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Masataka Kinjo - One of the best experts on this subject based on the ideXlab platform.
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A Quantitative Study of Internal and External Interactions of Homodimeric Glucocorticoid Receptor Using Fluorescence Cross-Correlation Spectroscopy in a Live Cell.
Scientific Reports, 2017Co-Authors: Manisha Tiwari, Shintaro Mikuni, Sho Oasa, Johtaro Yamamoto, Masataka KinjoAbstract:Glucocorticoid receptor (GRα) is a well-known ligand-dependent transcription-regulatory protein. The classic view is that unliganded GRα resides in the cytoplasm, relocates to the nucleus after ligand binding, and then associates with a specific DNA sequence, namely a glucocorticoid response element (GRE), to activate a specific gene as a homodimer. It is still a puzzle, however, whether GRα forms the homodimer in the cytoplasm or in the nucleus before DNA binding or after that. To quantify the homodimerization of GRα, we constructed the spectrally different fluorescent protein tagged hGRα and applied Fluorescence Cross-Correlation Spectroscopy. First, the dissociation constant (Kd) of mCherry2-fused hGRα or EGFP-fused hGRα was determined in vitro. Then, Kd of wild-type hGRα was found to be 3.00 μM in the nucleus, which was higher than that in vitro. Kd of a DNA-binding-deficient mutant was 3.51 μM in the nucleus. This similarity indicated that GRα homodimerization was not necessary for DNA binding but could take place on GRE by means of GRE as a scaffold. Moreover, cytoplasmic homodimerization was also observed using GRα mutated in the nuclear localization signal. These findings support the existence of a dynamic monomer pathway and regulation of GRα function both in the cytoplasm and nucleus.
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determination of the dissociation constant of the nfκb p50 p65 heterodimer in living cells using Fluorescence cross correlation Spectroscopy
Methods of Molecular Biology, 2015Co-Authors: Manisha Tiwari, Masataka KinjoAbstract:Fluorescence Cross-Correlation Spectroscopy (FCCS) is a promising technique for observing and quantifying protein-protein interactions in vitro and in vivo. FCCS has emerged as a useful tool for obtaining parameters of the concentration of labeled particles, their molecular dynamics, as well as the size of their complexes. This chapter discusses aspects of preparing a biological system for FCCS experiments and suggests practical advice for performing FCCS in living cells. Moreover, we describe the method of FCCS to determine the dissociation constant of a transcription factor dimer in the living cell.
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determination of dissociation constant of the nfκb p50 p65 heterodimer using Fluorescence cross correlation Spectroscopy in the living cell
Biochemical and Biophysical Research Communications, 2013Co-Authors: Manisha Tiwari, Shintaro Mikuni, Hideki Muto, Masataka KinjoAbstract:Abstract Two-laser-beam Fluorescence Cross-Correlation Spectroscopy (FCCS) is promising technique that provides quantitative information about the interactions of biomolecules. The p50/p65 heterodimer is the most abundant and well understood of the NFκB dimers in most cells. However, the quantitative value of affinity, namely the Kd, for the heterodimer in living cells is not known yet. To quantify the heterodimerization of the IPT domain of p50/p65 in the living cell, we used two-laser-beam FCCS. The Kd values of mCherry2- and EGFP-fused p50 and p65 were determined to be 0.46 μM in the cytoplasm and 1.06 μM in the nucleus of the living cell. These results suggest the different binding affinities of the p50/p65 heterodimer in the cytoplasm and nucleus of the living cell and different complex formation in each region.
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monitoring intracellular degradation of exogenous dna using diffusion properties
Journal of Controlled Release, 2010Co-Authors: Akira Sasaki, Masataka KinjoAbstract:Artificial nonviral gene vectors have the potential to improve the safety of gene therapy; however, such artificial vectors are less efficient for gene expression due to the existence of various barriers to delivery of exogenous DNAs to the nucleus in the living cell. Here we describe the degradation activities of cytoplasmic nucleases, which are involved as a barrier to efficient exogenous gene expression. The size and structure of degraded DNA were monitored by Fluorescence correlation Spectroscopy (FCS) and Fluorescence Cross-Correlation Spectroscopy (FCCS) in solution and in the cytoplasm of living cells. Differences in degradation by endo- and exonucleases were confirmed by differences in the size and structure of DNA. Moreover, we confirmed the influence of the exonuclease degradation in cytoplasm on the expression rate of DNA transfection with a cationic lipid. Based on a comparison of in vitro and cell measurements, we conclude that cytoplasmic degradation by exonucleases can be a considerable barrier against efficient gene delivery.
