Stoichiometric analysis of oligomeric states of three class-A GPCRs, chemokine-CXCR4, dopamine-D2, and prostaglandin-EP1 receptors, on living cells

2017 ◽  
Vol 23 (7-8) ◽  
pp. 650-658 ◽  
Author(s):  
Kenichi Kawano ◽  
Tetsuya Yagi ◽  
Nozomu Fukada ◽  
Yoshiaki Yano ◽  
Katsumi Matsuzaki
Keyword(s):  
Living Cells ◽  
Stoichiometric Analysis ◽  
Class A ◽  
Dopamine D2

Oligomerization of adenosine A2A and dopamine D2 receptors in living cells

2003 ◽  
Vol 306 (2) ◽  
pp. 544-549 ◽  
Author(s):  
Toshio Kamiya ◽  
Osamu Saitoh ◽  
Kazuaki Yoshioka ◽  
Hiroyasu Nakata
Keyword(s):  
Living Cells ◽  
D2 Receptors ◽  
Dopamine D2 Receptors ◽  
Dopamine D2 ◽  
Adenosine A2a

Stoichiometric Analysis of Oligomerization of Membrane Proteins on Living Cells Using Coiled-Coil Labeling and Spectral Imaging

2013 ◽  
Vol 85 (6) ◽  
pp. 3454-3461 ◽  
Author(s):  
Kenichi Kawano ◽  
Yoshiaki Yano ◽  
Kaoru Omae ◽  
Sayaka Matsuzaki ◽  
Katsumi Matsuzaki
Keyword(s):  
Membrane Proteins ◽  
Coiled Coil ◽  
Spectral Imaging ◽  
Living Cells ◽  
Stoichiometric Analysis

scholarly journals The Class-A GPCR Dopamine D2 Receptor Forms Transient Dimers Stabilized by Agonists: Detection by Single-Molecule Tracking

2017 ◽  
Vol 76 (1-2) ◽  
pp. 29-37 ◽  
Author(s):  
Rinshi S. Kasai ◽  
Shuichi V. Ito ◽  
Ryo M. Awane ◽  
Takahiro K. Fujiwara ◽  
Akihiro Kusumi
Keyword(s):  
Single Molecule ◽  
Resting State ◽  
Dopamine D2 Receptor ◽  
Critical Role ◽  
D2 Receptor ◽  
G Protein Coupled Receptors ◽  
Single Molecule Tracking ◽  
Class A ◽  
Dopamine D2 ◽  
G Protein Coupled

Abstract Whether class-A G-protein coupled receptors (GPCRs) exist and work as monomers or dimers has drawn extensive attention. A class-A GPCR dopamine D2 receptor (D2R) is involved in many physiological and pathological processes and diseases, indicating its critical role in proper functioning of neuronal circuits. In particular, D2R homodimers might play key roles in schizophrenia development and amphetamine-induced psychosis. Here, using single-molecule imaging, we directly tracked single D2R molecules in the plasma membrane at a physiological temperature of 37 °C, and unequivocally determined that D2R forms transient dimers with a lifetime of 68 ms in its resting state. Agonist addition prolonged the dimer lifetime by a factor of ~1.5, suggesting the possibility that transient dimers might be involved in signaling.

scholarly journals Detection of Heteromers Formed by Cannabinoid CB1, Dopamine D2, and Adenosine A2AG-Protein-Coupled Receptors by Combining Bimolecular Fluorescence Complementation and Bioluminescence Energy Transfer

2008 ◽  
Vol 8 ◽  
pp. 1088-1097 ◽  
Author(s):  
Gemma Navarro ◽  
Paulina Carriba ◽  
Jorge Gandí ◽  
Francisco Ciruela ◽  
Vicent Casadó ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Molecular Interactions ◽  
Living Cells ◽  
Bimolecular Fluorescence Complementation ◽  
Functional Interactions ◽  
Dopamine D2 ◽  
Fluorescence Complementation ◽  
Bimolecular Fluorescence

Functional interactions in signaling occur between dopamine D2(D2R) and cannabinoid CB1(CB1R) receptors, between CB1R and adenosine A2A(A2AR) receptors, and between D2R and A2AR. Furthermore, direct molecular interactions have been reported for the pairs CB1R-D2R, A2AR-D2R, and CB1R-A2AR. Here a combination of bimolecular fluorescence complementation and bioluminescence energy transfer techniques was used to identify the occurrence of D2R-CB1R-A2AR hetero-oligomers in living cells.

Fluorescence ratio imaging of dynamic intracellular ionic signals

1988 ◽  
Vol 46 ◽  
pp. 42-43
Author(s):  
R. Y. Tsien ◽  
A. Minta ◽  
M. Poenie ◽  
J.P.Y. Kao ◽  
A. Harootunian
Keyword(s):  
Binding Sites ◽  
Living Cells ◽  
Molecular Engineering ◽  
Ph Indicators ◽  
Highly Sensitive ◽  
Fluorescence Ratio Imaging ◽  
Ratio Imaging ◽  
Technical Advances ◽  
Indicator Dyes ◽  
Fluorescein Dyes

Recent technical advances now enable the continuous imaging of important ionic signals inside individual living cells with micron spatial resolution and subsecond time resolution. This methodology relies on the molecular engineering of indicator dyes whose fluorescence is strong and highly sensitive to ions such as Ca2+, H+, or Na+, or Mg2+. The Ca2+ indicators, exemplified by fura-2 and indo-1, derive their high affinity (Kd near 200 nM) and selectivity for Ca2+ to a versatile tetracarboxylate binding site3 modeled on and isosteric with the well known chelator EGTA. The most commonly used pH indicators are fluorescein dyes (such as BCECF) modified to adjust their pKa's and improve their retention inside cells. Na+ indicators are crown ethers with cavity sizes chosen to select Na+ over K+: Mg2+ indicators use tricarboxylate binding sites truncated from those of the Ca2+ chelators, resulting in a more compact arrangement of carboxylates to suit the smaller ion.

