A small-molecule approach to studying invasive mechanisms of Toxoplasma gondii.
AuthorsCarey KL, Westwood NJ, Mitchison TJ, Ward GE
JournalProc Natl Acad Sci U S A
PubMed ID15123807
'Toxoplasma gondii is the most common protozoan parasite of humans. Infection with T. gondii can lead to life-threatening disease as a result of repeated cycles of host cell invasion, parasite replication, and host cell lysis. Relatively little is known about the invasive mechanisms of T. gondii and related parasites within ... More
CD24 is expressed by myofiber synaptic nuclei and regulates synaptic transmission.
AuthorsJevsek M, Jaworski A, Polo-Parada L, Kim N, Fan J, Landmesser LT, Burden SJ
JournalProc Natl Acad Sci U S A
PubMed ID16606832
'The genes encoding several synaptic proteins, including acetylcholine receptors, acetylcholinesterase, and the muscle-specific kinase, MuSK, are expressed selectively by a small number of myofiber nuclei positioned near the synaptic site. Genetic analysis of mutant mice suggests that additional genes, expressed selectively by synaptic nuclei, might encode muscle-derived retrograde signals that ... More
Optimizing antibody immobilization strategies for the construction of protein microarrays.
AuthorsPeluso P, Wilson DS, Do D, Tran H, Venkatasubbaiah M, Quincy D, Heidecker B, Poindexter K, Tolani N, Phelan M, Witte K, Jung LS, Wagner P, Nock S
JournalAnal Biochem
PubMed ID12531195
'Antibody microarrays have the potential to revolutionize protein expression profiling. The intensity of specific signal produced on a feature of such an array is related to the amount of analyte that is captured from the biological mixture by the immobilized antibody (the "capture agent"). This in turn is a function ... 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
AuthorsKey TA, Foutz TD, Gurevich VV, Sklar LA, Prossnitz ER
JournalJ Biol Chem
PubMed ID12424254
'Arrestins regulate the signaling and endocytosis of many G protein-coupled receptors (GPCRs). It has been suggested that the functions of arrestins are dependent upon both the number and pattern of phosphorylation sites present in an activated GPCR. However, little is currently known about the relationships between the sites of receptor ... More
An actin-dependent conformational change in myosin.
AuthorsXiao M, Reifenberger JG, Wells AL, Baldacchino C, Chen LQ, Ge P, Sweeney HL, Selvin PR
JournalNat Struct Biol
PubMed ID12679807
'Conformational changes within myosin lead to its movement relative to an actin filament. Several crystal structures exist for myosin bound to various nucleotides, but none with bound actin. Therefore, the effect of actin on the structure of myosin is poorly understood. Here we show that the swing of smooth muscle ... More
Melanosomal sequestration of cytotoxic drugs contributes to the intractability of malignant melanomas.
'Multidrug resistance mechanisms underlying the intractability of malignant melanomas remain largely unknown. In this study, we demonstrate that the development of multidrug resistance in melanomas involves subcellular sequestration of intracellular cytotoxic drugs such as cis-diaminedichloroplatinum II (cisplatin; CDDP). CDDP is initially sequestered in subcellular organelles such as melanosomes, which significantly ... More
Exocytosis of IgG as mediated by the receptor, FcRn: an analysis at the single-molecule level.
AuthorsOber RJ, Martinez C, Lai X, Zhou J, Ward ES
JournalProc Natl Acad Sci U S A
PubMed ID15258288
'IgG transport within and across cells is essential for effective humoral immunity. Through a combination of biochemical and in vivo analyses, the MHC class I-related neonatal Fc receptor (FcRn) is known to play a central role in delivering IgGs within and across cells. However, little is known about the molecular ... 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
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
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
CNN-Gd(3+) enables cell nucleus molecular imaging of prostate cancer cells: the last 600 nm.