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reciprocal interaction with g actin and tropomyosin is essential for aquaporin 2 trafficking
Journal of Cell Biology, 2008Co-Authors: Yumi Noda, Saburo Horikawa, Eiichiro Kanda, Maho Yamashita, Hu Meng, Kayoko Eto, Michio Kuwahara, Keiji Hirai, Changi Pack, Masataka KinjoAbstract:Trafficking of water channel aquaporin-2 (AQP2) to the apical membrane and its vasopressin and protein kinase A (PKA)–dependent regulation in renal collecting ducts is critical for body water homeostasis. We previously identified an AQP2 binding protein complex including actin and tropomyosin-5b (TM5b). We show that dynamic interactions between AQP2 and the actin cytoskeleton are critical for initiating AQP2 apical targeting. Specific binding of AQP2 to G-actin in reconstituted liposomes is negatively regulated by PKA phosphorylation. Dual color Fluorescence Cross-Correlation Spectroscopy reveals local AQP2 interaction with G-actin in live epithelial cells at single-molecule resolution. Cyclic adenosine monophosphate signaling and AQP2 phosphorylation release AQP2 from G-actin. In turn, AQP2 phosphorylation increases its affinity to TM5b, resulting in reduction of TM5b bound to F-actin, subsequently inducing F-actin destabilization. RNA interference–mediated knockdown and overexpression of TM5b confirm its inhibitory role in apical trafficking of AQP2. These findings indicate a novel mechanism of channel protein trafficking, in which the channel protein itself critically regulates local actin reorganization to initiate its movement.
Atsushi Miyawaki - One of the best experts on this subject based on the ideXlab platform.
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a fluorescent variant of a protein from the stony coral montipora facilitates dual color single laser Fluorescence cross correlation Spectroscopy
Nature Biotechnology, 2006Co-Authors: Takako Kogure, Masataka Kinjo, Satoshi Karasawa, Toshio Araki, Kenta Saito, Atsushi MiyawakiAbstract:A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser Fluorescence Cross-Correlation Spectroscopy
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a fluorescent variant of a protein from the stony coral montipora facilitates dual color single laser Fluorescence cross correlation Spectroscopy
Nature Biotechnology, 2006Co-Authors: Takako Kogure, Masataka Kinjo, Satoshi Karasawa, Toshio Araki, Kenta Saito, Atsushi MiyawakiAbstract:Dual-color Fluorescence Cross-Correlation Spectroscopy (FCCS) is a promising technique for quantifying protein-protein interactions1,2,3,4,5. In this technique, two different fluorescent labels are excited and detected simultaneously within a common measurement volume. Difficulties in aligning two laser lines and emission crossover between the two fluorophores, however, make this technique complex. To overcome these limitations, we developed a fluorescent protein with a large Stokes shift. This protein, named Keima, absorbs and emits light maximally at 440 nm and 620 nm, respectively. Combining a monomeric version of Keima with cyan fluorescent protein allowed dual-color FCCS with a single 458-nm laser line and complete separation of the fluorescent protein emissions. This FCCS approach enabled sensitive detection of proteolysis by caspase-3 and the association of calmodulin with calmodulin-dependent enzymes. In addition, Keima and a spectral variant that emits maximally at 570 nm might facilitate simultaneous multicolor imaging with single-wavelength excitation.
Takako Kogure - One of the best experts on this subject based on the ideXlab platform.
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a fluorescent variant of a protein from the stony coral montipora facilitates dual color single laser Fluorescence cross correlation Spectroscopy
Nature Biotechnology, 2006Co-Authors: Takako Kogure, Masataka Kinjo, Satoshi Karasawa, Toshio Araki, Kenta Saito, Atsushi MiyawakiAbstract:A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser Fluorescence Cross-Correlation Spectroscopy
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a fluorescent variant of a protein from the stony coral montipora facilitates dual color single laser Fluorescence cross correlation Spectroscopy
Nature Biotechnology, 2006Co-Authors: Takako Kogure, Masataka Kinjo, Satoshi Karasawa, Toshio Araki, Kenta Saito, Atsushi MiyawakiAbstract:Dual-color Fluorescence Cross-Correlation Spectroscopy (FCCS) is a promising technique for quantifying protein-protein interactions1,2,3,4,5. In this technique, two different fluorescent labels are excited and detected simultaneously within a common measurement volume. Difficulties in aligning two laser lines and emission crossover between the two fluorophores, however, make this technique complex. To overcome these limitations, we developed a fluorescent protein with a large Stokes shift. This protein, named Keima, absorbs and emits light maximally at 440 nm and 620 nm, respectively. Combining a monomeric version of Keima with cyan fluorescent protein allowed dual-color FCCS with a single 458-nm laser line and complete separation of the fluorescent protein emissions. This FCCS approach enabled sensitive detection of proteolysis by caspase-3 and the association of calmodulin with calmodulin-dependent enzymes. In addition, Keima and a spectral variant that emits maximally at 570 nm might facilitate simultaneous multicolor imaging with single-wavelength excitation.
