Biomedical subjects

Roger Y Tsien

Publications and source records attributed to Roger Y Tsien.

At least 19 recordsLinked to original sources

PubMed2004

BI-1 regulates an apoptosis pathway linked to endoplasmic reticulum stress.

Bax inhibitor-1 (BI-1) is an evolutionarily conserved endoplasmic reticulum (ER) protein that suppresses cell death in both animal and plant cells. We characterized mice in which the bi-1 gene was ablated. Cells from BI-1-deficient mice, including fibroblasts, hepatocytes, and neurons, display selective hypersensitivity to apoptosis induced by ER stress agents (thapsigargin, tunicamycin, brefeldin A), but not to stimulators of mitochondrial or TNF/Fas-death receptor apoptosis pathways. Conversely, BI-1 overexpression protects against apoptosis induced by ER stress. BI-1-mediated protection from apoptosis induced by ER stress correlated with inhibition of Bax activation and translocation to mitochondria, preservation of mitochondrial membrane potential, and suppression of caspase activation. BI-1 overexpression also reduces releasable Ca(2+) from the ER. In vivo, bi-1(-/-) mice exhibit increased sensitivity to tissue damage induced by stimuli that trigger ER stress, including stroke and tunicamycin injection. Thus, BI-1 regulates a cell death pathway important for cytopreservation during ER stress.

Animals↗

PubMed2004

Bhc-cNMPs as either water-soluble or membrane-permeant photoreleasable cyclic nucleotides for both one- and two-photon excitation.

Cyclic nucleoside monophosphates (cNMPs) play key roles in many cellular regulatory processes, such as growth, differentiation, motility, and gene expression. Caged derivatives that can be activated by irradiation could be powerful tools for studying such diverse functions of intracellular second messengers, since the spatiotemporal dynamics of these molecules can be controlled by irradiation with appropriately focused light. Here we report the synthesis, photochemistry, and biological testing of 6-bromo-7-hydroxycoumarin-4-ylmethyl esters of cNMP (Bhc-cNMP) and their acetyl derivatives (Bhc-cNMP/Ac) as new caged second messengers. Irradiation of Bhc-cNMPs quantitatively produced the parent cNMPs with one-photon uncaging efficiencies (Phiepsilon) of up to one order of magnitude better than those of 2-nitrophenethyl (NPE) cNMPs. In addition, two-photon induced photochemical release of cNMP from Bhc-cNMPs (7 and 8) can be observed with the two-photon uncaging action cross-sections (delta(u)) of up to 2.28 GM (1 GM=10(-50) cm(4) s photon(-1)), which is the largest value among those of the reported Bhc-caged compounds. The wavelength dependence of the delta(u) values of 7 revealed that the peak wavelength was twice that of the one-photon absorption maximum. Bhc-cNMPs showed practically useful water solubility (nearly 500 microM), whereas 7-acetylated derivatives (Bhc-cNMPs/Ac) were expected to have a certain membrane permeability. Their advantages were demonstrated in two types of biological systems: the opening of cAMP-mediated transduction channels in newt olfactory receptor cells and cAMP-mediated motility responses in epidermal melanophores in scales from medaka fish. Both examples showed that Bhc and Bhc/Ac caged compounds have great potential for use in many cell biological applications.

Animals↗

PubMed2004

Functional fluorescent Ca2+ indicator proteins in transgenic mice under TET control.

Genetically encoded fluorescent calcium indicator proteins (FCIPs) are promising tools to study calcium dynamics in many activity-dependent molecular and cellular processes. Great hopes-for the measurement of population activity, in particular-have therefore been placed on calcium indicators derived from the green fluorescent protein and their expression in (selected) neuronal populations. Calcium transients can rise within milliseconds, making them suitable as reporters of fast neuronal activity. We here report the production of stable transgenic mouse lines with two different functional calcium indicators, inverse pericam and camgaroo-2, under the control of the tetracycline-inducible promoter. Using a variety of in vitro and in vivo assays, we find that stimuli known to increase intracellular calcium concentration (somatically triggered action potentials (APs) and synaptic and sensory stimulation) can cause substantial and rapid changes in FCIP fluorescence of inverse pericam and camgaroo-2.