AuthorsHeckl S, Debus J, Jenne J, Pipkorn R, Waldeck W, Spring H, Rastert R, von der Lieth CW, Braun K
JournalCancer Res
PubMed ID12460922
'Molecular imaging is defined as the characterization and measurement of biological processes at the cellular and molecular level. Molecular imaging, therefore, necessitates a sufficient amount of contrast agent within the cell. Consequently, we realized that the intracellular uptake and cell compartment specificity of the commonly used interstitial contrast agent gadolinium ... More
Fluorescence measurements of nucleotide association with the Na(+)/K(+)-ATPase.
AuthorsPratap PR, Mikhaylyants LO, Olden-Stahl N,
JournalBiochim Biophys Acta
PubMed ID19595797
'The Na(+)/K(+)-ATPase, a membrane-associated ion pump, uses energy from the hydrolysis of ATP to pump 3 Na(+) ions out of and 2 K(+) into cells. The dependence of ATP hydrolysis on ATP concentration was measured using a fluorescence coupled-enzyme assay. The dependence on concentration of nucleotide association with the ATPase ... More
Lipoprotein aggregation protects human monocyte-derived macrophages from OxLDL-induced cytotoxicity.
AuthorsAsmis R, Begley JG, Jelk J, Everson WV
JournalJ Lipid Res
PubMed ID15772426
'Oxidative modifications render low density lipoprotein cytotoxic and enhance its propensity to aggregate and fuse into particles similar to those found in atherosclerotic lesions. We showed previously that aggregation of oxidized LDL (OxLDL) promotes the transformation of human macrophages into lipid-laden foam cells (Asmis, R., and J. Jelk. 2000. Large ... More
Splicing promotes rapid and efficient mRNA export in mammalian cells.
AuthorsValencia P, Dias AP, Reed R,
JournalProc Natl Acad Sci U S A
PubMed ID18287003
'The numerous steps in protein gene expression are extensively coupled to one another through complex networks of physical and functional interactions. Indeed, >25 coupled reactions, often reciprocal, have been documented among such steps as transcription, capping, splicing, and polyadenylation. Coupling is usually not essential for gene expression, but instead enhances ... More
A transmembrane intracellular estrogen receptor mediates rapid cell signaling.
AuthorsRevankar CM, Cimino DF, Sklar LA, Arterburn JB, Prossnitz ER
JournalScience
PubMed ID15705806
'The steroid hormone estrogen regulates many functionally unrelated processes in numerous tissues. Although it is traditionally thought to control transcriptional activation through the classical nuclear estrogen receptors, it also initiates many rapid nongenomic signaling events. We found that of all G protein-coupled receptors characterized to date, GPR30 is uniquely localized ... 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
Three-dimensional structures of the flagellar dynein-microtubule complex by cryoelectron microscopy.
AuthorsOda T, Hirokawa N, Kikkawa M
JournalJ Cell Biol
PubMed ID17438074
The outer dynein arms (ODAs) of the flagellar axoneme generate forces needed for flagellar beating. Elucidation of the mechanisms underlying the chemomechanical energy conversion by the dynein arms and their orchestrated movement in cilia/flagella is of great importance, but the nucleotide-dependent three-dimensional (3D) movement of dynein has not yet been ... More
Transport to late endosomes is required for efficient reovirus infection.
AuthorsMainou BA, Dermody TS,
JournalJ Virol
PubMed ID22674975
Rab GTPases play an essential role in vesicular transport by coordinating the movement of various types of cargo from one cellular compartment to another. Individual Rab GTPases are distributed to specific organelles and thus serve as markers for discrete types of endocytic vesicles. Mammalian reovirus binds to cell surface glycans ... 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
Binding of laminin-1 to monosialoganglioside GM1 in lipid rafts is crucial for neurite outgrowth.
AuthorsIchikawa N, Iwabuchi K, Kurihara H, Ishii K, Kobayashi T, Sasaki T, Hattori N, Mizuno Y, Hozumi K, Yamada Y, Arikawa-Hirasawa E,
JournalJ Cell Sci
PubMed ID19118221
Laminin-1, an extracellular matrix molecule, promotes neurite outgrowth through the interaction of integrin and actin. Monosialoganglioside GM1 in the lipid rafts associates with and activates the NGF receptor TrkA, and enhances neurite outgrowth. However, the role of GM1 in laminin-1-induced neurite outgrowth was still unclear. Here, we describe that laminin-1 ... More
C-peptide is internalised in human endothelial and vascular smooth muscle cells via early endosomes.
