Inducer-modulated cooperative binding of the tetrameric CggR repressor to operator DNA.
AuthorsZorrilla S,Doan T,Alfonso C,Margeat E,Ortega A,Rivas G,Aymerich S,Royer CA,Declerck N
JournalBiophysical journal
PubMed ID17293407
The central glycolytic genes repressor (CggR) controls the transcription of the gapA operon encoding five key glycolytic enzymes in Bacillus subtilis. CggR recognizes a unique DNA target sequence comprising two direct repeats and fructose-1,6-bisphosphate (FBP) is the inducer that negatively controls this interaction. We present here analytical ultracentrifugation and fluorescence ... More
Activity of human IgG and IgA subclasses in immune defense against Neisseria meningitidis serogroup B.
AuthorsVidarsson G,van Der Pol WL,van Den Elsen JM,Vilé H,Jansen M,Duijs J,Morton HC,Boel E,Daha MR,Corthésy B,van De Winkel JG
JournalJournal of immunology (Baltimore, Md. : 1950)
PubMed ID11342648
Single dose intranasal administration of retinal autoantigen generates a rapid accumulation and cell activation in draining lymph node and spleen: implications for tolerance therapy.
AuthorsDick AD, Sharma V, Liversidge J
JournalBr J Ophthalmol
PubMed ID11466262
BACKGROUND/AIMS: A single intranasal delivery of retinal autoantigen suppresses effectively experimental autoimmune uveoretinitis (EAU). To further unravel underlying mechanisms the authors wished to determine, firstly, the kinetics of antigen delivery and, secondly, the early cellular responses involved in the initial stages of nasal mucosal tolerance induction. METHODS: Flow cytometry, cell ... More
Reconstituted syntaxin1a/SNAP25 interacts with negatively charged lipids as measured by lateral diffusion in planar supported bilayers.
AuthorsWagner ML, Tamm LK
JournalBiophys J
PubMed ID11423412
According to the soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein (SNAP) receptor hypothesis (SNARE hypothesis), interactions between target SNAREs and vesicle SNAREs (t- and v-SNAREs) are required for membrane fusion in intracellular vesicle transport and exocytosis. The precise role of the SNAREs in tethering, docking, and fusion is still disputed. Biophysical measurements ... More
Tubulin equilibrium unfolding followed by time-resolved fluorescence and fluorescence correlation spectroscopy.
AuthorsSánchez SA, Brunet JE, Jameson DM, Lagos R, Monasterio O
JournalProtein Sci
PubMed ID14691224
The pathway for the in vitro equilibrium unfolding of the tubulin heterodimer by guanidinium chloride (GdmCl) has been studied using several spectroscopic techniques, specifically circular dichroism (CD), two-photon Fluorescence Correlation Spectroscopy (FCS), and time-resolved fluorescence, including lifetime and dynamic polarization. The results show that tubulin unfolding is characterized by distinct ... More
Segregation of nitrogen fixation and oxygenic photosynthesis in the marine cyanobacterium Trichodesmium.
AuthorsBerman-Frank I, Lundgren P, Chen YB, Küpper H, Kolber Z, Bergman B, Falkowski P
JournalScience
PubMed ID11711677
'In the modern ocean, a significant amount of nitrogen fixation is attributed to filamentous, nonheterocystous cyanobacteria of the genus Trichodesmium. In these organisms, nitrogen fixation is confined to the photoperiod and occurs simultaneously with oxygenic photosynthesis. Nitrogenase, the enzyme responsible for biological N2 fixation, is irreversibly inhibited by oxygen in ... More
Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation.
'We discovered that many proteins located in the kinetochore outer domain, but not the inner core, are depleted from kinetochores and accumulate at spindle poles when ATP production is suppressed in PtK1 cells, and that microtubule depolymerization inhibits this process. These proteins include the microtubule motors CENP-E and cytoplasmic dynein, ... More
KIFC3, a microtubule minus end-directed motor for the apical transport of annexin XIIIb-associated Triton-insoluble membranes.
AuthorsNoda Y, Okada Y, Saito N, Setou M, Xu Y, Zhang Z, Hirokawa N
JournalJ Cell Biol
PubMed ID11581287
'We have identified and characterized a COOH-terminal motor domain-type kinesin superfamily protein (KIFC), KIFC3, in the kidney. KIFC3 is a minus end-directed microtubule motor protein, therefore it accumulates in regions where minus ends of microtubules assemble. In polarized epithelial cells, KIFC3 is localized on membrane organelles immediately beneath the apical ... More
Compaction of the Escherichia coli nucleoid caused by Cyt1Aa.
AuthorsManasherob R, Zaritsky A, Metzler Y, Ben-Dov E, Itsko M, Fishov I
JournalMicrobiology
PubMed ID14663087
'Compaction of the Escherichia coli nucleoid in the cell''s centre was associated with the loss of colony-forming ability; these effects were caused by induction of Cyt1Aa, the cytotoxic 27 kDa protein from Bacillus thuringiensis subsp. israelensis. Cyt1Aa-affected compaction of the nucleoids was delayed but eventually more intense than compaction caused ... More
Differential localization of the centromere-specific proteins in the major centromeric satellite of Arabidopsis thaliana.
AuthorsShibata F, Murata M
JournalJ Cell Sci
PubMed ID15161939
'The 180 bp family of tandem repetitive sequences, which constitutes the major centromeric satellite in Arabidopsis thaliana, is thought to play important roles in kinetochore assembly. To assess the centromere activities of the 180 bp repeats, we performed indirect fluorescence immunolabeling with antibodies against phosphorylated histone H3 at Ser10, HTR12 ... More
The dibenzodioxocin lignin substructure is abundant in the inner part of the secondary wall in Norway spruce and silver birch xylem.
