Regulatory T cell development in the absence of functional Foxp3.
AuthorsLin W, Haribhai D, Relland LM, Truong N, Carlson MR, Williams CB, Chatila TA
JournalNat Immunol
PubMed ID17273171
'Although the development of regulatory T cells (T(reg) cells) in the thymus is defined by expression of the lineage marker Foxp3, the precise function of Foxp3 in T(reg) cell lineage commitment is unknown. Here we examined T(reg) cell development and function in mice with a Foxp3 allele that directs expression ... More
Targeting quantum dots to surface proteins in living cells with biotin ligase.
AuthorsHowarth M, Takao K, Hayashi Y, Ting AY
JournalProc Natl Acad Sci U S A
PubMed ID15897449
'Escherichia coli biotin ligase site-specifically biotinylates a lysine side chain within a 15-amino acid acceptor peptide (AP) sequence. We show that mammalian cell surface proteins tagged with AP can be biotinylated by biotin ligase added to the medium, while endogenous proteins remain unmodified. The biotin group then serves as a ... More
Receptor (CD155)-dependent endocytosis of poliovirus and retrograde axonal transport of the endosome.
AuthorsOhka S, Matsuda N, Tohyama K, Oda T, Morikawa M, Kuge S, Nomoto A
JournalJ Virol
PubMed ID15194795
'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 mice carrying the human PV receptor (hPVR/CD155) gene. Here, we demonstrated by using an immunoelectron microscope that PV particles exist on vesicle structures in nerve ... More
Signal propagation in Drosophila central neurons.
AuthorsGouwens NW, Wilson RI,
JournalJ Neurosci
PubMed ID19439602
'Drosophila is an important model organism for investigating neural development, neural morphology, neurophysiology, and neural correlates of behaviors. However, almost nothing is known about how electrical signals propagate in Drosophila neurons. Here, we address these issues in antennal lobe projection neurons, one of the most well studied classes of Drosophila ... More
Surface expression and endocytosis of the human cytomegalovirus-encoded chemokine receptor US28 is regulated by agonist-independent phosphorylation.
AuthorsMokros T, Rehm A, Droese J, Oppermann M, Lipp M, Höpken UE
JournalJ Biol Chem
PubMed ID12244063
'Human cytomegalovirus encodes the G protein-coupled chemokine receptor homologue US28 that binds several CC chemokines and sequesters extracellular chemokines from the environment of infected cells. Mechanistically, it has been shown that US28 undergoes rapid constitutive receptor endocytosis and recycling. Monoclonal antibodies were raised that allowed the characterization of a ligand-independent ... More
Biotinylated theta-toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells.
AuthorsSugii S, Reid PC, Ohgami N, Shimada Y, Maue RA, Ninomiya H, Ohno-Iwashita Y, Chang TY
JournalJ Lipid Res
PubMed ID12562855
'BCtheta is a proteolytically nicked and biotinylated derivative of a cholesterol binding protein perfringolysin O (theta-toxin), and has been used to detect cholesterol-rich domains at the plasma membrane (PM). Here we show that by modifying the cell fixation condition, BCtheta can also be used to detect cholesterol-rich domains intracellularly. When ... More
Cryptomonad algal phycobiliproteins as fluorochromes for extracellular and intracellular antigen detection by flow cytometry.
AuthorsTelford WG, Moss MW, Morseman JP, Allnutt FC
JournalCytometry
PubMed ID11309804
'BACKGROUND: Phycobiliproteins play an important role in fluorescent labeling, particularly for flow cytometry. The spectral properties of R-phycoerythrin (R-PE) and allophycocyanin (APC) have made them the dominant reagents in this class of fluorochromes. In this study, we evaluate a lesser-known but potentially important series of low-molecular weight cryptomonad-derived phycobiliproteins (commercially ... More
Fluorescence microscopy methods in the study of protein structure and function.