AuthorsLuppi P, Geng X, Cifarelli V, Drain P, Trucco M,
JournalDiabetologia
PubMed ID19662378
AIMS/HYPOTHESIS: There is increasing evidence that C-peptide exerts intracellular effects in a variety of cells and could be beneficial in patients with type 1 diabetes. Exactly how C-peptide achieves these effects, however, is unknown. Recent reports showed that C-peptide internalised in the cytoplasm of HEK-293 and Swiss 3T3 cells, where ... More
Effect of mannose chain length on targeting of glucocerebrosidase for enzyme replacement therapy of Gaucher disease.
Recombinant human glucocerebrosidase (imiglucerase, Cerezyme) is used in enzyme replacement therapy for Gaucher disease. Complex oligosaccharides present on Chinese hamster ovary cell-expressed glucocerebrosidase (GCase) are enzymatically remodeled into a mannose core, facilitating mannose receptor-mediated uptake into macrophages. Alternative expression systems could be used to produce GCase containing larger oligomannose structures, ... More
Prions hijack tunnelling nanotubes for intercellular spread.
AuthorsGousset K, Schiff E, Langevin C, Marijanovic Z, Caputo A, Browman DT, Chenouard N, de Chaumont F, Martino A, Enninga J, Olivo-Marin JC, Männel D, Zurzolo C,
JournalNat Cell Biol
PubMed ID19198598
In variant Creutzfeldt-Jakob disease, prions (PrP(Sc)) enter the body with contaminated foodstuffs and can spread from the intestinal entry site to the central nervous system (CNS) by intercellular transfer from the lymphoid system to the peripheral nervous system (PNS). Although several means and different cell types have been proposed to ... 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
Fluorescent DNA hybridization probe preparation using amine modification and reactive dye coupling.
AuthorsCox WG, Singer VL,
JournalBiotechniques
PubMed ID14740493
Fluorescent nucleic acid hybridization probes traditionally have been generated by enzymatic incorporation of dye-labeled nucleotides, even though incorporation efficiency is low and variable from dye to dye. Alternatively, 5-(3-aminoallyl)-2'-deoxyuridine 5'-triphosphate (aa-dUTP) is enzymatically incorporated to generate amine-modified DNA, which is then chemically labeled with an amine-reactive fluorescent dye. We optimized ... More
Vertical silicon nanowires as a universal platform for delivering biomolecules into living cells.
AuthorsShalek AK, Robinson JT, Karp ES, Lee JS, Ahn DR, Yoon MH, Sutton A, Jorgolli M, Gertner RS, Gujral TS, Macbeath G, Yang EG, Park H,
JournalProc Natl Acad Sci U S A
PubMed ID20080678
A generalized platform for introducing a diverse range of biomolecules into living cells in high-throughput could transform how complex cellular processes are probed and analyzed. Here, we demonstrate spatially localized, efficient, and universal delivery of biomolecules into immortalized and primary mammalian cells using surface-modified vertical silicon nanowires. The method relies ... 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
Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes.
AuthorsMarras SA, Kramer FR, Tyagi S
JournalNucleic Acids Res
PubMed ID12409481
An important consideration in the design of oligonucleotide probes for homogeneous hybridization assays is the efficiency of energy transfer between the fluorophore and quencher used to label the probes. We have determined the efficiency of energy transfer for a large number of combinations of commonly used fluorophores and quenchers. We ... More
Quantitative analysis and characterization of biofunctionalized fluorescent silica particles.
AuthorsCorrie SR, Lawrie GA, Trau M
JournalLangmuir
PubMed ID16519476
A strategy for the production and subsequent characterization of biofunctionalized silica particles is presented. The particles were engineered to produce a bifunctional material capable of both (a) the attachment of fluorescent dyes for particle encoding and (b) the sequential modification of the surface of the particles to couple oligonucleotide probes. ... More
Following tracks of hemichannels.