Animals↗

PubMed2004

Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators.

Changes in the redox equilibrium of cells influence a host of cell functions. Alterations in the redox equilibrium are precipitated by changing either the glutathione/glutathione-disulfide ratio (GSH/GSSG) and/or the reduced/oxidized thioredoxin ratio. Redox-sensitive green fluorescent proteins (GFP) allow real time visualization of the oxidation state of the indicator. Ratios of fluorescence from excitation at 400 and 490 nm indicate the extent of oxidation and thus the redox potential while canceling out the amount of indicator and the absolute optical sensitivity. Because the indicator is genetically encoded, it can be targeted to specific proteins or organelles of interest and expressed in a wide variety of cells and organisms. We evaluated roGFP1 (GFP with mutations C48S, S147C, and Q204C) and roGFP2 (the same plus S65T) with physiologically or toxicologically relevant oxidants both in vitro and in living mammalian cells. Furthermore, we investigated the response of the redox probes under physiological redox changes during superoxide bursts in macrophage cells, hyperoxic and hypoxic conditions, and in responses to H(2)O(2)-stimulating agents, e.g. epidermal growth factor and lysophosphatidic acid.

Cloning, Molecular↗

PubMed2004

Imaging tri-fusion multimodality reporter gene expression in living subjects.

Imaging reporter gene expression in living subjects with various imaging modalities is a rapidly accelerating area of research. Applications of these technologies to cancer research, gene therapy, and transgenic models are rapidly expanding. We report construction and testing of several triple fusion reporter genes compatible with bioluminescence, fluorescence and positron emission tomography (PET) imaging. A triple fusion reporter vector harboring a bioluminescence synthetic Renilla luciferase (hrl) reporter gene, a reporter gene encoding the monomeric red fluorescence protein (mrfp1), and a mutant herpes simplex virus type 1 sr39 thymidine kinase [HSV1-truncated sr39tk (ttk); a PET reporter gene] was found to preserve the most activity for each protein component and was therefore investigated in detail. After validating the activities of all three proteins encoded by the fusion gene in cell culture, we imaged living mice bearing 293T cells transiently expressing the hrl-mrfp-ttk vector by microPET and using a highly sensitive cooled charge-coupled device camera compatible with both bioluminescence and fluorescence imaging. A lentiviral vector carrying the triple fusion reporter gene was constructed and used to isolate stable expressers by fluorescence-activated cell sorting. These stable 293T cells were further used to show good correlation (R(2) approximately 0.74-0.85) of signal from each component by imaging tumor xenografts in living mice with all three modalities. Furthermore, metastases of a human melanoma cell line (A375M) stably expressing the triple fusion were imaged by microPET and optical technologies over a 40-50-day time period in living mice. Imaging of reporter gene expression from single cells to living animals with the help of a single tri-fusion reporter gene will have the potential to accelerate translational cancer research.

Animals↗

PubMed2004

Activity-dependent regulation of dendritic synthesis and trafficking of AMPA receptors.

Regulation of AMPA receptor (AMPAR) trafficking is important for neural plasticity. Here we examined the trafficking and synthesis of the GluR1 and GluR2 subunits using ReAsH-EDT(2) and FlAsH-EDT(2) staining. Activity blockade of rat cultured neurons increased dendritic GluR1, but not GluR2, levels. Examination of transected dendrites revealed that both AMPAR subunits were synthesized in dendrites and that activity blockade enhanced dendritic synthesis of GluR1 but not GluR2. In contrast, acute pharmacological manipulations increased dendritic synthesis of both subunits. AMPARs synthesized in dendrites were inserted into synaptic plasma membranes and, after activity blockade, the electrophysiological properties of native synaptic AMPARs changed in the manner predicted by the imaging experiments. In addition to providing a novel mechanism for synaptic modifications, these results point out the advantages of using FlAsH-EDT(2) and ReAsH-EDT(2) for studying the trafficking of newly synthesized proteins in local cellular compartments such as dendrites.

