Imaging endoplasmic reticulum calcium with a fluorescent biosensor in transgenic mice

2004 ◽  
Vol 287 (4) ◽  
pp. C932-C938 ◽  
Author(s):  
Manami Hara ◽  
Vytautas Bindokas ◽  
James P. Lopez ◽  
Kelly Kaihara ◽  
Luis R. Landa ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Transgenic Mice ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Β Cells ◽  
Transgenic Expression ◽  
Endoplasmic Reticulum Calcium ◽  
Transgenic Organisms

The use of biosynthetic fluorescent sensors is an important new approach for imaging Ca2+ in cells. Genetically encoded indicators based on green fluorescent protein, calmodulin, and fluorescence resonance energy transfer (FRET) have been utilized to measure Ca2+ in nonmammalian transgenic organisms and provide information about the organization and regulation of Ca2+ signaling events in vivo. However, expression of biosynthetic FRET-based Ca2+ indicators in transgenic mammals has proven to be problematic. Here, we report transgenic expression of an endoplasmic reticulum (ER) Ca2+ biosensor in mouse pancreas. We targeted expression of a yellow cameleon3.3er (YC3.3er) transgene with mouse insulin I promoter. YC3.3er protein expression was limited to pancreatic β-cells within islets of Langerhans and absent in the exocrine pancreas and other tissues. Animals developed and matured normally; sensor expression was unaffected by age. Glucose tolerance in transgenic mice was also unaffected, indicating the transgenic biosensor did not impair endocrine pancreas function. ER Ca2+ responses after administration of thapsigargin, carbachol, and glucose were measured in individual β-cells of intact islets using confocal microscopy and confirmed the function of the biosensor. We conclude that controlling transgene transcription with a cell-specific promoter permits transgenic expression of FRET-based Ca2+ sensors in mammals and that this approach will facilitate real-time optical imaging of signal transduction events in living tissues.

scholarly journals Measuring ligand-dependent and ligand-independent interactions between nuclear receptors and associated proteins using Bioluminescence Resonance Energy Transfer (BRET2)

2006 ◽  
Vol 4 (1) ◽  
pp. nrs.04021 ◽  
Author(s):  
Kristen L. Koterba ◽  
Brian G. Rowan
Keyword(s):  
Energy Transfer ◽  
Protein Interactions ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Renilla Luciferase ◽  
Receptor Protein ◽  
Protein Protein Interactions

Bioluminescent resonance energy transfer (BRET2) is a recently developed technology for the measurement of protein-protein interactions in a live, cell-based system. BRET2 is characterized by the efficient transfer of excited energy between a bioluminescent donor molecule (Renilla luciferase) and a fluorescent acceptor molecule (a mutant of Green Fluorescent Protein (GFP2)). The BRET2 assay offers advantages over fluorescence resonance energy transfer (FRET) because it does not require an external light source thereby eliminating problems of photobleaching and autoflourescence. The absence of contamination by light results in low background that permits detection of very small changes in the BRET2 signal. BRET2 is dependent on the orientation and distance between two fusion proteins and therefore requires extensive preliminary standardization experiments to conclude a positive BRET2 signal independent of variations in protein titrations and arrangement in tertiary structures. Estrogen receptor (ER) signaling is modulated by steroid receptor coactivator 1 (SRC-1). To establish BRET2 in a ligand inducible system we used SRC-1 as the donor moiety and ER as the acceptor moiety. Expression and functionality of the fusion proteins were assessed by transient transfection in HEK-293 cells followed by Western blot analysis and measurement of ER-dependent reporter gene activity. These preliminary determinations are required prior to measuring nuclear receptor protein-protein interactions by BRET2. This article describes in detail the BRET2 methodology for measuring interaction between full-length ER and coregulator proteins in real-time, in an in vivo environment.

scholarly journals The in vivo mechanics of the magnetotactic backbone as revealed by correlative FLIM-FRET and STED microscopy

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Erika Günther ◽  
André Klauß ◽  
Mauricio Toro-Nahuelpan ◽  
Dirk Schüler ◽  
Carsten Hille ◽  
...  
Keyword(s):  
Magnetic Particles ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Stimulated Emission ◽  
Fluorescence Lifetime Imaging ◽  
Sted Microscopy