AuthorsKukkola EM, Koutaniemi S, Pöllänen E, Gustafsson M, Karhunen P, Lundell TK, Saranpää P, Kilpeläinen I, Teeri TH, Fagerstedt KV
JournalPlanta
PubMed ID13680231
'A specific condensed lignin substructure, dibenzodioxocin, was immunolocalized in differentiating cell walls of Norway spruce ( Picea abies (L.) H. Karsten) and silver birch ( Betula pendula Roth) xylem. A fluorescent probe, Alexa 488 was used as a marker on the dibenzodioxocin-specific secondary antibody. For the detection of this lignin ... More
Persistent cAMP-signals triggered by internalized G-protein-coupled receptors.
'G-protein-coupled receptors (GPCRs) are generally thought to signal to second messengers like cyclic AMP (cAMP) from the cell surface and to become internalized upon repeated or prolonged stimulation. Once internalized, they are supposed to stop signaling to second messengers but may trigger nonclassical signals such as mitogen-activated protein kinase (MAPK) ... More
Haptoglobin-related protein mediates trypanosome lytic factor binding to trypanosomes.
AuthorsDrain J, Bishop JR, Hajduk SL
JournalJ Biol Chem
PubMed ID11352898
'Trypanosome lytic factor (TLF-1) is an unusual high density lipoprotein (HDL) found in human serum that is toxic to Trypanosoma brucei brucei and may be critical in preventing human infections by this parasite. TLF-1 is composed of four major apolipoproteins: apolipoprotein AI, apolipoprotein AII, paraoxonase, and the primate-specific haptoglobin-related protein ... 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
Viral nanoparticles as tools for intravital vascular imaging.
AuthorsLewis JD, Destito G, Zijlstra A, Gonzalez MJ, Quigley JP, Manchester M, Stuhlmann H
JournalNat Med
PubMed ID16501571
'A significant impediment to the widespread use of noninvasive in vivo vascular imaging techniques is the current lack of suitable intravital imaging probes. We describe here a new strategy to use viral nanoparticles as a platform for the multivalent display of fluorescent dyes to image tissues deep inside living organisms. ... 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
Apoptosis of adherent cells by recruitment of caspase-8 to unligated integrins.
AuthorsStupack DG, Puente XS, Boutsaboualoy S, Storgard CM, Cheresh DA
JournalJ Cell Biol
PubMed ID11684710
'Integrin-mediated adhesion promotes cell survival in vitro, whereas integrin antagonists induce apoptosis of adherent cells in vivo. Here, we demonstrate that cells adherent within a three-dimensional extracellular matrix undergo apoptosis due to expression of unligated integrins, the beta subunit cytoplasmic domain, or its membrane proximal sequence KLLITIHDRKEF. Integrin-mediated death requires ... More
Deconvolving single-molecule intensity distributions for quantitative microscopy measurements.
AuthorsMutch SA, Fujimoto BS, Kuyper CL, Kuo JS, Bajjalieh SM, Chiu DT,
JournalBiophys J
PubMed ID17259276
'In fluorescence microscopy, images often contain puncta in which the fluorescent molecules are spatially clustered. This article describes a method that uses single-molecule intensity distributions to deconvolve the number of fluorophores present in fluorescent puncta as a way to "count" protein number. This method requires a determination of the correct ... More
Two-photon fluorescence absorption and emission spectra of dyes relevant for cell imaging.
AuthorsBestvater F, Spiess E, Stobrawa G, Hacker M, Feurer T, Porwol T, Berchner-Pfannschmidt U, Wotzlaw C, Acker H
JournalJ Microsc
PubMed ID12423261
'Two-photon absorption and emission spectra for fluorophores relevant in cell imaging were measured using a 45 fs Ti:sapphire laser, a continuously tuneable optical parametric amplifier for the excitation range 580-1150 nm and an optical multichannel analyser. The measurements included DNA stains, fluorescent dyes coupled to antibodies as well as organelle ... More
Intracellular and extracellular cathepsin B facilitate invasion of MCF-10A neoT cells through reconstituted extracellular matrix in vitro.
AuthorsPremzl A, Zavasnik-Bergant V, Turk V, Kos J
JournalExp Cell Res
PubMed ID12581740
'Lysosomal cysteine proteinase cathepsin B is implicated in remodeling the extracellular matrix, a crucial step in the process of tumor cell invasion. In this study the contributions of intracellular and extracellular cathepsin B activities in the invasion of ras-transformed human breast epithelial cells, MCF-10A neoT, were assessed using specific cathepsin ... More
VASP governs actin dynamics by modulating filament anchoring.
AuthorsTrichet L, Campàs O, Sykes C, Plastino J
JournalBiophys J
PubMed ID17098798
'Actin filament dynamics at the cell membrane are important for cell-matrix and cell-cell adhesions and the protrusion of the leading edge. Since actin filaments must be connected to the cell membrane to exert forces but must also detach from the membrane to allow it to move and evolve, the balance ... 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
Serum amyloid A is a ligand for scavenger receptor class B type I and inhibits high density lipoprotein binding and selective lipid uptake.
AuthorsCai L, de Beer MC, de Beer FC, van der Westhuyzen DR
JournalJ Biol Chem
PubMed ID15561721
'Serum amyloid A is an acute phase protein that is carried in the plasma largely as an apolipoprotein of high density lipoprotein (HDL). In this study we investigated whether SAA is a ligand for the HDL receptor, scavenger receptor class B type I (SR-BI), and how SAA may influence SR-BI-mediated ... More
Physical interactions of the peroxisomal targeting signal 1 receptor pex5p, studied by fluorescence correlation spectroscopy.
AuthorsWang D, Visser NV, Veenhuis M, van der Klei IJ
JournalJ Biol Chem
PubMed ID12930827
'We have studied Hansenula polymorpha Pex5p and Pex8p using fluorescence correlation spectroscopy (FCS). Pex5p is the Peroxisomal Targeting Signal 1 (PTS1) receptor and Pex8p is an intraperoxisomal protein. Both proteins are essential for PTS1 protein import and have been shown to physically interact. We used FCS to analyze the molecular ... More
The kinetics of phagosome maturation as a function of phagosome/lysosome fusion and acquisition of hydrolytic activity.
