Mapping of adherens junction components using microscopic resonance energy transfer imaging

1995 ◽  
Vol 108 (3) ◽  
pp. 1051-1062 ◽  
Author(s):  
Z. Kam ◽  
T. Volberg ◽  
B. Geiger
Keyword(s):  
Energy Transfer ◽  
High Resolution ◽  
Resonance Energy Transfer ◽  
Focal Adhesions ◽  
Resonance Energy ◽  
Ccd Camera ◽  
The Other ◽  
Molecular Organization ◽  
Other Hand ◽  
Immunofluorescence Labeling

Quantitative microscopic imaging of resonance energy transfer (RET) was applied for immunological high resolution proximity mapping of several cytoskeletal components of cell adhesions. To conduct this analysis, a microscopic system was developed, consisting of a highly stable field illuminator, computer-controlled filter wheels for rapid multiple-color imaging and a sensitive, high resolution CCD camera, enabling quantitative data recording and processing. Using this system, we have investigated the spatial inter-relationships and organization of four adhesion-associated proteins, namely vinculin, talin, alpha-actinin and actin. Cultured chick lens cells were double labeled for each of the junctional molecules, using fluorescein- and rhodamine-conjugated antibodies or phalloidin. RET images were acquired with fluorescein excitation and rhodamine emission filter setting, corrected for fluorescein and rhodamine fluorescence, and normalized to the fluorescein image. The results pointed to high local densities of vinculin, talin and F-actin in focal adhesions, manifested by mean RET values of 15%, 12% and 10%, respectively. On the other hand, relatively low values (less than 1%) were observed following double immunofluorescence labeling of the same cells for alpha-actinin. Double indirect labeling for pairs of these four proteins (using fluorophore-conjugated antibodies or phalloidin) resulted in RET values of 5% or lower, except for the pair alpha-actinin and actin, which yielded significantly higher values (13-15%). These results suggest that despite their overlapping staining patterns, at the level of resolution of the light microscope, the plaque proteins vinculin and talin are not homogeneously interspersed at the molecular level but form segregated clusters. alpha-Actinin, on the other hand, does not appear to form such clusters but, rather, closely interacts with actin. We discuss here the conceptual and applicative aspects of RET measurements and the implications of the results on the subcellular molecular organization of adherens-type junctions.

A novel homogeneous bioluminescence resonance energy transfer element for biomolecular detection with CCD camera or CMOS device

Luminescence ◽  
2008 ◽  
Vol 23 (1) ◽  
pp. 22-27 ◽  
Author(s):  
Brian Filanoski ◽  
Shiva K. Rastogi ◽  
Eric Cameron ◽  
Nirankar N. Mishra ◽  
Wusi Maki ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Ccd Camera ◽  
Bioluminescence Resonance Energy Transfer ◽  
Biomolecular Detection

scholarly journals Dual-color metal-induced and Förster resonance energy transfer for cell nanoscopy

2018 ◽  
Vol 29 (7) ◽  
pp. 846-851 ◽  
Author(s):  
Anna M. Chizhik ◽  
Carina Wollnik ◽  
Daja Ruhlandt ◽  
Narain Karedla ◽  
Alexey I. Chizhik ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Focal Adhesions ◽  
Resonance Energy ◽  
Forster Resonance Energy Transfer ◽  
Förster Resonance Energy Transfer ◽  
Fluorescence Lifetime Imaging ◽  
Dual Color ◽  
Structural Details ◽  
Förster Resonance

We report a novel method, dual-color axial nanometric localization by metal-­induced energy transfer, and combine it with Förster resonance energy transfer (FRET) for resolving structural details in cells on the molecular level. We demonstrate the capability of this method on cytoskeletal elements and adhesions in human mesenchymal stem cells. Our approach is based on fluorescence-lifetime-imaging microscopy and allows for precise determination of the three-dimensional architecture of stress fibers anchoring at focal adhesions, thus yielding crucial information to understand cell–matrix mechanics. In addition to resolving nanometric structural details along the z-axis, we use FRET to gain precise information on the distance between actin and vinculin at focal adhesions.

scholarly journals Application of Advanced Light Microscopy to the Study of HIV and Its Interactions with the Host

