scholarly journals Endocytosis of Connexin 36 is Mediated by Interaction with Caveolin-1

2020 ◽  
Vol 21 (15) ◽  
pp. 5401
Author(s):  
Anna Kotova ◽  
Ksenia Timonina ◽  
Georg R. Zoidl
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Pharmacological Interventions ◽  
Caveolin 1 ◽  
Connexin 36 ◽  
Cell Surface Biotinylation ◽  
Junctional Protein ◽  
Gap Junctional ◽  
Microscopy Techniques ◽  
Dependent Pathway

The gap junctional protein connexin 36 (Cx36) has been co-purified with the lipid raft protein caveolin-1 (Cav-1). The relevance of an interaction between the two proteins is unknown. In this study, we explored the significance of Cav-1 interaction in the context of intracellular and membrane transport of Cx36. Coimmunoprecipitation assays and Förster resonance energy transfer analysis (FRET) were used to confirm the interaction between the two proteins in the Neuro 2a cell line. We found that the Cx36 and Cav-1 interaction was dependent on the intracellular calcium levels. By employing different microscopy techniques, we demonstrated that Cav-1 enhances the vesicular transport of Cx36. Pharmacological interventions coupled with cell surface biotinylation assays and FRET analysis revealed that Cav-1 regulates membrane localization of Cx36. Our data indicate that the interaction between Cx36 and Cav-1 plays a role in the internalization of Cx36 by a caveolin-dependent pathway.

scholarly journals Functional Expression of the Murine Connexin 36 Gene Coding for a Neuron-Specific Gap Junctional Protein

2000 ◽  
Vol 176 (3) ◽  
pp. 249-262 ◽  
Author(s):  
B. Teubner ◽  
J. Degen ◽  
G. Söhl ◽  
M. Güldenagel ◽  
F.F. Bukauskas ◽  
...  
Keyword(s):  
Functional Expression ◽  
Connexin 36 ◽  
Gene Coding ◽  
Junctional Protein ◽  
Gap Junctional

scholarly journals Functional Expression of the Murine Connexin 36 Gene Coding for a Neuron-Specific Gap Junctional Protein

2000 ◽  
Vol 176 (3) ◽  
pp. 249-262 ◽  
Author(s):  
B. Teubner ◽  
J. Degen ◽  
G. Söhl ◽  
M. Güldenagel ◽  
F.F. Bukauskas ◽  
...  
Keyword(s):  
Functional Expression ◽  
Connexin 36 ◽  
Gene Coding ◽  
Junctional Protein ◽  
Gap Junctional

Advances in Microscopy Techniques

2011 ◽  
Vol 135 (2) ◽  
pp. 255-263
Author(s):  
Daniel B Schmolze ◽  
Clive Standley ◽  
Kevin E Fogarty ◽  
Andrew H Fischer
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Near Field ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Molecular Techniques ◽  
Live Cell ◽  
Stimulated Emission ◽  
Structured Illumination ◽  
Microscopy Techniques

Abstract Context.—Advances in microscopy enable visualization of a broad range of new morphologic features. Objective.—To review and illustrate advances in microscopy with relevance to pathologists. Data Sources.—Literature review and new observations. Results.—Fluorescence microscopy enables multiantigen detection; allows novel optical-sectioning techniques, with some advantages compared to paraffin sectioning; and permits live-cell imaging. Live-cell imaging allows pathologists to move from a period when all diagnostic expertise was reliant on interpreting static images to a period when cellular dynamics can play a role in diagnosis. New techniques have bypassed by about 100-fold what had long been believed to be a limit to the resolution of light microscopy. Fluorescence resonance energy transfer (FRET) appears capable of visualizing diagnostically relevant molecular events in living or fixed cells that are immeasurable by other molecular techniques. We describe applications of 2-photon microscopy, FRET, structured illumination, and the subdiffraction techniques of near-field microscopy, photoactivated localization microscopy, stochastic optical reconstruction microscopy, and stimulated emission depletion microscopy. Conclusion.—New microscopy techniques present opportunities for pathologists to develop improved diagnostic tests.

scholarly journals Muscarinic receptor-mediated bronchoconstriction is coupled to caveolae in murine airways

2010 ◽  
Vol 298 (5) ◽  
pp. L626-L636 ◽  
Author(s):  
Heike Schlenz ◽  
Wolfgang Kummer ◽  
Gitte Jositsch ◽  
Jürgen Wess ◽  
Gabriela Krasteva
Keyword(s):  
Smooth Muscle ◽  
Signaling Pathways ◽  
Laser Scanning ◽  
Cardiac Myocyte ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Caveolin 1