AbstractProtein interaction and protein imaging strongly benefit from the advancements in time-resolved and superresolution fluorescence microscopic techniques. However, the techniques were typically applied separately and ex vivo because of technical challenges and the absence of suitable fluorescent protein pairs. Here, we show correlative in vivo fluorescence lifetime imaging microscopy Förster resonance energy transfer (FLIM-FRET) and stimulated emission depletion (STED) microscopy to unravel protein mechanics and structure in living cells. We use magnetotactic bacteria as a model system where two proteins, MamJ and MamK, are used to assemble magnetic particles called magnetosomes. The filament polymerizes out of MamK and the magnetosomes are connected via the linker MamJ. Our system reveals that bacterial filamentous structures are more fragile than the connection of biomineralized particles to this filament. More importantly, we anticipate the technique to find wide applicability for the study and quantification of biological processes in living cells and at high resolution.

scholarly journals Activation Function 1 of Glucocorticoid Receptor Binds TATA-Binding Protein in Vitro and in Vivo

2006 ◽  
Vol 20 (6) ◽  
pp. 1218-1230 ◽  
Author(s):  
Alicja J. Copik ◽  
M. Scott Webb ◽  
Aaron L. Miller ◽  
Yongxin Wang ◽  
Raj Kumar ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Glucocorticoid Receptor ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Activation Function ◽  
Tata Binding Protein

Abstract The mechanism through which the glucocorticoid receptor (GR) stimulates transcription is still unclear, although it is clear that the GR affects assembly of the transcriptional machinery. The binding of the TATA-binding protein (TBP) to the TATA-box is accepted as essential in this process. It is known that the GR can interact in vitro with TBP, but the direct interaction of TBP with GR has not been previously characterized quantitatively and has not been appreciated as an important step in assembling the transcriptional complex. Herein, we demonstrate that the TBP-GR interaction is functionally significant by characterizing the association of TBP and GR in vitro by a combination of techniques and confirming the role of this interaction in vivo. Combined analysis, using native gel electrophoresis, sedimentation equilibrium, and isothermal microcalorimetry titrations, characterize the stoichiometry, affinity, and thermodynamics of the TBP-GR interaction. TBP binds recombinant GR activation function 1 (AF1) with a 1:2 stoichiometry and a dissociation constant in the nanomolar range. In vivo fluorescence resonance energy transfer experiments, using fluorescently labeled TBP and various GR constructs, transiently transfected into CV-1 cells, show GR-TBP interactions, dependent on AF1. AF1-deletion variants showed fluorescence resonance energy transfer efficiencies on the level of coexpressed cyan fluorescent protein and yellow fluorescent protein, indicating that the interaction is dependent on AF1 domain. To demonstrate the functional role of the in vivo GR-TBP interaction, increased amounts of TBP expressed in vivo stimulated expression of GR-driven reporters and endogenous genes, and the effect was also specifically dependent on AF1.

scholarly journals The organization of engaged and quiescent translocons in the endoplasmic reticulum of mammalian cells

2004 ◽  
Vol 164 (7) ◽  
pp. 997-1007 ◽  
Author(s):  
Erik L. Snapp ◽  
Gretchen A. Reinhart ◽  
Brigitte A. Bogert ◽  
Jennifer Lippincott-Schwartz ◽  
Ramanujan S. Hegde
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Signal Recognition Particle ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cellular Environment ◽  
Essential Components ◽  
Nascent Chain ◽  
Functional Components

Protein translocons of the mammalian endoplasmic reticulum are composed of numerous functional components whose organization during different stages of the transport cycle in vivo remains poorly understood. We have developed generally applicable methods based on fluorescence resonance energy transfer (FRET) to probe the relative proximities of endogenously expressed translocon components in cells. Examination of substrate-engaged translocons revealed oligomeric assemblies of the Sec61 complex that were associated to varying degrees with other essential components including the signal recognition particle receptor TRAM and the TRAP complex. Remarkably, these components not only remained assembled but also had a similar, yet distinguishable, organization both during and after nascent chain translocation. The persistence of preassembled and complete translocons between successive rounds of transport may facilitate highly efficient translocation in vivo despite temporal constraints imposed by ongoing translation and a crowded cellular environment.