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 223
Author(s):  
Saveez Saffarian
Keyword(s):  
Human Immunodeficiency Virus ◽  
Energy Transfer ◽  
High Resolution ◽  
Light Microscopy ◽  
Single Molecule ◽  
Total Internal Reflection ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Immunodeficiency Virus ◽  
Microscopy Techniques

This review highlights the significant observations of human immunodeficiency virus (HIV) assembly, release and maturation made possible with advanced light microscopy techniques. The advances in technology which now enables these light microscopy measurements are discussed with special emphasis on live imaging approaches including Total Internal Reflection Fluorescence (TIRF), high-resolution light microscopy techniques including PALM and STORM and single molecule measurements, including Fluorescence Resonance Energy Transfer (FRET). The review concludes with a discussion on what new insights and understanding can be expected from these measurements.

Abstract 16905: Fluorescence Resonance Energy Transfer Reveals that Cardiac Calcium ATPase Dimerizes and Forms a Complex with Phospholamban in a 2:1 Stoichiometry

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Daniel Blackwell ◽  
Seth L Robia
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Calcium Atpase ◽  
The Other ◽  
Fret Efficiency ◽  
Fluorescence Resonance

The sarco/endoplasmic reticulum calcium ATPase (SERCA) has been proposed to form functional dimers in vitro. In order to investigate whether SERCA forms homo-dimers in live cells, we fused canine SERCA2a to cerulean (Cer) or yellow fluorescent protein (YFP), and quantified SERCA-SERCA interactions by fluorescence resonance energy transfer (FRET). SERCA-SERCA FRET efficiency was dependent on the labeling position of the fluorescent protein tags, with the highest FRET efficiency achieved when the respective fluorescent proteins were fused to SERCA N-termini. FRET was reduced by competition with unlabeled SERCA, suggesting that the observed FRET was due to specific protein-protein interactions. Progressive photobleaching of YFP showed that Cer intensity increased linearly with decreasing YFP intensity, suggesting that the stoichiometry of the SERCA complex is a dimer. In contrast, a control experiment with phospholamban (PLB) oligomer showed a non-linear YFP/Cer relationship, consistent with its well-known pentameric stoichiometry. We also investigated whether SERCA dimers could interact with PLB, the regulatory binding partner of SERCA. Interestingly, while average maximal FRET was 28% between SERCA and PLB, fluorescence lifetime measurements revealed two different lifetimes, consistent with two different populations of FRET donors. One population showed very low FRET, while the other population exhibited high FRET- approximately double the measured average maximal FRET efficiency. The data are consistent with a single PLB bound to each SERCA homo-dimer; in this regulatory complex one SERCA protomer is in close proximity to PLB (50 Å), while the other is too far away to participate in FRET with PLB.

MODULATION OF FRET THROUGH THE SPECTRAL-OVERLAP STRATEGY

2012 ◽  
Vol 05 (03) ◽  
pp. 1250014 ◽  
Author(s):  
HAIBO YU ◽  
YI XIAO
Keyword(s):  
Energy Transfer ◽  
Absorption Spectra ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
The Other ◽  
Biological Sensing ◽  
Spectral Overlap ◽  
Donor And Acceptor ◽  
Major Factors ◽  
Ratiometric Sensors

Fluorescence resonance energy transfer (FRET) is an important photophysical mechanism which finds many applications in biophotonics, particularly in biological sensing and imaging. It is well known, there are two major factors that determine the efficiency of FRET. One is the distance between the donor and the acceptor, and the other is the overlap of the donor's emission and the acceptor's absorption spectra. However, while the distance-modulation of FRET is very popular, the spectral-overlap-modulation draws much less attention. In this talk, I would like to illustrate the importance of the strategy of spectral-overlap-modulation. The presentation will include our works on the construction of highly efficient FRET systems featuring the short and rigid linker between the donor and acceptor moieties, and several typical examples of spectral-overlap-modulation strategy applied in the development of ratiometric sensors.

scholarly journals An enhanced molecular tension sensor based on bioluminescence resonance energy transfer (BRET)