AuthorsYates RM, Hermetter A, Russell DG
JournalTraffic
PubMed ID15813751
'Professional phagocytes function at the hinge of innate and acquired immune responses by internalizing particulate material that is digested and sampled within the phagosome of the cell. Despite intense interest, assays to measure phagosome maturation remain insensitive and few in number. In this current study, we describe three novel assays ... More
Analysis of proteins stained by Alexa dyes.
AuthorsHuang S, Wang H, Carroll CA, Hayes SJ, Weintraub ST, Serwer P
JournalElectrophoresis
PubMed ID15004835
'Alexa dye staining of proteins is used for the fluorescence microscopy of single particles that are sometimes multimolecular protein complexes. To characterize the staining, post-staining determination must be made of which protein(s) in a complex have been Alexa-stained. The present communication describes the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis ... More
Temporally resolved interactions between antigen-stimulated IgE receptors and Lyn kinase on living cells.
AuthorsLarson DR, Gosse JA, Holowka DA, Baird BA, Webb WW
JournalJ Cell Biol
PubMed ID16275755
'Upon cross-linking by antigen, the high affinity receptor for immunoglobulin E (IgE), FcepsilonRI, is phosphorylated by the Src family tyrosine kinase Lyn to initiate mast cell signaling, leading to degranulation. Using fluorescence correlation spectroscopy (FCS), we observe stimulation-dependent associations between fluorescently labeled IgE-FcepsilonRI and Lyn-EGFP on individual cells. We also ... More
Spectroscopic investigation of a FRET molecular beacon containing two fluorophores for probing DNA/RNA sequences.
AuthorsJockusch S, Martí AA, Turro NJ, Li Z, Li X, Ju J, Stevens N, Akins DL
JournalPhotochem Photobiol Sci
PubMed ID16685327
'We report the design, synthesis, and characterization of a molecular beacon (MB) consisting of two fluorescent dyes (Alexa 488 and RedX) for DNA and RNA analysis. In the absence of the target DNA or RNA the MB is in its stem-closed form and shows efficient energy transfer from the donor ... More
Direct stochastic optical reconstruction microscopy with standard fluorescent probes.
Authorsvan de Linde S, Löschberger A, Klein T, Heidbreder M, Wolter S, Heilemann M, Sauer M,
JournalNat Protoc
PubMed ID21720313
'Direct stochastic optical reconstruction microscopy (dSTORM) uses conventional fluorescent probes such as labeled antibodies or chemical tags for subdiffraction resolution fluorescence imaging with a lateral resolution of ~20 nm. In contrast to photoactivated localization microscopy (PALM) with photoactivatable fluorescent proteins, dSTORM experiments start with bright fluorescent samples in which the ... More
In vivo passage of albumin from the aqueous humor into the lens.
AuthorsSabah JR, Davidson H, McConkey EN, Takemoto L
JournalMol Vis
PubMed ID15073582
'PURPOSE: To determine if albumin, the major protein component of the aqueous humor, passes into the lens in vivo. METHODS: Rat albumin was covalently-labeled with Alexa 488 fluorophore, purified by gel permeation chromatography, then injected into the aqueous chamber of living rats. At 5 min postinjection, lenses were removed and ... 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
A fluorescence assay for peptide translocation into mitochondria.
AuthorsMartinez-Caballero S, Peixoto PM, Kinnally KW, Campo ML
JournalAnal Biochem
PubMed ID17240346
'Translocation of the presequence is an early event in import of preproteins across the mitochondrial inner membrane by the TIM23 complex. Import of signal peptides, whose sequences mimic mitochondrial import presequences, was measured using a novel, qualitative, fluorescence assay in about 1h. This peptide assay was used in conjunction with ... More
Single-molecule investigation of the T4 bacteriophage DNA polymerase holoenzyme: multiple pathways of holoenzyme formation.
'In T4 bacteriophage, the DNA polymerase holoenzyme is responsible for accurate and processive DNA synthesis. The holoenzyme consists of DNA polymerase gp43 and clamp protein gp45. To form a productive holoenzyme complex, clamp loader protein gp44/62 is required for the loading of gp45, along with MgATP, and also for the ... More
Ultrasensitive detection of pathological prion protein aggregates by dual-color scanning for intensely fluorescent targets.
AuthorsBieschke J, Giese A, Schulz-Schaeffer W, Zerr I, Poser S, Eigen M, Kretzschmar H
JournalProc Natl Acad Sci U S A
PubMed ID10805803
'A definite diagnosis of prion diseases such as Creutzfeldt-Jakob disease (CJD) relies on the detection of pathological prion protein (PrP(Sc)). However, no test for PrP(Sc) in cerebrospinal fluid (CSF) has been available thus far. Based on a setup for confocal dual-color fluorescence correlation spectroscopy, a technique suitable for single molecule ... More
Polymerization kinetics of ADP- and ADP-Pi-actin determined by fluorescence microscopy.
AuthorsFujiwara I, Vavylonis D, Pollard TD
JournalProc Natl Acad Sci U S A
PubMed ID17517656
'We used fluorescence microscopy to determine how polymerization of Mg-ADP-actin depends on the concentration of phosphate. From the dependence of the elongation rate on the actin concentration and direct observations of depolymerizing filaments, we measured the polymerization rate constants of ADP-actin and ADP-P(i)-actin. Saturating phosphate reduces the critical concentration for ... More
Coiled-coil tag--probe system for quick labeling of membrane receptors in living cell.