'As more and more proteins specific to hair cells are discovered, it becomes imperative to understand their structure and how that contributes to their function. The fluorescence microscopic methods described here can be employed to provide information on protein-protein interactions, whether homomeric or heteromeric, and on protein conformation. Here, we ... More
Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidin.
AuthorsHowarth M, Ting AY,
JournalNat Protoc
PubMed ID18323822
'This protocol describes a simple and efficient way to label specific cell surface proteins with biophysical probes on mammalian cells. Cell surface proteins tagged with a 15-amino acid peptide are biotinylated by Escherichia coli biotin ligase (BirA), whereas endogenous proteins are not modified. The biotin group then allows sensitive and ... 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
Neurogenesis in the neocortex of adult primates.
AuthorsGould E, Reeves AJ, Graziano MS, Gross CG
JournalScience
PubMed ID10521353
In primates, prefrontal, inferior temporal, and posterior parietal cortex are important for cognitive function. It is shown that in adult macaques, new neurons are added to these three neocortical association areas, but not to a primary sensory area (striate cortex). The new neurons appeared to originate in the subventricular zone ... More
Transformation of olfactory representations in the Drosophila antennal lobe.
AuthorsWilson RI, Turner GC, Laurent G
JournalScience
PubMed ID14684826
Molecular genetics has revealed a precise stereotypy in the projection of primary olfactory sensory neurons onto secondary neurons. A major challenge is to understand how this mapping translates into odor responses in these second-order neurons. We investigated this question in Drosophila using whole-cell recordings in vivo. We observe that monomolecular ... More
In vivo assembly of nanoparticle components to improve targeted cancer imaging.
AuthorsPerrault SD, Chan WC,
JournalProc Natl Acad Sci U S A
PubMed ID20534561
Many small molecular anticancer agents are often ineffective at detecting or treating cancer due to their poor pharmacokinetics. Using nanoparticles as carriers can improve this because their large size reduces clearance and improves retention within tumors, but it also slows their rate of transfer from circulation into the tumor interstitium. ... More
Ezrin tunes the magnitude of humoral immunity.
AuthorsPore D, Parameswaran N, Matsui K, Stone MB, Saotome I, McClatchey AI, Veatch SL, Gupta N,
Journal
PubMed ID24043890
Ezrin is a member of the ezrin-radixin-moesin family of membrane-actin cytoskeleton cross-linkers that participate in a variety of cellular processes. In B cells, phosphorylation of ezrin at different sites regulates multiple processes, such as lipid raft coalescence, BCR diffusion, microclustering, and endosomal JNK activation. In this study, we generated mice ... More
The NALP3 inflammasome is involved in the innate immune response to amyloid-beta.
AuthorsHalle A, Hornung V, Petzold GC, Stewart CR, Monks BG, Reinheckel T, Fitzgerald KA, Latz E, Moore KJ, Golenbock DT,
JournalNat Immunol
PubMed ID18604209
The fibrillar peptide amyloid-beta (A beta) has a chief function in the pathogenesis of Alzheimer's disease. Interleukin 1 beta (IL-1 beta) is a key cytokine in the inflammatory response to A beta. Insoluble materials such as crystals activate the inflammasome formed by the cytoplasmic receptor NALP3, which results in the ... More
Paneth cell trypsin is the processing enzyme for human defensin-5.
AuthorsGhosh D, Porter E, Shen B, Lee SK, Wilk D, Drazba J, Yadav SP, Crabb JW, Ganz T, Bevins CL
JournalNat Immunol
PubMed ID12021776
The antimicrobial peptide human alpha-defensin 5 (HD5) is expressed in Paneth cells, secretory epithelial cells in the small intestine. Unlike other characterized defensins, HD5 is stored in secretory vesicles as a propeptide. The storage quantities of HD5 are approximately 90 450 microg per cm2 of mucosal surface area, which is ... More
Improved double immunofluorescence for confocal laser scanning microscopy.
