncogenicity and limits imatinib response in mouse models of Bcr-Abl-induced acute lymphoblastic leukemia.
AuthorsWilliams RT, Roussel MF, Sherr CJ
JournalProc Natl Acad Sci U S A
PubMed ID16618932
Mouse bone marrow cells transduced with retroviral vectors encoding either of two oncogenic Bcr-Abl isoforms (p210(Bcr-Abl) and p185(Bcr-Abl)) induce B cell lympholeukemias when transplanted into lethally irradiated mice. If the activity of the Arf tumor suppressor is compromised, these donor cells initiate a much more highly aggressive and rapidly fatal ... More
Intracellular phospho-protein staining techniques for flow cytometry: monitoring single cell signaling events.
AuthorsKrutzik PO, Nolan GP
JournalCytometry A
PubMed ID14505311
BACKGROUND: Recent advances in intracellular staining techniques, cytometer technology, fluorescent reagents, and antibody production have expanded the number of intracellular antigens that can be analyzed by flow cytometry. Measurement of protein phosphorylation with phospho-specific antibodies has given insight into kinase signaling cascades. However, available techniques for phospho-epitope staining can differ ... More
A functional RNAi screen for regulators of receptor tyrosine kinase and ERK signalling.
AuthorsFriedman A, Perrimon N
JournalNature
PubMed ID17086199
Receptor tyrosine kinase (RTK) signalling through extracellular-signal-regulated kinases (ERKs) has pivotal roles during metazoan development, underlying processes as diverse as fate determination, differentiation, proliferation, survival, migration and growth. Abnormal RTK/ERK signalling has been extensively documented to contribute to developmental disorders and disease, most notably in oncogenic transformation by mutant RTKs ... More
Application of spectral imaging microscopy in cytomics and fluorescence resonance energy transfer (FRET) analysis.
AuthorsEcker RC, de Martin R, Steiner GE, Schmid JA
JournalCytometry A
PubMed ID15170596
BACKGROUND: Specific signal detection has been a fundamental issue in fluorescence microscopy. In the context of tissue samples, this problem has been even more pronounced, with respect to spectral overlap and autofluorescence. METHODS: Recent improvements in confocal laser scanning microscopy combine sophisticated hardware to obtain fluorescence emission spectra on a ... More
Exploring the regulation of tRNA distribution on the genomic scale.
AuthorsDittmar KA, Mobley EM, Radek AJ, Pan T
JournalJ Mol Biol
PubMed ID15001350
Though up to 20% of the total RNA in bacterial cells is tRNA, the regulation of tRNA distribution on the genomic level remains unclear. tRNA distribution is governed by four processes: transcription, processing of precursor tRNA, degradation of precursor tRNA and degradation of mature tRNA. To elucidate the relationship between ... More
Analysis of human telomerase activity and function by two color single molecule coincidence fluorescence spectroscopy.
AuthorsRen X, Li H, Clarke RW, Alves DA, Ying L, Klenerman D, Balasubramanian S
JournalJ Am Chem Soc
PubMed ID16608333
Telomerase is a nonclassical DNA polymerase that uses its integral RNA as a template to synthesize telomeric repeats onto chromosome ends. The molecular mechanism of telomerase is unique and involves a translocation step after the synthesis of each telomeric repeat. To directly measure the enzymatic turnover of substrate and the ... More
Synergistic and differential modulation of immune responses by Hsp60 and lipopolysaccharide.
AuthorsOsterloh A, Kalinke U, Weiss S, Fleischer B, Breloer M
JournalJ Biol Chem
PubMed ID17164250
Activation of professional antigen-presenting cells (APC) is a crucial step in the initiation of an efficient immune response. In this study we show that Hsp60 mediates immune stimulation by different mechanisms, dependent and independent of lipopolysaccharide (LPS). We have demonstrated earlier that both, Hsp60 and LPS, increase antigen-specific interferon (IFN) ... More
Time-resolved Forster resonance energy transfer assays for the binding of nucleotide and protein substrates to p38alpha protein kinase.