AuthorsDermietzel R, Meier C, Bukauskas F, Spray DC
JournalCell Commun Adhes
PubMed ID14681038
It has been suggested that plasma membrane-bound hemichannels perform physiological and pathophysiological functions per se. Such functions require the presence of hemichannels on the cell surface and their accessibility to the extracellular environment for at least some limited period of time. We have previously shown that hemichannels can be labeled ... More
A fluorescence resonance energy transfer sensor for the beta-domain of metallothionein.
AuthorsHong SH, Maret W
JournalProc Natl Acad Sci U S A
PubMed ID12618543
We have designed a nanosensor to study the potential function of metallothionein (MT) in metal transfer and its interactions with redox partners and ligands by attaching two fluorescent probes to recombinant human MT. The specific labeling takes advantage of two different modification reactions. One is based on the fact that ... More
Different transport routes for high density lipoprotein and its associated free sterol in polarized hepatic cells.
AuthorsWüstner D, Mondal M, Huang A, Maxfield FR
JournalJ Lipid Res
PubMed ID14679167
We analyzed the intracellular transport of HDL and its associated free sterol in polarized human hepatoma HepG2 cells. Using pulse-chase protocols, we demonstrated that HDL labeled with Alexa 488 at the apolipoprotein (Alexa 488-HDL) was internalized by a scavenger receptor class B type I (SR-BI)-dependent process at the basolateral membrane ... More
The low density lipoprotein receptor-related protein modulates protease activity in the brain by mediating the cellular internalization of both neuroserpin and neuroserpin-tissue-type plasminogen activator complexes.
AuthorsMakarova A, Mikhailenko I, Bugge TH, List K, Lawrence DA, Strickland DK
JournalJ Biol Chem
PubMed ID14522960
Proteases contribute to a variety of processes in the brain; consequently, their activity is carefully regulated by protease inhibitors, such as neuroserpin. This inhibitor is thought to be secreted by axons at synaptic regions where it controls tissue-type plasminogen activator (tPA) activity. Mechanisms regulating neuroserpin are not known, and the ... More
Persistent cAMP signaling by thyrotropin (TSH) receptors is not dependent on internalization.
AuthorsNeumann S, Geras-Raaka E, Marcus-Samuels B, Gershengorn MC
JournalFASEB J
PubMed ID20538910
Evidence was presented that thyrotropin [thyroid-stimulating hormone (TSH)]-stimulated persistent cAMP signaling is dependent on receptor (with G-protein a subunits and adenylyl cyclase) internalization. Because it is not clear whether G proteins and adenylyl cyclase internalize with receptors, we tested whether persistent cAMP signaling by TSH receptor (TSHR) is dependent on ... More
Demonstration of intermediate cells during human prostate epithelial differentiation in situ and in vitro using triple-staining confocal scanning microscopy.
In human prostate epithelium, morphologically basal and luminal cells can be discriminated. The basal cell layer that putatively contains progenitor cells of the secretory epithelium is characterized by the expression of keratins (K) 5 and 14. Luminal cells represent the secretory compartment of the epithelium and express K8 and 18. ... More
Charge- and size-based separation of macromolecules using ultrathin silicon membranes.
Commercial ultrafiltration and dialysis membranes have broad pore size distributions and are over 1,000 times thicker than the molecules they are designed to separate, leading to poor size cut-off properties, filtrate loss within the membranes, and low transport rates. Nanofabricated membranes have great potential in molecular separation applications by offering ... More
T7 RNA polymerase-induced bending of promoter DNA is coupled to DNA opening.
AuthorsTang GQ, Patel SS
JournalBiochemistry
PubMed ID16605261
To initiate transcription, T7 RNA polymerase (RNAP) forms a specific complex with its promoter DNA and melts several base pairs near the initiation site to form an open complex. Previous gel electrophoresis studies have indicated that the promoter DNA in the initiation complex is bent [Ujvari, A., and Martin, C. ... More