AuthorsYano Y, Yano A, Oishi S, Sugimoto Y, Tsujimoto G, Fujii N, Matsuzaki K,
JournalACS Chem Biol
PubMed ID18533657
'The specific labeling of proteins in living cells using a genetically encodable tag and a small synthetic probe targeting the tag has been craved as an alternative to widely used larger fluorescent proteins. We describe a rapid method with a small tag (21 amino acids) for the fluorescence labeling of ... 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
Changes of phagocytic capacity in basic fibroblast growth factor-transfected iris pigment epithelial cells in rats.
AuthorsSakuragi M, Tomita H, Abe T, Tamai M
JournalCurr Eye Res
PubMed ID11803480
'PURPOSE: To evaluate differences of phagocytic capacities in rat retinal pigment epithelial (RPE) cells, iris pigment epithelial (IPE) cells, and basic fibroblast growth factor (bFGF) cDNA-transfected IPE (bFGF-IPE) cells in vitro. METHODS: The RPE cells and IPE cells were isolated from adult Long Evans rats'' eyes. The bFGF cDNA was ... More
Fluorescence assays for F-pili and their application.
'Conjugative pili are extracellular filaments elaborated by Gram-negative bacteria expressing certain type IV secretion systems. They are required at the earliest stages of conjugal DNA transfer to establish specific and secure cell-cell contacts. Conjugative pili also serve as adsorption organelles for both RNA and DNA bacteriophages. Beyond these facts, the ... More
Toxicity of organic fluorophores used in molecular imaging: literature review.
AuthorsAlford R, Simpson HM, Duberman J, Hill GC, Ogawa M, Regino C, Kobayashi H, Choyke PL,
JournalMol Imaging
PubMed ID20003892
'Fluorophores are potentially useful for in vivo cancer diagnosis. Using relatively inexpensive and portable equipment, optical imaging with fluorophores permits real-time detection of cancer. However, fluorophores can be toxic and must be investigated before they can be administered safely to patients. A review of published literature on the toxicity of ... More
A kinome-wide siRNA screen identifies multiple roles for protein kinases in hypoxic stress adaptation, including roles for IRAK4 and GAK in protection against apoptosis in VHL-/- renal carcinoma cells, despite activation of the NF-?B pathway.
AuthorsPan J, Zhang J, Hill A, Lapan P, Berasi S, Bates B, Miller C, Haney S,
JournalJ Biomol Screen
PubMed ID23591012
'Hypoxia induces changes to cancer cells that make them more resistant to treatment. We have looked at signaling pathways that facilitate these changes by screening the human kinome for effects on hypoxic responses in SW480 colon cancer cells. Hits identified in the screen were examined for effects on multiple molecular ... 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
Remodeling of the bacterial RNA polymerase supramolecular complex in response to environmental conditions.
AuthorsVerma S, Xiong Y, Mayer MU, Squier TC
JournalBiochemistry
PubMed ID17319694
'Directed binding of RNA polymerase to distinct promoter elements controls transcription and promotes adaptive responses to changing environmental conditions. To identify proteins that modulate transcription, we have expressed a tagged alpha-subunit of RNA polymerase in Shewanella oneidensis under controlled growth conditions, isolated the protein complex using newly developed multiuse affinity ... More
Optical properties of Alexa 488 and Cy5 immobilized on a glass surface.
AuthorsWang L, Gaigalas AK, Reipa V
JournalBiotechniques
PubMed ID15679095
'The absorption and emission spectra were measured for Cy5 and Alexa 488 fluorophores confined on a glass surface. The data were obtained using fluorometry and spectroscopic ellipsometry. Red shifts of the surface-immobilized fluorophore absorption spectra relative to the fluorophore spectra in aqueous solution were observed using both methods. We interpret ... More
Conformational transitions as determinants of specificity for the DNA methyltransferase EcoRI.
AuthorsYoungblood B, Reich NO
JournalJ Biol Chem
PubMed ID16845123
'Changes in DNA bending and base flipping in a previously characterized specificity-enhanced M.EcoRI DNA adenine methyltransferase mutant suggest a close relationship between precatalytic conformational transitions and specificity (Allan, B. W., Garcia, R., Maegley, K., Mort, J., Wong, D., Lindstrom, W., Beechem, J. M., and Reich, N. O. (1999) J. Biol. ... More
Zona pellucida protein binding ability of porcine sperm during epididymal maturation and the acrosome reaction.
AuthorsBurkin HR, Miller DJ
JournalDev Biol
PubMed ID10885749
'In many mammals, the first interaction between gametes during fertilization occurs when sperm contact the zona pellucida surrounding the egg. Although porcine sperm first contact the zona pellucida via their plasma membrane, the regions of the sperm surface that display zona receptors have not been determined. We have used the ... More
Interdependent assembly of specific regulatory lipids and membrane fusion proteins into the vertex ring domain of docked vacuoles.
AuthorsFratti RA, Jun Y, Merz AJ, Margolis N, Wickner W
JournalJ Cell Biol
PubMed ID15611334
'Membrane microdomains are assembled by lipid partitioning (e.g., rafts) or by protein-protein interactions (e.g., coated vesicles). During docking, yeast vacuoles assemble "vertex" ring-shaped microdomains around the periphery of their apposed membranes. Vertices are selectively enriched in the Rab GTPase Ypt7p, the homotypic fusion and vacuole protein sorting complex (HOPS)-VpsC Rab ... More
Viewing dynamic assembly of molecular complexes by multi-wavelength single-molecule fluorescence.
AuthorsFriedman LJ, Chung J, Gelles J
JournalBiophys J
PubMed ID16698779
'Complexes of macromolecules that transiently self-assemble, perform a particular function, and then dissociate are a recurring theme in biology. Such systems often have a large number of possible assembly/disassembly intermediates and complex, highly branched reaction pathways. Measuring the single-step kinetic parameters in these reactions would help to identify the functionally ... More
Evidence for a Trypanosoma brucei lipoprotein scavenger receptor.