AuthorsZhang WX, Wang R, Wisniewski D, Marcy AI, LoGrasso P, Lisnock JM, Cummings RT, Thompson JE
JournalAnal Biochem
PubMed ID15979553
We have developed assays for the binding of nucleotide and protein substrates to p38alpha protein kinase based on time-resolved Forster resonance energy transfer. p38alpha was biotinylated by addition of a sequence that targets biotin to a single lysine when coexpressed with biotin ligase in Escherichia coli, allowing formation of a ... More
Chromatin Fiber Invasion and Nucleosome Displacement by the Rap1 Transcription Factor.
AuthorsMivelaz M, Cao AM, Kubik S, Zencir S, Hovius R, Boichenko I, Stachowicz AM, Kurat CF, Shore D, Fierz B
JournalMol Cell
PubMed ID31761495
'Pioneer transcription factors (pTFs) bind to target sites within compact chromatin, initiating chromatin remodeling and controlling the recruitment of downstream factors. The mechanisms by which pTFs overcome the chromatin barrier are not well understood. Here, we reveal, using single-molecule fluorescence, how the yeast transcription factor Rap1 invades and remodels chromatin. ... More
Direct comparison of clathrin-mediated endocytosis in budding and fission yeast reveals conserved and evolvable features.
'Conserved proteins drive clathrin-mediated endocytosis (CME), which from yeast to humans involves a burst of actin assembly. To gain mechanistic insights into this process, we performed a side-by-side quantitative comparison of CME in two distantly related yeast species. Though endocytic protein abundance in' ... More
A peptide tag-specific nanobody enables high-quality labeling for dSTORM imaging.
AuthorsVirant D, Traenkle B, Maier J, Kaiser PD, Bodenhöfer M, Schmees C, Vojnovic I, Pisak-Lukáts B, Endesfelder U, Rothbauer U
JournalNat Commun
PubMed ID29500346
'Dense fluorophore labeling without compromising the biological target is crucial for genuine super-resolution microscopy. Here we introduce a broadly applicable labeling strategy for fixed and living cells utilizing a short peptide tag-specific nanobody (BC2-tag/bivBC2-Nb). BC2-tagging of ectopically introduced or endogenous proteins does not interfere with the examined structures and bivBC2-Nb ... More
Single molecule characterization of individual extracellular vesicles from pancreatic cancer.
AuthorsLennon KM, Wakefield DL, Maddox AL, Brehove MS, Willner AN, Garcia-Mansfield K, Meechoovet B, Reiman R, Hutchins E, Miller MM, Goel A, Pirrotte P, Van Keuren-Jensen K, Jovanovic-Talisman T
JournalJ Extracell Vesicles
PubMed ID31741725
'Biofluid-accessible extracellular vesicles (EVs) may represent a new means to improve the sensitivity and specificity of detecting disease. However, current methods to isolate EVs encounter challenges when they are used to select specific populations. Moreover, it has been difficult to comprehensively characterize heterogeneous EV populations at the single vesicle level. ... More
Patient-Derived Head and Neck Cancer Organoids Recapitulate EGFR Expression Levels of Respective Tissues and Are Responsive to EGFR-Targeted Photodynamic Therapy.
AuthorsDriehuis E, Spelier S, Beltrán Hernández I, de Bree R, M Willems S, Clevers H, Oliveira S
JournalJ Clin Med
PubMed ID31694307
Patients diagnosed with head and neck squamous cell carcinoma (HNSCC) are currently treated with surgery and/or radio- and chemotherapy. Despite these therapeutic interventions, 40% of patients relapse, urging the need for more effective therapies. In photodynamic therapy (PDT), a light-activated photosensitizer produces reactive oxygen species that ultimately lead to cell ... More