Action Potentials↗

PubMed2004

Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators.

Current methods for determining ambient redox potential in cells are labor-intensive and generally require destruction of tissue. This precludes single cell or real time studies of changes in redox poise that result from metabolic processes or environmental influences. By substitution of surface-exposed residues on the Aequorea victoria green fluorescent protein (GFP) with cysteines in appropriate positions to form disulfide bonds, reduction-oxidation-sensitive GFPs (roGFPs) have been created. roGFPs have two fluorescence excitation maxima at about 400 and 490 nm and display rapid and reversible ratiometric changes in fluorescence in response to changes in ambient redox potential in vitro and in vivo. Crystal structure analyses of reduced and oxidized crystals of roGFP2 at 2.0- and 1.9-A resolution, respectively, reveal in the oxidized state a highly strained disulfide and localized main chain structural changes that presumably account for the state-dependent spectral changes. roGFP1 has been targeted to the mitochondria in HeLa cells. Fluorometric measurements on these cells using a fluorescence microscope or in cell suspension using a fluorometer reveal that the roGFP1 probe is in dynamic equilibrium with the mitochondrial redox status and responds to membrane-permeable reductants and oxidants. The roGFP1 probe reports that the matrix space in HeLa cell mitochondria is highly reducing, with a midpoint potential near -360 mV (assuming mitochondrial pH approximately 8.0 at 37 degrees C). In other work (C. T. Dooley, T. M. Dore, G. Hanson, W. C. Jackson, S. J. Remington, and R. Y. Tsien, submitted for publication), it is shown that the cytosol of HeLa cells is also unusually reducing but somewhat less so than the mitochondrial matrix.

Cell Line↗

PubMed2003

Reversing cerebellar long-term depression.

The discovery of a postsynaptically expressed form of cerebellar parallel fiber-Purkinje cell long-term potentiation (LTP) raises the question whether this is the long-sought resetting mechanism for long-term depression (LTD). Extracellular monitoring of PC spikes enables stable prolonged recordings of parallel fiber-Purkinje cell synaptic efficacy. LTD, saturated by repeated induction protocols, can be reversed by a single round of postsynaptic LTP or nitric oxide (NO), enabling LTD to be reinduced. Conversely, after postsynaptic LTP has been saturated, one round of LTD permits fresh postsynaptic LTP. By contrast, after saturation of LTD, induction of presynaptic LTP or application of forskolin leaves LTD still saturated. Likewise, presynaptic LTP cannot be reversed by LTD. Therefore postsynaptic LTP mediated by NO without postsynaptic Ca2+ elevation, unlike presynaptic LTP mediated by cAMP, is a true counterbalance to LTD mediated by coincidence of NO plus postsynaptic Ca2+

Animals↗

PubMed2003

Aptamers switch on fluorescence of triphenylmethane dyes.

MG and SRB aptamers, which are short RNA sequences originally selected only for binding to malachite green or sulforhodamine B, can greatly enhance the fluorescence of normally nonfluorescent triphenylmethane dyes. MG aptamer enhances the quantum yields of malachite green (MG) and a novel rigidized derivative, indolinyl malachite green (IMG) by >2000-fold. SRB aptamer brightens patent blue V and VF by >90-fold. These enhancements are specific because MG aptamer has no effect on patent blue dyes and SRB aptamer has little or no effect on MG and IMG. Such sequence-specific fluorescence labeling of short RNA motifs is a first step toward genetically encodable fusion tags for imaging selected RNAs in vitro and in cells.

Fluorescence↗

PubMed2003

Imaging Tetrahymena ribozyme splicing activity in single live mammalian cells.