AuthorsGreen HP, Del Pilar Molina Portela M, St Jean EN, Lugli EB, Raper J
JournalJ Biol Chem
PubMed ID12401813
'African trypanosomes are lipid auxotrophs that live in the bloodstream of their human and animal hosts. Trypanosomes require lipoproteins in addition to other serum components in order to multiply under axenic culture conditions. Delipidation of the lipoproteins abrogates their capacity to support trypanosome growth. Both major classes of serum lipoproteins, ... More
The carboxyl terminus of the alpha-subunit of the amiloride-sensitive epithelial sodium channel binds to F-actin.
AuthorsMazzochi C, Bubien JK, Smith PR, Benos DJ
JournalJ Biol Chem
PubMed ID16356937
'The activity of the amiloride-sensitive epithelial sodium channel (ENaC) is modulated by F-actin. However, it is unknown if there is a direct interaction between alpha-ENaC and actin. We have investigated the hypothesis that the actin cytoskeleton directly binds to the carboxyl terminus of alpha-ENaC using a combination of confocal microscopy, ... More
Adenovirus-facilitated nuclear translocation of adeno-associated virus type 2.
AuthorsXiao W, Warrington KH, Hearing P, Hughes J, Muzyczka N
JournalJ Virol
PubMed ID12388712
'We examined cytoplasmic trafficking and nuclear translocation of adeno-associated virus type 2 (AAV) by using Alexa Fluor 488-conjugated wild-type AAV, A20 monoclonal antibody immunocytochemistry, and subcellular fractionation techniques followed by DNA hybridization. Our results indicated that in the absence of adenovirus (Ad), AAV enters the cell rapidly and escapes from ... More
Internalization of exogenously added memapsin 2 (beta-secretase) ectodomain by cells is mediated by amyloid precursor protein.
'Memapsin 2 (beta-secretase) is the protease that initiates cleavage of amyloid precursor protein (APP) leading to the production of amyloid-beta (Abeta) peptide and the onset of Alzheimer''s disease. Both APP and memapsin 2 are Type I transmembrane proteins and are endocytosed into endosomes where APP is cleaved by memapsin 2. ... More
Isolation of anti-CD22 Fv with high affinity by Fv display on human cells.
AuthorsHo M, Nagata S, Pastan I
JournalProc Natl Acad Sci U S A
PubMed ID16763048
'In vitro antibody affinity maturation has generally been achieved by display of mouse or human antibodies on the surface of microorganisms (phage, bacteria, and yeast). However, problems with protein folding, posttranslational modification, and codon usage still limit the number of improved antibodies that can be obtained. An ideal system would ... More
An anti-transferrin receptor-avidin fusion protein exhibits both strong proapoptotic activity and the ability to deliver various molecules into cancer cells.
AuthorsNg PP, Dela Cruz JS, Sorour DN, Stinebaugh JM, Shin SU, Shin DS, Morrison SL, Penichet ML
JournalProc Natl Acad Sci U S A
PubMed ID12149472
'We have developed an antibody fusion protein (anti-rat TfR IgG3-Av) with the ability to deliver different molecules into cancer cells. It consists of avidin genetically fused to the C(H)3 region of a human IgG3 specific for the rat transferrin receptor. It forms strong, noncovalent interactions with biotinylated molecules such as ... More
Zero mode waveguides for single-molecule spectroscopy on lipid membranes.
'Zero mode waveguides (ZMWs), subwavelength optical nanostructures with dimensions ranging from 50 to 200 nm, have been used to study systems involving ligand-receptor interactions. We show that under proper conditions, lipid membranes will invaginate into the nanostructures, which confine optical excitation to subattoliter volumes. Fluorescence correlation spectroscopy (FCS) was used ... More
Diffusion of single cardiac ryanodine receptors in lipid bilayers is decreased by annexin 12.
AuthorsPeng S, Publicover NG, Airey JA, Hall JE, Haigler HT, Jiang D, Chen SR, Sutko JL
JournalBiophys J
PubMed ID14695258
'Diffusion of cardiac ryanodine receptors (RyR2) in lipid bilayers was characterized. RyR2 location was monitored by imaging fluo-3 fluorescence due to Ca2+ flux through RyR2 channels or fluorescence from RyR2 conjugated with Alexa 488 or containing green fluorescent protein. Single channel currents were recorded to ensure that functional channels were ... More
Functional and structural characterization of synthetic HIV-1 Vpr that transduces cells, localizes to the nucleus, and induces G2 cell cycle arrest.
AuthorsHenklein P, Bruns K, Sherman MP, Tessmer U, Licha K, Kopp J, de Noronha CM, Greene WC, Wray V, Schubert U
JournalJ Biol Chem
PubMed ID10903315
'Human immunodeficiency virus (HIV) Vpr contributes to nuclear import of the viral pre-integration complex and induces G(2) cell cycle arrest. We describe the production of synthetic Vpr that permitted the first studies on the structure and folding of the full-length protein. Vpr is unstructured at neutral pH, whereas under acidic ... More
Two-photon fluorescence coincidence analysis: rapid measurements of enzyme kinetics.
AuthorsHeinze KG, Rarbach M, Jahnz M, Schwille P
JournalBiophys J
PubMed ID12202390
'Dual-color fluorescence cross-correlation analysis is a powerful tool for probing interactions of different fluorescently labeled molecules in aqueous solution. The concept is the selective observation of coordinated spontaneous fluctuations in two separate detection channels that unambiguously reflect the existence of physical or chemical linkages among the different fluorescent species. It ... More
Crystal structure of the complete integrin alphaVbeta3 ectodomain plus an alpha/beta transmembrane fragment.
'We determined the crystal structure of 1TM-alphaVbeta3, which represents the complete unconstrained ectodomain plus short C-terminal transmembrane stretches of the alphaV and beta3 subunits. 1TM-alphaVbeta3 is more compact and less active in solution when compared with DeltaTM-alphaVbeta3, which lacks the short C-terminal stretches. The structure reveals a bent conformation and ... More
Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling.