AuthorsKumar RK, Chapple CC, Hunter N
JournalJ Histochem Cytochem
PubMed ID10449542
Reliable double immunofluorescence labeling for confocal laser scanning microscopy requires good separation of the signals generated by the fluorochromes. We have successfully overcome the limitation of a single argon ion laser in achieving effective excitation of dyes with well-separated emission spectra by employing the novel sulfonated rhodamine fluorochromes designated Alexa ... More
Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase.
AuthorsChen I, Howarth M, Lin W, Ting AY
JournalNat Methods
PubMed ID15782206
We report a highly specific, robust and rapid new method for labeling cell surface proteins with biophysical probes. The method uses the Escherichia coli enzyme biotin ligase (BirA), which sequence-specifically ligates biotin to a 15-amino-acid acceptor peptide (AP). We report that BirA also accepts a ketone isostere of biotin as ... More
gp130 signaling in proopiomelanocortin neurons mediates the acute anorectic response to centrally applied ciliary neurotrophic factor.
Ciliary neurotrophic factor (CNTF) exerts anorectic effects by overcoming leptin resistance via activation of hypothalamic neurons. However, the exact site of CNTF action in the hypothalamus has not yet been identified. Using Cre-loxP-mediated recombination in vivo, we have selectively ablated the common cytokine signaling chain gp130, which is required for ... More
Enzymatic activity of the Src homology 2 domain-containing inositol phosphatase is regulated by a plasma membrane location.
AuthorsPhee H, Jacob A, Coggeshall KM
JournalJ Biol Chem
PubMed ID10764758
The negative regulatory role of the Src homology 2 domain-containing inositol 5-phosphatase (SHIP) has been invoked in a variety of receptor-mediated signaling pathways. In B lymphocytes, co-clustering of antigen receptor surface immunoglobulin with FcgammaRIIb promotes the negative effects of SHIP, but how SHIP activity is regulated is unknown. To explore ... More
Protein micropatterns using a pH-responsive polymer and light.
AuthorsChristman KL, Maynard HD
JournalLangmuir
PubMed ID16114947
Protein and peptide microarrays are popular candidates for medical diagnostics because of the possibility for high sensitivity and simultaneous marker screening. To realize the potential of these arrays, new strategies for ligand patterning are needed. We report a method for patterning proteins that utilizes a pH-responsive polymer, deep ultraviolet (DUV) ... More
Submicron streptavidin patterns for protein assembly.
AuthorsChristman KL, Requa MV, Enriquez-Rios VD, Ward SC, Bradley KA, Turner KL, Maynard HD
JournalLangmuir
PubMed ID16893251
Micron and submicron-scale features of aldehyde functionality were fabricated in polymer films by photolithography to develop a platform for protein immobilization and assembly at a biologically relevant scale. Films containing the pH-reactive polymer poly(3,3'-diethoxypropyl methacrylate) and a photoacid generator (PAG) were patterned from 500 nm to 40 mum by exposure ... More
Peptide tags for labeling membrane proteins in live cells with multiple fluorophores.
AuthorsMcCann CM, Bareyre FM, Lichtman JW, Sanes JR
JournalBiotechniques
PubMed ID16018556
We describe a method to label specific membrane proteins with fluorophores for live imaging. Fusion proteins are generated that incorporate into their extracellular domains short peptide sequences (13-38 amino acids) recognized with high affinity and specificity by protein ligands, alpha-bungarotoxin (BTX), or streptavidin (SA). Many fluorophore- and enzyme-conjugated derivatives of ... More
DPSS yellow-green 561-nm lasers for improved fluorochrome detection by flow cytometry.
AuthorsTelford W, Murga M, Hawley T, Hawley R, Packard B, Komoriya A, Haas F, Hubert C
JournalCytometry A
PubMed ID16163703
INTRODUCTION: Blue-green 488-nm laser sources are widespread in flow cytometry but suffer some drawbacks for cell analysis, including their excitation of endogenous proteins (resulting in high cellular autofluorescence) and their less-than-optimal coincidence with the excitation maxima of commonly used fluorochromes, including the phycoerythrins (PE). Longer wavelength lasers such as green ... More