Cholinergic bronchoconstriction is mediated by M2 and M3 muscarinic receptors (MR). In heart and urinary bladder, MR are linked to caveolin-1 or -3, the structural proteins of caveolae. Caveolae are cholesterol-rich, omega-shaped invaginations of the plasma membrane. They provide a scaffold for multiple G protein receptors and membrane-bound enzymes, thereby orchestrating signaling into the cell interior. Hence, we hypothesized that airway MR signaling pathways are coupled to caveolae as well. To address this issue, we determined the distribution of caveolin isoforms and MR subtype M2R in murine and human airways and investigated protein-protein associations by fluorescence resonance energy transfer (FRET)-confocal laser scanning microscopy (CLSM) analysis in immunolabeled murine tissue sections. Bronchoconstrictor responses of murine bronchi were recorded in lung-slice preparations before and after caveolae disruption by methyl-β-cyclodextrin, with efficiency of this treatment being validated by electron microscopy. KCl-induced bronchoconstriction was unaffected after treatment, demonstrating functional integrity of the smooth muscle. Caveolae disruption decreased muscarine-induced bronchoconstriction in wild-type and abolished it in M2R−/− and M3R−/− mice. Thus M2R and M3R signaling pathways require intact caveolae. Furthermore, we identified a presumed skeletal and cardiac myocyte-specific caveolin isoform, caveolin-3, in human and murine bronchial smooth muscle and found it to be associated with M2R in situ. In contrast, M2R was not associated with caveolin-1, despite an in situ association of caveolin-1 and caveolin-3 that was detected. Here, we demonstrated that M2R- and M3R-mediated bronchoconstriction is caveolae-dependent. Since caveolin-3 is directly associated with M2R, we suggest caveolin-3 as novel regulator of M2R-mediated signaling.

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.

Evidence that GPVI is Expressed as a Mixture of Monomers and Dimers, and that the D2 Domain is not Essential for GPVI Activation

2021 ◽  
Author(s):  
Joanne C. Clark ◽  
Raluca A. I. Neagoe ◽  
Malou Zuidscherwoude ◽  
Deirdre M. Kavanagh ◽  
Alexandre Slater ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Single Molecule ◽  
Fluorescence Correlation Spectroscopy ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Glycoprotein Vi ◽  
D2 Domain ◽  
Microscopy Techniques

AbstractCollagen has been proposed to bind to a unique epitope in dimeric glycoprotein VI (GPVI) and the number of GPVI dimers has been reported to increase upon platelet activation. However, in contrast, the crystal structure of GPVI in complex with collagen-related peptide (CRP) showed binding distinct from the site of dimerization. Further fibrinogen has been reported to bind to monomeric but not dimeric GPVI. In the present study, we have used the advanced fluorescence microscopy techniques of single-molecule microscopy, fluorescence correlation spectroscopy (FCS) and bioluminescence resonance energy transfer (BRET), and mutagenesis studies in a transfected cell line model to show that GPVI is expressed as a mixture of monomers and dimers and that dimerization through the D2 domain is not critical for activation. As many of these techniques cannot be applied to platelets to resolve this issue, due to the high density of GPVI and its anucleate nature, we used Förster resonance energy transfer (FRET) to show that endogenous GPVI is at least partially expressed as a dimer on resting and activated platelet membranes. We propose that GPVI may be expressed as a monomer on the cell surface and it forms dimers in the membrane through diffusion, giving rise to a mixture of monomers and dimers. We speculate that the formation of dimers facilitates ligand binding through avidity.

Structural dynamics of P-type ATPase ion pumps

2019 ◽  
Vol 47 (5) ◽  
pp. 1247-1257 ◽  
Author(s):  
Mateusz Dyla ◽  
Sara Basse Hansen ◽  
Poul Nissen ◽  
Magnus Kjaergaard
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
New Technologies ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Dynamics Simulations ◽  
Types Of Information ◽  
P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

Quantum Dot Bioconjugates as Energy Donors in Fluorescence Resonance Energy Transfer Assays

2003 ◽  
Vol 773 ◽  
Author(s):  
Aaron R. Clapp ◽  
Igor L. Medintz ◽  
J. Matthew Mauro ◽  
Hedi Mattoussi
Keyword(s):  
Energy Transfer ◽  
Quantum Dot ◽  
Organic Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Genetically Engineered ◽  
Molar Ratio ◽  
Binding Assays ◽  
Specific Sequence ◽  
Donor Acceptor

AbstractLuminescent CdSe-ZnS core-shell quantum dot (QD) bioconjugates were used as energy donors in fluorescent resonance energy transfer (FRET) binding assays. The QDs were coated with saturating amounts of genetically engineered maltose binding protein (MBP) using a noncovalent immobilization process, and Cy3 organic dyes covalently attached at a specific sequence to MBP were used as energy acceptor molecules. Energy transfer efficiency was measured as a function of the MBP-Cy3/QD molar ratio for two different donor fluorescence emissions (different QD core sizes). Apparent donor-acceptor distances were determined from these FRET studies, and the measured distances are consistent with QD-protein conjugate dimensions previously determined from structural studies.

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