AuthorsKrutzik PO, Nolan GP
JournalNat Methods
PubMed ID16628206
'Flow cytometry allows high-content, multiparameter analysis of single cells, making it a promising tool for drug discovery and profiling of intracellular signaling. To add high-throughput capacity to flow cytometry, we developed a cell-based multiplexing technique called fluorescent cell barcoding (FCB). In FCB, each sample is labeled with a different signature, ... More
Rapid isolation of high-affinity protein binding peptides using bacterial display.
AuthorsBessette PH, Rice JJ, Daugherty PS
JournalProtein Eng Des Sel
PubMed ID15531628
'A robust bacterial display methodology was developed that allows the rapid isolation of peptides that bind to arbitrarily selected targets with high affinity. To demonstrate the utility of this approach, a large library (5 x 10(10) clones) was constructed composed of random 15-mer peptide insertions constrained within a flexible, surface ... More
Interaction of dihydrofolate reductase with methotrexate: ensemble and single-molecule kinetics.
'The thermodynamics and kinetics of the interaction of dihydrofolate reductase (DHFR) with methotrexate have been studied by using fluorescence, stopped-flow, and single-molecule methods. DHFR was modified to permit the covalent addition of a fluorescent molecule, Alexa 488, and a biotin at the N terminus of the molecule. The fluorescent molecule ... 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
Monitoring the permeability of the nuclear envelope during the cell cycle.
AuthorsLénárt P, Ellenberg J,
JournalMethods
PubMed ID16343937
'In animal organisms the nuclear envelope (NE) dis-assembles during cell division resulting in complete intermixing of cytoplasmic and nuclear compartments. This leads to the activation of many mitotic enzymes, which were kept away from their substrates or regulators by nuclear or cytoplasmic sequestration in interphase. Nuclear envelope breakdown (NEBD) is ... More
Subdiffraction-resolution fluorescence microscopy reveals a domain of the centrosome critical for pericentriolar material organization.
AuthorsMennella V, Keszthelyi B, McDonald KL, Chhun B, Kan F, Rogers GC, Huang B, Agard DA,
JournalNat Cell Biol
PubMed ID23086239
'As the main microtubule-organizing centre in animal cells, the centrosome has a fundamental role in cell function. Surrounding the centrioles, the pericentriolar material (PCM) provides a dynamic platform for nucleating microtubules. Although the importance of the PCM is established, its amorphous electron-dense nature has made it refractory to structural investigation. ... More
Differential impact of caveolae and caveolin-1 scaffolds on the membrane raft proteome.
AuthorsZheng YZ, Boscher C, Inder KL, Fairbank M, Loo D, Hill MM, Nabi IR, Foster LJ,
JournalMol Cell Proteomics
PubMed ID21753190
'Caveolae, a class of cholesterol-rich lipid rafts, are smooth invaginations of the plasma membrane whose formation in nonmuscle cells requires caveolin-1 (Cav1). The recent demonstration that Cav1-associated cavin proteins, in particular PTRF/cavin-1, are also required for caveolae formation supports a functional role for Cav1 independently of caveolae. In tumor cells ... 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
Spatial organization of bacteriorhodopsin in model membranes. Light-induced mobility changes.
AuthorsKahya N, Wiersma DA, Poolman B, Hoekstra D
JournalJ Biol Chem
PubMed ID12167614
'Bacteriorhodopsin is a proton-transporting membrane protein in Halophilic archaea, and it is considered a prototype of membrane transporters and a model for G-protein-coupled receptors. Oligomerization of the protein has been reported, but it is unknown whether this feature is correlated with, for instance, light activation. Here, we have addressed this ... More
Printing proteins as microarrays for high-throughput function determination.
AuthorsMacBeath G, Schreiber SL
JournalScience
PubMed ID10976071
'Systematic efforts are currently under way to construct defined sets of cloned genes for high-throughput expression and purification of recombinant proteins. To facilitate subsequent studies of protein function, we have developed miniaturized assays that accommodate extremely low sample volumes and enable the rapid, simultaneous processing of thousands of proteins. A ... More
Cholesterol depletion induces large scale domain segregation in living cell membranes.
AuthorsHao M, Mukherjee S, Maxfield FR
JournalProc Natl Acad Sci U S A
PubMed ID11698680
'Local inhomogeneities in lipid composition play a crucial role in regulation of signal transduction and membrane traffic. Nevertheless, most evidence for microdomains in cells remains indirect, and the nature of membrane inhomogeneities has been difficult to characterize. We used lipid analogs and lipid-anchored proteins with varying fluidity preferences to examine ... 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
Optimizing primer--probe design for fluorescent PCR.
AuthorsProudnikov D, Yuferov V, Zhou Y, LaForge KS, Ho A, Kreek MJ
JournalJ Neurosci Methods
PubMed ID12581847
'TaqMan, a variation of fluorescent PCR, is a powerful tool for gene expression and polymorphism studies. Here we describe the design and evaluation of 27 new TaqMan primer-probe sets for rat genes that play a key role in neural signaling. These newly designed and synthesized probes were tested and then ... 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
FRET measurements of cell-traction forces and nano-scale clustering of adhesion ligands varied by substrate stiffness.
AuthorsKong HJ, Polte TR, Alsberg E, Mooney DJ
JournalProc Natl Acad Sci U S A
PubMed ID15767572
'The mechanical properties of cell adhesion substrates regulate cell phenotype, but the mechanism of this relation is currently unclear. It may involve the magnitude of traction force applied by the cell, and/or the ability of the cells to rearrange the cell adhesion molecules presented from the material. In this study, ... More
Fluorescence resonance energy transfer studies on anthrax lethal toxin.