Application of FRAP and digitized fluorescence microscopy to problems in cell membrane dynamics

1988 ◽  
Vol 46 ◽  
pp. 118-119
Author(s):  
K. Jacobson ◽  
A. Ishihara ◽  
B. Holifield ◽  
F. Zhang
Keyword(s):  
Fluorescence Microscopy ◽  
Cell Biology ◽  
Extended Period ◽  
Dynamic Structure ◽  
Living Cells ◽  
Membrane Dynamics ◽  
Molecular Cell Biology ◽  
Image Averaging ◽  
Single Living Cells ◽  
Single Living

Our laboratory is concerned with understanding the dynamic structure of the plasma membrane with particular reference to the movement of membrane constituents during cell locomotion. In addition to the standard tools of molecular cell biology, we employ both fluorescence recovery after photo- bleaching (FRAP) and digitized fluorescence microscopy (DFM) to investigate individual cells. FRAP allows the measurement of translational mobility of membrane and cytoplasmic molecules in small regions of single, living cells. DFM is really a new form of light microscopy in that the distribution of individual classes of ions, molecules, and macromolecules can be followed in single, living cells. By employing fluorescent antibodies to defined antigens or fluorescent analogs of cellular constituents as well as ultrasensitive, electronic image detectors and video image averaging to improve signal to noise, fluorescent images of living cells can be acquired over an extended period without significant fading and loss of cell viability.

Multimode light microscopy and fluorescence-based reagents as tools for the study of chemical and molecular dynamics of living cells and tissues

1992 ◽  
Vol 50 (1) ◽  
pp. 418-419
Author(s):  
D. L. Taylor
Keyword(s):  
Living Cells ◽  
Cellular Level ◽  
Molecular Techniques ◽  
Cellular Functions ◽  
Macromolecular Assemblies ◽  
Cell Functions ◽  
Molecular Events ◽  
Yield Information ◽  
Full Knowledge ◽  
Structural Methods

Cells function through the complex temporal and spatial interplay of ions, metabolites, macromolecules and macromolecular assemblies. Biochemical approaches allow the investigator to define the components and the solution chemical reactions that might be involved in cellular functions. Static structural methods can yield information concerning the 2- and 3-D organization of known and unknown cellular constituents. Genetic and molecular techniques are powerful approaches that can alter specific functions through the manipulation of gene products and thus identify necessary components and sequences of molecular events. However, full knowledge of the mechanism of particular cell functions will require direct measurement of the interplay of cellular constituents. Therefore, there has been a need to develop methods that can yield chemical and molecular information in time and space in living cells, while allowing the integration of information from biochemical, molecular and genetic approaches at the cellular level.

4-dimensional, high-resolution imaging of living cells

1992 ◽  
Vol 50 (1) ◽  
pp. 416-417
Author(s):  
Shinya Inoué
Keyword(s):  
Light Microscope ◽  
Living Cells ◽  
Live Cells ◽  
Video Tape ◽  
Active Living ◽  
High Resolution Imaging ◽  
3 Dimensional ◽  
Dynamic Architecture ◽  
Resolution Imaging ◽  
Disk Memory

This paper reports progress of our effort to rapidly capture, and display in time-lapsed mode, the 3-dimensional dynamic architecture of active living cells and developing embryos at the highest resolution of the light microscope. Our approach entails: (A) real-time video tape recording of through-focal, ultrathin optical sections of live cells at the highest resolution of the light microscope; (B) repeat of A at time-lapsed intervals; (C) once each time-lapsed interval, an image at home focus is recorded onto Optical Disk Memory Recorder (OMDR); (D) periods of interest are selected using the OMDR and video tape records; (E) selected stacks of optical sections are converted into plane projections representing different view angles (±4 degrees for stereo view, additional angles when revolving stereos are desired); (F) analysis using A - D.

Modulated polarization microscopy displays the dynamics of cytoskeletal structures in living, motile cells

1995 ◽  
Vol 53 ◽  
pp. 902-903
Author(s):  
J. R. Kuhn ◽  
M. Poenie
Keyword(s):  
Mitotic Spindle ◽  
Actin Filaments ◽  
Cell Shape ◽  
Dynamic Structure ◽  
Living Cells ◽  
Cytoskeletal Proteins ◽  
Polarization Microscopy ◽  
Video Enhancement ◽  
Ultrastructural Studies ◽  
Motile Cells

Cell shape and movement are controlled by elements of the cytoskeleton including actin filaments an microtubules. Unfortunately, it is difficult to visualize the cytoskeleton in living cells and hence follow it dynamics. Immunofluorescence and ultrastructural studies of fixed cells while providing clear images of the cytoskeleton, give only a static picture of this dynamic structure. Microinjection of fluorescently Is beled cytoskeletal proteins has proved useful as a way to follow some cytoskeletal events, but long terry studies are generally limited by the bleaching of fluorophores and presence of unassembled monomers.Polarization microscopy has the potential for visualizing the cytoskeleton. Although at present, it ha mainly been used for visualizing the mitotic spindle. Polarization microscopy is attractive in that it pro vides a way to selectively image structures such as cytoskeletal filaments that are birefringent. By combing ing standard polarization microscopy with video enhancement techniques it has been possible to image single filaments. In this case, however, filament intensity depends on the orientation of the polarizer and analyzer with respect to the specimen.

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