Calmodulin-dependent binding to the NHE1 cytosolic tail mediates activation of the Na+/H+ exchanger by Ca2+ and endothelin

2013 ◽  
Vol 305 (11) ◽  
pp. C1161-C1169 ◽  
Author(s):  
Xiuju Li ◽  
Daniel Prins ◽  
Marek Michalak ◽  
Larry Fliegel
Keyword(s):  
Amino Acid ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Dependent ◽  
Wild Type ◽  
Membrane Domain ◽  
Plasma Membrane Protein ◽  
Nhe1 Activity

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a ubiquitous plasma membrane protein that regulates intracellular pH by removing a single proton (H+) in exchange for one extracellular Na+. The human protein contains a ∼500-amino acid membrane domain and a regulatory, ∼315-amino acid cytosolic domain. NHE1 is activated by a number of hormones including endothelin (ET) and by Ca2+. The regulatory tail possesses an inhibitory calmodulin (CaM)-binding domain, and inhibition of NHE1 is relieved by binding of a Ca2+-CaM complex. We examined the dynamics of ET-1 and Ca2+ regulation of binding to NHE1 in vivo. CFP was linked to the NHE1 protein cytoplasmic COOH terminus. This was stably transfected into AP-1 cells that are devoid of their own NHE1 protein. The protein was expressed and targeted properly and retained NHE1 activity comparable to the wild-type protein. We examined the in vivo coupling of NHE1 to CaM by Förster resonance energy transfer using CaM linked to the fluorescent protein Venus. CaM interaction with NHE1 was dynamic. Removal of serum reduced CaM interaction with NHE1. Addition of the Ca2+ ionophore ionomycin increased the interaction between CaM and NHE1. We expressed an ET receptor in AP-1 cells and also found a time-dependent association of NHE1 with CaM in vivo that was dependent on ET treatment. The results are the first demonstration of the in vivo association of NHE1 and CaM through ET-dependent signaling pathways.

scholarly journals Fluorescent sensors for activity and regulation of the nitrate transceptor CHL1/NRT1.1 and oligopeptide transporters

2014 ◽  
Author(s):  
Cheng Hsun Ho ◽  
Wolf B. Frommer
Keyword(s):  
Fluorescent Protein ◽  
New Technology ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Peptide Transport ◽  
Transport Activity ◽  
Proton Coupling ◽  
Activity Sensors

To monitor nitrate and peptide transport activity in vivo, we converted the dual-affinity nitrate transceptor CHL1/NRT1.1/NPF6.3 and four related oligopeptide transporters PTR1, 2, 4 and 5 into fluorescence activity sensors (NiTrac1, PepTrac). Substrate addition to yeast expressing transporter fusions with yellow fluorescent protein and mCerulean triggered substrate-dependent donor quenching or resonance energy transfer. Fluorescence changes were nitrate/peptide-specific, respectively. Like CHL1, NiTrac1 had biphasic kinetics. Mutation of T101A eliminated high-affinity transport and blocked the fluorescence response to low nitrate. NiTrac was used for characterizing side chains considered important for substrate interaction, proton coupling, and regulation. We observed a striking correlation between transport activity and sensor output. Coexpression of NiTrac with known calcineurin-like proteins (CBL1, 9; CIPK23) and candidates identified in an interactome screen (CBL1, KT2, WNKinase 8) blocked NiTrac1 responses, demonstrating the suitability for in vivo analysis of activity and regulation. The new technology is applicable in plant and medical research.

scholarly journals Diazepam Accelerates GABAAR Synaptic Exchange and Alters Intracellular Trafficking

2019 ◽  
Author(s):  
Joshua M. Lorenz-Guertin ◽  
Matthew J. Bambino ◽  
Sabyasachi Das ◽  
Susan T. Weintraub ◽  
Tija C. Jacob
Keyword(s):  
Cortical Neurons ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Adult Brain ◽  
Scaffolding Protein ◽  
Gamma Aminobutyric Acid ◽  
Inhibitory Neurotransmitter