Petra Schwille - One of the best experts on this subject based on the ideXlab platform.
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membrane targeting of the spir formin actin nucleator complex requires a sequential handshake of polar interactions
Journal of Biological Chemistry, 2015Co-Authors: Janine Tittel, Thomas Weidemann, Petra Schwille, Tobias Welz, Aleksander Czogalla, Susanne Dietrich, Annette Samolwolf, Markos Schulte, Eugen KerkhoffAbstract:Spir and formin (FMN)-type actin nucleators initiate actin polymerization at vesicular membranes necessary for long range vesicular transport processes. Here we studied in detail the membrane binding properties and protein/protein interactions that govern the assembly of the membrane-associated Spir·FMN complex. Using biomimetic membrane models we show that binding of the C-terminal Spir-2 FYVE-type zinc finger involves both the presence of negatively charged lipids and hydrophobic contributions from the turret loop that intrudes the lipid bilayer. In solution, we uncovered a yet unknown intramolecular interaction between the Spir-2 FYVE-type domain and the N-terminal kinase non-catalytic C-lobe domain (KIND) that could not be detected in the membrane-bound state. Interestingly, we found that the intramolecular Spir-2 FYVE/KIND and the trans-regulatory Fmn-2-FSI/Spir-2-KIND interactions are competitive. We therefore characterized co-expressed Spir-2 and Fmn-2 fluorescent protein fusions in living cells by Fluorescence Cross-Correlation Spectroscopy. The data corroborate a model according to which Spir-2 exists in two different states, a cytosolic monomeric conformation and a membrane-bound state in which the KIND domain is released and accessible for subsequent Fmn-2 recruitment. This sequence of interactions mechanistically couples membrane binding of Spir to the recruitment of FMN, a pivotal step for initiating actin nucleation at vesicular membranes.
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Dynamics and interaction of interleukin-4 receptor subunits in living cells.
Biophysical journal, 2014Co-Authors: Hetvi Gandhi, Kristina Kurgonaite, Petra Schwille, Christian Bökel, Remigiusz Worch, Martin Hintersteiner, Thomas WeidemannAbstract:It has long been established that dimerization of Interleukin-4 receptor (IL-4R) subunits is a pivotal step for JAK/STAT signal transduction. However, ligand-induced complex formation at the surface of living cells has been challenging to observe. Here we report an experimental assay employing trisNTA dyes for orthogonal, external labeling of eGFP-tagged receptor constructs that allows the quantification of receptor heterodimerization by dual-color Fluorescence Cross-Correlation Spectroscopy. Fluorescence Cross-Correlation Spectroscopy analysis at the plasma membrane shows that IL-4R subunit dimerization is indeed a strictly ligand-induced process. Under conditions of saturating cytokine occupancy, we determined intramembrane dissociation constants (Kd,2D) of 180 and 480 receptors per μm2 for the type-2 complexes IL-4:IL-4Rα/IL-13Rα1 and IL-13:IL-13Rα1/IL-4Rα, respectively. For the lower affinity type-1 complex IL-4:IL-4Rα/IL-2Rγ, we estimated a Kd,2D of ∼1000 receptors per μm2. The receptor densities required for effective dimerization thus exceed the typical, average expression levels by several orders of magnitude. In addition, we find that all three receptor subunits accumulate rapidly within a subpopulation of early sorting and recycling endosomes stably anchored just beneath the plasma membrane (cortical endosomes, CEs). The receptors, as well as labeled IL-4 and trisNTA ligands are specifically trafficked into CEs by a constitutive internalization mechanism. This may compensate for the inherent weak affinities that govern ligand-induced receptor dimerization at the plasma membrane. Consistently, activated receptors are also concentrated at the CEs. Our observations thus suggest that receptor trafficking may play an important role for the regulation of IL-4R-mediated JAK/STAT signaling.