2019 ◽  
Author(s):  
Eric J. Aird ◽  
Kassidy J. Tompkins ◽  
Wendy R. Gordon
Keyword(s):  
Energy Transfer ◽  
Dynamic Range ◽  
Resonance Energy Transfer ◽  
Focal Adhesions ◽  
Resonance Energy ◽  
Cellular Protein ◽  
Bioluminescence Resonance Energy Transfer ◽  
Tension Sensor ◽  
Focal Adhesion Protein

ABSTRACTMolecular tension sensors measure piconewton forces experienced by individual proteins in the context of the cellular microenvironment. Current genetically-encoded tension sensors use FRET to report on extension of an elastic peptide encoded in a cellular protein of interest. Here we present the development and characterization of a new type of molecular tension sensor based on bioluminescence resonance energy transfer (BRET) which exhibits more desirable spectral properties and an enhanced dynamic range compared to other molecular tension sensors. Moreover, it avoids many disadvantages of FRET measurements in cells, including heating of the sample, autofluorescence, photobleaching, and corrections of direct acceptor excitation. We benchmark the sensor by inserting it into the canonical mechanosensing focal adhesion protein vinculin, observing highly resolved gradients of tensional changes across focal adhesions. We anticipate that the BRET-TS will expand the toolkit available to study mechanotransduction at a molecular level and allow potential extension to an in vivo context.

scholarly journals High-Resolution Temperature−Concentration Diagram of α-Synuclein Conformation Obtained from a Single Förster Resonance Energy Transfer Image in a Microfluidic Device

2009 ◽  
Vol 81 (16) ◽  
pp. 6929-6935 ◽  
Author(s):  
Virginia Vandelinder ◽  
Allan Chris M. Ferreon ◽  
Yann Gambin ◽  
Ashok A. Deniz ◽  
Alex Groisman
Keyword(s):  
Energy Transfer ◽  
High Resolution ◽  
Microfluidic Device ◽  
Resonance Energy Transfer ◽  
Resonance Energy

scholarly journals Förster Resonance Energy Transfer Structural Kinetic Studies of Cardiac Thin Filament Deactivation

2009 ◽  
Vol 284 (24) ◽  
pp. 16432-16441 ◽  
Author(s):  
Jun Xing ◽  
Jayant J. Jayasundar ◽  
Yexin Ouyang ◽  
Wen-Ji Dong
Keyword(s):  
Energy Transfer ◽  
Conformational Change ◽  
Thin Filament ◽  
Regulatory Region ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
The Other ◽  
Structural Transitions ◽  
Inhibitory Region ◽  
Kinetics Of

Cardiac thin filament deactivation is initiated by Ca2+ dissociation from troponin C (cTnC), followed by multiple structural changes of thin filament proteins. These structural transitions are the molecular basis underlying the thin filament regulation of cardiac relaxation, but the detailed mechanism remains elusive. In this study Förster resonance energy transfer (FRET) was used to investigate the dynamics and kinetics of the Ca2+-induced conformational changes of the cardiac thin filaments, specifically the closing of the cTnC N-domain, the cTnC-cTnI (troponin I) interaction, and the cTnI-actin interaction. The cTnC N-domain conformational change was examined by monitoring FRET between a donor (AEDANS) attached to one cysteine residue and an acceptor (DDPM) attached the other cysteine of the mutant cTnC(L13C/N51C). The cTnC-cTnI interaction was investigated by monitoring the distance changes from residue 89 of cTnC to residues 151 and 167 of cTnI, respectively. The cTnI-actin interaction was investigated by monitoring the distance changes from residues 151 and 167 of cTnI to residue 374 of actin. FRET Ca2+ titrations and stopped-flow kinetic measurements show that different thin filament structural transitions have different Ca2+ sensitivities and Ca2+ dissociation-induced kinetics. The observed structural transitions involving the regulatory region and the mobile domain of cTnI occurred at fast kinetic rates, whereas the kinetics of the structural transitions involving the cTnI inhibitory region was slow. Our results suggest that the thin filament deactivation upon Ca2+ dissociation is a two-step process. One step involves rapid binding of the mobile domain of cTnI to actin, which is kinetically coupled with the conformational change of the N-domain of cTnC and the dissociation of the regulatory region of cTnI from cTnC. The other step involves switching the inhibitory region of cTnI from interacting with cTnC to interacting with actin. The latter processes may play a key role in regulating cross-bridge kinetics.

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