Tetrahymena ribozymes hold promise for repairing genetic disorders but are largely limited by their modest splicing efficiency and low production of final therapeutic proteins. Ribozyme evolution in intact living mammalian cells would greatly facilitate the discovery of new ribozyme variants with high in vivo activity, but no such strategies have been reported. Here we present a study using a new reporter enzyme, beta-lactamase, to report splicing activity in single living cells and perform high-throughput screening with flow cytometry. The reporter ribozyme constructs consist of the self-splicing Tetrahymena thermophila group I intron ribozyme that is inserted into the ORF of the mRNA of beta-lactamase. The splicing activity in single living cells can be readily detected quantitatively and visualized. Individual cells have shown considerable heterogeneity in ribozyme activity. Screening of Tetrahymena ribozymes with insertions in the middle of the L1 loop led to identification of better variants with at least 4-fold more final in vivo activity than the native sequence. Our work has provided a new reporter system that allows high-throughput screening with flow cytometry of single living mammalian cells for a direct and facile in vivo selection of desired ribozyme variants.

Animals↗

PubMed2003

Genetically targeted chromophore-assisted light inactivation.

Studies of protein function would be facilitated by a general method to inactivate selected proteins in living cells noninvasively with high spatial and temporal precision. Chromophore-assisted light inactivation (CALI) uses photochemically generated, reactive oxygen species to inactivate proteins acutely, but its use has been limited by the need to microinject dye-labeled nonfunction-blocking antibodies. We now demonstrate CALI of connexin43 (Cx43) and alpha1C L-type calcium channels, each tagged with one or two small tetracysteine (TC) motifs that specifically bind the membrane-permeant, red biarsenical dye, ReAsH. ReAsH-based CALI is genetically targeted, requires no antibodies or microinjection, and inactivates each protein by approximately 90% in <30 s of widefield illumination. Similar light doses applied to Cx43 or alpha1C tagged with green fluorescent protein (GFP) had negligible to slight effects with or without ReAsH exposure, showing the expected molecular specificity. ReAsH-mediated CALI acts largely via singlet oxygen because quenchers or enhancers of singlet oxygen respectively inhibit or enhance CALI.

Calcium Channels, L-Type↗

PubMed2003

All-optical histology using ultrashort laser pulses.

As a means to automate the three-dimensional histological analysis of brain tissue, we demonstrate the use of femtosecond laser pulses to iteratively cut and image fixed as well as fresh tissue. Cuts are accomplished with 1 to 10 microJ pulses to ablate tissue with micron precision. We show that the permeability, immunoreactivity, and optical clarity of the tissue is retained after pulsed laser cutting. Further, samples from transgenic mice that express fluorescent proteins retained their fluorescence to within microns of the cut surface. Imaging of exogenous or endogenous fluorescent labels down to 100 microm or more below the cut surface is accomplished with 0.1 to 1 nJ pulses and conventional two-photon laser scanning microscopy. In one example, labeled projection neurons within the full extent of a neocortical column were visualized with micron resolution. In a second example, the microvasculature within a block of neocortex was measured and reconstructed with micron resolution.

Animals↗

PubMed2003

A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C.

Signals transduced by kinases depend on the extent and duration of substrate phosphorylation. We generated genetically encoded fluorescent reporters for PKC activity that reversibly respond to stimuli activating PKC. Specifically, phosphorylation of the reporter expressed in mammalian cells causes changes in fluorescence resonance energy transfer (FRET), allowing real time imaging of phosphorylation resulting from PKC activation. Targeting of the reporter to the plasma membrane, where PKC is activated, reveals oscillatory phosphorylation in HeLa cells in response to histamine. Each oscillation in substrate phosphorylation follows a calcium oscillation with a lag of approximately 10 s. Novel FRET-based reporters for PKC translocation, phosphoinositide bisphosphate conversion to IP3, and diacylglycerol show that in HeLa cells the oscillatory phosphorylations correlate with Ca2+-controlled translocation of conventional PKC to the membrane without oscillations of PLC activity or diacylglycerol. However, in MDCK cells stimulated with ATP, PLC and diacylglycerol fluctuate together with Ca2+ and phosphorylation. Thus, specificity of PKC signaling depends on the local second messenger-controlled equilibrium between kinase and phosphatase activities to result in strict calcium-controlled temporal regulation of substrate phosphorylation.

Adenosine Triphosphate↗

PubMed2003

Detection of calcium transients in Drosophila mushroom body neurons with camgaroo reporters.