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correcting for artifacts from spectral cross talk and imperfect detection volume overlap in dual color Fluorescence cross correlation Spectroscopy
Biophysical Journal, 2012Co-Authors: Kirsten Bacia, Stefan Werner, Zdenek Petrasek, Petra SchwilleAbstract:Dual-color Fluorescence Cross-Correlation Spectroscopy (dcFCCS) allows to quantitatively assess the interactions of mobile molecules labeled with distinct fluorophores.One major artifact risk in dcFCCS is a false-positive or overestimated Cross-Correlation amplitude arising from spectral cross-talk. Cross-talk can be reduced or prevented by fast alternating excitation, but the technology is not easily implemented in standard commercial setups. We devised an experimental strategy that does not require specialized hard- and software for recognizing and correcting for cross-talk in standard dcFCCS.Another major artifact risk in dcFCCS is a false-negative or underestimated Cross-Correlation amplitude arising from an imperfect detection volume overlap. Samples based on fluorophores conjugated to oligonucleotides that have been traditionally used for dcFCCS calibration typically suffer from incomplete labeling. Using these samples, it is difficult to attribute a reduced Cross-Correlation amplitude to the imperfection of the calibration sample or to that of the setup. We therefore developed a new Cross-Correlation calibration standard with a predictable degree of labeling.View Large Image | View Hi-Res Image | Download PowerPoint Slide
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Fluorescence correlation Spectroscopy and Fluorescence cross correlation Spectroscopy reveal the cytoplasmic origination of loaded nuclear risc in vivo in human cells
Nucleic Acids Research, 2008Co-Authors: Thomas Ohrt, Jorg Mutze, Wolfgang Staroske, Lasse Weinmann, Julia Hock, Karin Crell, Gunter Meister, Petra SchwilleAbstract:Studies of RNA interference (RNAi) provide evidence that in addition to the well-characterized cytoplasmic mechanisms, nuclear mechanisms also exist. The mechanism by which the nuclear RNA-induced silencing complex (RISC) is formed in mammalian cells, as well as the relationship between the RNA silencing pathways in nuclear and cytoplasmic compartments is still unknown. Here we show by applying Fluorescence correlation and Cross-Correlation Spectroscopy (FCS/FCCS) in vivo that two distinct RISC exist: a large ∼3 MDa complex in the cytoplasm and a 20-fold smaller complex of ∼158 kDa in the nucleus. We further show that nuclear RISC, consisting only of Ago2 and a short RNA, is loaded in the cytoplasm and imported into the nucleus. The loaded RISC accumulates in the nucleus depending on the presence of a target, based on an miRNA-like interaction with impaired cleavage of the cognate RNA. Together, these results suggest a new RISC shuttling mechanism between nucleus and cytoplasm ensuring concomitant gene regulation by small RNAs in both compartments.
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dual color Fluorescence cross correlation Spectroscopy for multicomponent diffusional analysis in solution
Biophysical Journal, 1997Co-Authors: Petra Schwille, Franzjosef Meyeralmes, Rudolf RiglerAbstract:The present paper describes a new experimental scheme for following diffusion and chemical reaction systems of fluorescently labeled molecules in the nanomolar concentration range by Fluorescence correlation analysis. In the dual-color Fluorescence Cross-Correlation Spectroscopy provided here, the concentration and diffusion characteristics of two fluorescent species in solution as well as their reaction product can be followed in parallel. By using two differently labeled reaction partners, the selectivity to investigate the temporal evolution of reaction product is significantly increased compared to ordinary one-color Fluorescence autocorrelation systems. Here we develop the theoretical and experimental basis for carrying out measurements in a confocal dual-beam Fluorescence correlation Spectroscopy setup and discuss conditions that are favorable for Cross-Correlation analysis. The measurement principle is explained for carrying out DNA-DNA renaturation kinetics with two differently labeled complementary strands. The concentration of the reaction product can be directly determined from the Cross-Correlation amplitude.
Toshio Araki - One of the best experts on this subject based on the ideXlab platform.
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a fluorescent variant of a protein from the stony coral montipora facilitates dual color single laser Fluorescence cross correlation Spectroscopy
Nature Biotechnology, 2006Co-Authors: Takako Kogure, Masataka Kinjo, Satoshi Karasawa, Toshio Araki, Kenta Saito, Atsushi MiyawakiAbstract:A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser Fluorescence Cross-Correlation Spectroscopy
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a fluorescent variant of a protein from the stony coral montipora facilitates dual color single laser Fluorescence cross correlation Spectroscopy
Nature Biotechnology, 2006Co-Authors: Takako Kogure, Masataka Kinjo, Satoshi Karasawa, Toshio Araki, Kenta Saito, Atsushi MiyawakiAbstract:Dual-color Fluorescence Cross-Correlation Spectroscopy (FCCS) is a promising technique for quantifying protein-protein interactions1,2,3,4,5. In this technique, two different fluorescent labels are excited and detected simultaneously within a common measurement volume. Difficulties in aligning two laser lines and emission crossover between the two fluorophores, however, make this technique complex. To overcome these limitations, we developed a fluorescent protein with a large Stokes shift. This protein, named Keima, absorbs and emits light maximally at 440 nm and 620 nm, respectively. Combining a monomeric version of Keima with cyan fluorescent protein allowed dual-color FCCS with a single 458-nm laser line and complete separation of the fluorescent protein emissions. This FCCS approach enabled sensitive detection of proteolysis by caspase-3 and the association of calmodulin with calmodulin-dependent enzymes. In addition, Keima and a spectral variant that emits maximally at 570 nm might facilitate simultaneous multicolor imaging with single-wavelength excitation.