Despite 50+ years of clinical use as anxiolytics, anti-convulsants, and sedative/hypnotic agents, the mechanisms underlying benzodiazepine (BZD) tolerance are poorly understood. BZDs potentiate the actions of gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the adult brain, through positive allosteric modulation of γ2 subunit containing GABA type A receptors (GABAARs). Here we define key molecular events impacting γ2 GABAAR and the inhibitory synapse gephyrin scaffold following initial sustained BZD exposure in vitro and in vivo. Using immunofluorescence and biochemical experiments, we found that cultured cortical neurons treated with the classical BZD, diazepam (DZP), presented no substantial change in surface or synaptic levels of γ2-GABAARs. In contrast, both γ2 and the postsynaptic scaffolding protein gephyrin showed diminished total protein levels following a single DZP treatment in vitro and in mouse cortical tissue. We further identified DZP treatment enhanced phosphorylation of gephyrin Ser270 and increased generation of gephyrin cleavage products. Selective immunoprecipitation of γ2 from cultured neurons revealed enhanced ubiquitination of this subunit following DZP exposure. To assess novel trafficking responses induced by DZP, we employed a γ2 subunit containing an N terminal fluorogen-activating peptide (FAP) and pH-sensitive green fluorescent protein (γ2pHFAP). Live-imaging experiments using γ2pHFAP GABAAR expressing neurons identified enhanced lysosomal targeting of surface GABAARs and increased overall accumulation in vesicular compartments in response to DZP. Using fluorescence resonance energy transfer (FRET) measurements between γ2 and γ2 subunits within a GABAAR in neurons, we identified reductions in synaptic clusters of this subpopulation of surface BZD sensitive receptor. Moreover, we found DZP simultaneously enhanced synaptic exchange of both γ2-GABAARs and gephyrin using fluorescence recovery after photobleaching (FRAP) techniques. Finally we provide the first proteomic analysis of the BZD sensitive GABAAR interactome in DZP vs. vehicle treated mice. Collectively, our results indicate DZP exposure elicits down-regulation of gephyrin scaffolding and BZD sensitive GABAAR synaptic availability via multiple dynamic trafficking processes.

scholarly journals Discovery of Enzyme Modulators via High-Throughput Time-Resolved FRET in Living Cells

2014 ◽  
Vol 19 (2) ◽  
pp. 215-222 ◽  
Author(s):  
Simon J. Gruber ◽  
Razvan L. Cornea ◽  
Ji Li ◽  
Kurt C. Peterson ◽  
Tory M. Schaaf ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dependent Manner ◽  
Chemical Library ◽  
Time Resolved ◽  
Endoplasmic Reticulum Calcium ◽  
Green Fluorescent

We have used a “two-color” SERCA (sarco/endoplasmic reticulum calcium ATPase) biosensor and a unique high-throughput fluorescence lifetime plate reader (FLT-PR) to develop a high-precision live-cell assay designed to screen for small molecules that perturb SERCA structure. A SERCA construct, in which red fluorescent protein (RFP) was fused to the N terminus and green fluorescent protein (GFP) to an interior loop, was stably expressed in an HEK cell line that grows in monolayer or suspension. Fluorescence resonance energy transfer (FRET) from GFP to RFP was measured in the FLT-PR, which increases precision 30-fold over intensity-based plate readers without sacrificing throughput. FRET was highly sensitive to known SERCA modulators. We screened a small chemical library and identified 10 compounds that significantly affected two-color SERCA FLT. Three of these compounds reproducibly lowered FRET and inhibited SERCA in a dose-dependent manner. This assay is ready for large-scale HTS campaigns and is adaptable to many other targets.

Molecular probes: insights into design and analysis from computational and physical chemistry

2008 ◽  
Vol 36 (1) ◽  
pp. 46-50 ◽  
Author(s):  
Felicity L. Mitchell ◽  
Gabriel E. Marks ◽  
Elena V. Bichenkova ◽  
Kenneth T. Douglas ◽  
Richard A. Bryce
Keyword(s):  
Physical Chemistry ◽  
Energy Transfer ◽  
Experimental Approach ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Molecular Probes ◽  
Green Fluorescent ◽  
Nanoscale Level

The application of new molecular diagnostics to probe cellular process in vivo is leading to a greater understanding of molecular cytology at a sub-nanoscale level and is opening the way to individualized medicines. We review here three distinct fluorescence-based molecular probes, HyBeacons™, split-probe exciplexes and GFP (green fluorescent protein)-based FRET (fluorescence resonance energy transfer) systems. Through this, we highlight the insights into the mechanism and design that a combined computational and experimental approach can yield.

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