Camgaroos are yellow fluorescent protein derivatives that hold promise as transgenically encoded calcium sensors in behaving animals. We expressed two versions of camgaroo in Drosophila mushroom bodies using the galactosidase-4 (GAL4) system. Potassium depolarization of brains expressing the reporters produces a robust increase in fluorescence that is blocked by removing extracellular calcium or by antagonists of voltage-dependent calcium channels. The fluorescence increase is not attributable to cytoplasmic alkalization; depolarization induces a slight acidification of the cytoplasm of mushroom body neurons. Acetylcholine applied near the dendrites of the mushroom body neurons induces a rapid and ipsilateral-specific fluorescence increase in the mushroom body axons that is blocked by antagonists of calcium channels or nicotinic acetylcholine receptors. Fluorescence was observed to increase in all three classes of mushroom body neurons, indicating that all types respond to cholinergic innervation.

Acetylcholine↗

PubMed2003

Imagining imaging's future.

Imaging specific molecules and their interactions in space and time will be essential to understand how genomes create cells, how cells constitute organisms and how errant cells cause disease. Molecular imaging must be extended and applied from nanometre to metre scales and from milliseconds to days. This quest will require input from physics, chemistry, and the genetics and biochemistry of diverse organisms with useful talents.

Animals↗

PubMed2002

Overview of the Alliance for Cellular Signaling.

The Alliance for Cellular Signaling is a large-scale collaboration designed to answer global questions about signalling networks. Pathways will be studied intensively in two cells--B lymphocytes (the cells of the immune system) and cardiac myocytes--to facilitate quantitative modelling. One goal is to catalyse complementary research in individual laboratories; to facilitate this, all alliance data are freely available for use by the entire research community.

B-Lymphocytes↗

PubMed2002

A monomeric red fluorescent protein.

All coelenterate fluorescent proteins cloned to date display some form of quaternary structure, including the weak tendency of Aequorea green fluorescent protein (GFP) to dimerize, the obligate dimerization of Renilla GFP, and the obligate tetramerization of the red fluorescent protein from Discosoma (DsRed). Although the weak dimerization of Aequorea GFP has not impeded its acceptance as an indispensable tool of cell biology, the obligate tetramerization of DsRed has greatly hindered its use as a genetically encoded fusion tag. We present here the stepwise evolution of DsRed to a dimer and then either to a genetic fusion of two copies of the protein, i.e., a tandem dimer, or to a true monomer designated mRFP1 (monomeric red fluorescent protein). Each subunit interface was disrupted by insertion of arginines, which initially crippled the resulting protein, but red fluorescence could be rescued by random and directed mutagenesis totaling 17 substitutions in the dimer and 33 in mRFP1. Fusions of the gap junction protein connexin43 to mRFP1 formed fully functional junctions, whereas analogous fusions to the tetramer and dimer failed. Although mRFP1 has somewhat lower extinction coefficient, quantum yield, and photostability than DsRed, mRFP1 matures >10 times faster, so that it shows similar brightness in living cells. In addition, the excitation and emission peaks of mRFP1, 584 and 607 nm, are approximately 25 nm red-shifted from DsRed, which should confer greater tissue penetration and spectral separation from autofluorescence and other fluorescent proteins.

Amino Acid Sequence↗

PubMed2002

A new form of cerebellar long-term potentiation is postsynaptic and depends on nitric oxide but not cAMP.

Long-term depression (LTD) at cerebellar parallel fiber (PF)-Purkinje cell synapses must be balanced by long-term potentiation (LTP) to prevent saturation and allow reversal of motor learning. The only previously analyzed form of cerebellar LTP is induced by 4-8 Hz PF stimulation and requires cAMP but not nitric oxide. It is a poor candidate to reverse LTD because it is presynaptically expressed whereas LTD is postsynaptic. We now characterize a new form of LTP induced by 1 Hz PF stimulation for at least 300 s. This LTP is postsynaptically expressed, enhanced by chelating postsynaptic Ca(2+), and depends on nitric oxide but not cAMP or cGMP, making it a plausible anti-Hebbian counterpart to Hebbian LTD.

Animals↗