AuthorsCroney JC, Cunningham KM, Collier RJ, Jameson DM
JournalFEBS Lett
PubMed ID12935906
'Anthrax lethal toxin is a binary bacterial toxin consisting of two proteins, protective antigen (PA) and lethal factor (LF), that self-assemble on receptor-bearing eukaryotic cells to form toxic, non-covalent complexes. PA(63), a proteolytically activated form of PA, spontaneously oligomerizes to form ring-shaped heptamers that bind LF and translocate it into ... More
Analysis of retrograde transport in motor neurons reveals common endocytic carriers for tetanus toxin and neurotrophin receptor p75NTR.
AuthorsLalli G, Schiavo G
JournalJ Cell Biol
PubMed ID11807088
'Axonal retrograde transport is essential for neuronal growth and survival. However, the nature and dynamics of the membrane compartments involved in this process are poorly characterized. To shed light on this pathway, we established an experimental system for the visualization and the quantitative study of retrograde transport in living motor ... More
H-type dimer formation of fluorophores: a mechanism for activatable, in vivo optical molecular imaging.
AuthorsOgawa M, Kosaka N, Choyke PL, Kobayashi H,
JournalACS Chem Biol
PubMed ID19480464
'In vivo molecular imaging with target-specific activatable '
Accumulation of Mad2-Cdc20 complex during spindle checkpoint activation requires binding of open and closed conformers of Mad2 in Saccharomyces cerevisiae.
AuthorsNezi L, Rancati G, De Antoni A, Pasqualato S, Piatti S, Musacchio A
JournalJ Cell Biol
PubMed ID16818718
'The spindle assembly checkpoint (SAC) coordinates mitotic progression with sister chromatid alignment. In mitosis, the checkpoint machinery accumulates at kinetochores, which are scaffolds devoted to microtubule capture. The checkpoint protein Mad2 (mitotic arrest deficient 2) adopts two conformations: open (O-Mad2) and closed (C-Mad2). C-Mad2 forms when Mad2 binds its checkpoint ... More
Visualization of galectin-3 oligomerization on the surface of neutrophils and endothelial cells using fluorescence resonance energy transfer.
AuthorsNieminen J, Kuno A, Hirabayashi J, Sato S
JournalJ Biol Chem
PubMed ID17082191
'Galectin-3, a member of the galectin family of carbohydrate binding proteins, is widely expressed, particularly in cells involved in the immune response. Galectin-3 has also been indicated to play a role in various biological activities ranging from cell repression to cell activation and adhesion and has, thus, been recognized as ... More
Production of Alexa Fluor 488-labeled reovirus and characterization of target cell binding, competence, and immunogenicity of labeled virions.
AuthorsFecek RJ, Busch R, Lin H, Pal K, Cunningham CA, Cuff CF
JournalJ Immunol Methods
PubMed ID16822520
'Respiratory enteric orphan virus (reovirus) has been used to study many aspects of the biology and genetics of viruses, viral infection, pathogenesis, and the immune response to virus infection. This report describes the functional activity of virus labeled with Alexa Fluor 488, a stable fluorescent dye. Matrix assisted laser desorption-time ... More
Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes.
'Live cell imaging is a powerful method to study protein dynamics at the cell surface, but conventional imaging probes are bulky, or interfere with protein function, or dissociate from proteins after internalization. Here, we report technology for covalent, specific tagging of cellular proteins with chemical probes. Through rational design, we ... More
AuthorsSabirsh A, Wetterholm A, Bristulf J, Leffler H, Haeggström JZ, Owman C
JournalJ Lipid Res
PubMed ID15805550
'Leukotriene B4 (LTB4) is a potent lipid mediator of inflammation that acts primarily via a seven-transmembrane-spanning, G-protein-coupled receptor denoted BLT1. Here, we describe the synthesis and characterization of fluorescent analogs of LTB4 that are easy to produce, inexpensive, and without the disadvantages of a radioligand. Fluorescent LTB4 is useful for ... More
Single-molecule and transient kinetics investigation of the interaction of dihydrofolate reductase with NADPH and dihydrofolate.
'The interaction of dihydrofolate (H(2)F) and NADPH with a fluorescent derivative of H(2)F reductase (DHFR) was studied by using transient and single-molecule techniques. The fluorescent moiety Alexa 488 was attached to the structural loop that closes over the substrates after they are bound. Fluorescence quenching was found to accompany the ... More
Differential regulation of doxorubicin-induced mitochondrial dysfunction and apoptosis by Bcl-2 in mammary adenocarcinoma (MTLn3) cells.
AuthorsHuigsloot M, Tijdens IB, Mulder GJ, van de Water B
JournalJ Biol Chem
PubMed ID12107157
'Various anticancer drugs cause mitochondrial perturbations in association with apoptosis. Here we investigated the involvement of caspase- and Bcl-2-dependent pathways in doxorubicin-induced mitochondrial perturbations and apoptosis. For this purpose, we set up a novel three-color flow cytometric assay using rhodamine 123, annexin V-allophycocyanin, and propidium iodide to assess the involvement ... 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
Small-molecule mimics of an alpha-helix for efficient transport of proteins into cells.
AuthorsOkuyama M, Laman H, Kingsbury SR, Visintin C, Leo E, Eward KL, Stoeber K, Boshoff C, Williams GH, Selwood DL
JournalNat Methods
PubMed ID17220893
'We designed and synthesized small-molecule mimics of an alpha-helical peptide protein transduction domain (PTD). These small-molecule carriers, which we termed SMoCs, are easily coupled to biomolecules, and efficiently deliver dye molecules and recombinant proteins into a variety of cell types. We designed the SMoCs using molecular modeling techniques. As an ... More
Binding of Plasmodium falciparum-infected erythrocytes to soluble platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31): frequent recognition by clinical isolates.
AuthorsHeddini A, Chen Q, Obiero J, Kai O, Fernandez V, Marsh K, Muller WA, Wahlgren M
JournalAm J Trop Med Hyg
PubMed ID11504407
'Platelet-endothelial cell adhesion molecule-1 or CD31 (PECAM-1/CD31) is a receptor recognized by Plasmodium falciparum-parasitized erythrocytes (pRBCs). Fluorescence-labeled soluble recombinant PECAM-1/CD31 (sPECAM-1/CD31) is shown to bind to the surface of P. falciparum-infected erythrocytes on up to 70% of the cells. Binding is blocked by the addition of the unlabeled receptor in ... More
Receptor-dependent and -independent axonal retrograde transport of poliovirus in motor neurons.
AuthorsOhka S, Sakai M, Bohnert S, Igarashi H, Deinhardt K, Schiavo G, Nomoto A,
JournalJ Virol
PubMed ID19244317
'Poliovirus (PV), when injected intramuscularly into the calf, is incorporated into the sciatic nerve and causes an initial paralysis of the inoculated limb in transgenic (Tg) mice carrying the human PV receptor (hPVR/CD155) gene. We have previously demonstrated that a fast retrograde axonal transport process is required for PV dissemination ... More
Optically directed molecular transport and 3D isoelectric positioning of amphoteric biomolecules.
AuthorsHafeman DG, Harkins JB, Witkowski CE, Lewis NS, Warmack RJ, Brown GM, Thundat T
JournalProc Natl Acad Sci U S A
PubMed ID16618926
'We demonstrate the formation of charged molecular packets and their transport within optically created electrical force-field traps in a pH-buffered electrolyte. We call this process photoelectrophoretic localization and transport (PELT). The electrolyte is in contact with a photoconductive semiconductor electrode and a counterelectrode that are connected through an external circuit. ... More
Editing antigen presentation: antigen transfer between human B lymphocytes and macrophages mediated by class A scavenger receptors.
AuthorsHarvey BP, Quan TE, Rudenga BJ, Roman RM, Craft J, Mamula MJ,
JournalJ Immunol
PubMed ID18768860
'B lymphocytes can function independently as efficient APCs. However, our previous studies demonstrate that both dendritic cells and macrophages are necessary to propagate immune responses initiated by B cell APCs. This finding led us to identify a process in mice whereby Ag-specific B cells transfer Ag to other APCs. In ... 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
Cortical constriction during abscission involves helices of ESCRT-III-dependent filaments.
AuthorsGuizetti J, Schermelleh L, Mäntler J, Maar S, Poser I, Leonhardt H, Müller-Reichert T, Gerlich DW,
JournalScience
PubMed ID21310966
'After partitioning of cytoplasmic contents by cleavage furrow ingression, animal cells remain connected by an intercellular bridge, which subsequently splits by abscission. Here, we examined intermediate stages of abscission in human cells by using live imaging, three-dimensional structured illumination microscopy, and electron tomography. We identified helices of 17-nanometer-diameter filaments, which ... 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
Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations.
AuthorsSekar RB, Periasamy A
JournalJ Cell Biol
PubMed ID12615908
The current advances in fluorescence microscopy, coupled with the development of new fluorescent probes, make fluorescence resonance energy transfer (FRET) a powerful technique for studying molecular interactions inside living cells with improved spatial (angstrom) and temporal (nanosecond) resolution, distance range, and sensitivity and a broader range of biological applications. ... More
Sharper low-power STED nanoscopy by time gating.
AuthorsVicidomini G, Moneron G, Han KY, Westphal V, Ta H, Reuss M, Engelhardt J, Eggeling C, Hell SW,
JournalNat Methods
PubMed ID21642963
Applying pulsed excitation together with time-gated detection improves the fluorescence on-off contrast in continuous-wave stimulated emission depletion (CW-STED) microscopy, thus revealing finer details in fixed and living cells using moderate light intensities. This method also enables super-resolution fluorescence correlation spectroscopy with CW-STED beams, as demonstrated by quantifying the dynamics of ... More
AuthorsIngaramo M, York AG, Wawrzusin P, Milberg O, Hong A, Weigert R, Shroff H, Patterson GH,
Journal
PubMed ID24706872
Multifocal structured illumination microscopy (MSIM) provides a twofold resolution enhancement beyond the diffraction limit at sample depths up to 50 µm, but scattered and out-of-focus light in thick samples degrades MSIM performance. Here we implement MSIM with a microlens array to enable efficient two-photon excitation. Two-photon MSIM gives resolution-doubled images ... More
Molecular perspective of antigen-mediated mast cell signaling.
AuthorsDavey AM, Krise KM, Sheets ED, Heikal AA,
JournalJ Biol Chem
PubMed ID18093971
Antigen-mediated cross-linking of the high affinity receptor for IgE (Fc epsilon RI), in the plasma membrane of mast cells, is the first step in the allergic immune response. This event triggers the phosphorylation of specific tyrosines in the cytoplasmic segments of the beta and gamma subunits of Fc epsilon RI ... More
Functional delivery of a cytosolic tRNA into mutant mitochondria of human cells.
AuthorsMahata B, Mukherjee S, Mishra S, Bandyopadhyay A, Adhya S
JournalScience
PubMed ID17053148
Many maternally inherited and incurable neuromyopathies are caused by mutations in mitochondrial (mt) transfer RNA (tRNA) genes. Kinetoplastid protozoa, including Leishmania, have evolved specialized systems for importing nucleus-encoded tRNAs into mitochondria. We found that the Leishmania RNA import complex (RIC) could enter human cells by a caveolin-1-dependent pathway, where it ... More
Electrochemical release of fluorescently labeled thiols from patterned gold surfaces.
AuthorsGhaly T, Wildt BE, Searson PC,
JournalLangmuir
PubMed ID19902935
Reductive desorption of alkanethiols is a tool for spatially and temporally controlled release of small molecules or particles from individually addressable gold electrodes. Here we report on the dynamics of release using fluorophore-terminated C6 or C11 thiols. We show that the release kinetics for C6 thiols are determined solely by ... More