scholarly journals AKAP79/150 recruits the transcription factor NFAT to regulate signaling to the nucleus by neuronal L-type Ca2+ channels

2019 ◽  
Vol 30 (14) ◽  
pp. 1743-1756 ◽  
Author(s):  
Jonathan G. Murphy ◽  
Kevin C. Crosby ◽  
Philip J. Dittmer ◽  
William A. Sather ◽  
Mark L. Dell’Acqua
Keyword(s):  
Hippocampal Neurons ◽  
Leucine Zipper ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Activated T Cells ◽  
Signaling Complex ◽  
Electrophysiological Recordings ◽  
C Terminus ◽  
Anchoring Protein

In neurons, regulation of activity-dependent transcription by the nuclear factor of activated T-cells (NFAT) depends upon Ca2+ influx through voltage-gated L-type calcium channels (LTCC) and NFAT translocation to the nucleus following its dephosphorylation by the Ca2+-dependent phosphatase calcineurin (CaN). CaN is recruited to the channel by A-kinase anchoring protein (AKAP) 79/150, which binds to the LTCC C-terminus via a modified leucine-zipper (LZ) interaction. Here we sought to gain new insights into how LTCCs and signaling to NFAT are regulated by this LZ interaction. RNA interference–mediated knockdown of endogenous AKAP150 and replacement with human AKAP79 lacking its C-terminal LZ domain resulted in loss of depolarization-stimulated NFAT signaling in rat hippocampal neurons. However, the LZ mutation had little impact on the AKAP–LTCC interaction or LTCC function, as measured by Förster resonance energy transfer, Ca2+ imaging, and electrophysiological recordings. AKAP79 and NFAT coimmunoprecipitated when coexpressed in heterologous cells, and the LZ mutation disrupted this association. Critically, measurements of NFAT mobility in neurons employing fluorescence recovery after photobleaching and fluorescence correlation spectroscopy provided further evidence for an AKAP79 LZ interaction with NFAT. These findings suggest that the AKAP79/150 LZ motif functions to recruit NFAT to the LTCC signaling complex to promote its activation by AKAP-anchored calcineurin.

scholarly journals Live Cell FRET Imaging Reveals Amyloid β-Peptide Oligomerization in Hippocampal Neurons

2021 ◽  
Author(s):  
Yang Gao ◽  
Stefan Wennmalm ◽  
Bengt Winblad ◽  
Sophia Schedin-Weiss ◽  
Lars Tjernberg
Keyword(s):  
Hippocampal Neurons ◽  
Resonance Energy Transfer ◽  
Amyloid Β ◽  
Resonance Energy ◽  
Live Cell ◽  
Amyloid Β Peptide ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Late Endosomes ◽  
Aβ Oligomerization

Amyloid β-peptide (Aβ) oligomerization is believed to contribute to the neuronal dysfunction in Alzheimer disease (AD). Despite decades of research, many details of Aβ oligomerization in neurons still need to be revealed. Förster Resonance Energy Transfer (FRET) is a simple but effective way to study molecular interactions. Here we use a confocal microscope with a sensitive Airyscan detector for FRET detection. By live cell FRET imaging, we detect Aβ42 oligomerization in primary neurons. The neurons were incubated with fluorescently labelled Aβ42 in the cell culture medium for 24 hours. Aβ42 were internalized and oligomerized into the lysosomes/late endosomes in a concentration-dependent manner. Both the cellular uptake and intracellular oligomerization of Aβ42 were significantly higher than for Aβ40. These findings provide a better understanding of Aβ42 oligomerization in neurons.

scholarly journals Allosteric modulation of the SARS-CoV-2 spike conformation

2021 ◽  
Author(s):  
Marco A Diaz-Salinas ◽  
Qi Li ◽  
Monir Ejemel ◽  
Yang Wang ◽  
James B Munro
Keyword(s):  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Structural Data ◽  
Correlation Spectroscopy ◽  
Host Cells ◽  
Parallel Solution ◽  
Real Time Analysis ◽  
Multiple Conformations

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells through binding to angiotensin-converting enzyme 2 (ACE2), which is mediated by the receptor-binding domain (RBD) of the viral spike (S) glycoprotein. Structural data and real-time analysis of conformational dynamics have shown that S can adopt multiple conformations, which mediate the exposure of the ACE2-binding site in the RBD. Here, using single-molecule Förster resonance energy transfer (smFRET) imaging we report the effects of ACE2 and antibody binding on the conformational dynamics of S from the Wuhan-1 strain and the B.1 variant (D614G). We found that antibodies that target diverse epitopes, including those distal to the RBD, stabilize the RBD in a position competent for ACE2 binding. Parallel solution-based binding experiments using fluorescence correlation spectroscopy (FCS) indicated antibody-mediated enhancement of ACE2 binding. These findings inform on novel strategies for therapeutic antibody cocktails.

scholarly journals Heart failure leads to altered β2-adrenoceptor/cyclic adenosine monophosphate dynamics in the sarcolemmal phospholemman/Na,K ATPase microdomain

2018 ◽  
Vol 115 (3) ◽  
pp. 546-555 ◽  
Author(s):  
Zeynep Bastug-Özel ◽  
Peter T Wright ◽  
Axel E Kraft ◽  
Davor Pavlovic ◽  
Jacqueline Howie ◽  
...  
Keyword(s):  
Heart Failure ◽  
Ventricular Myocytes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Adult Rat ◽  
C Terminus ◽  
Complex Forms ◽  
Cardiac Excitation

Abstract Aims Cyclic adenosine monophosphate (cAMP) regulates cardiac excitation–contraction coupling by acting in microdomains associated with sarcolemmal ion channels. However, local real time cAMP dynamics in such microdomains has not been visualized before. We sought to directly monitor cAMP in a microdomain formed around sodium–potassium ATPase (NKA) in healthy and failing cardiomyocytes and to better understand alterations of cAMP compartmentation in heart failure. Methods and results A novel Förster resonance energy transfer (FRET)-based biosensor termed phospholemman (PLM)-Epac1 was developed by fusing a highly sensitive cAMP sensor Epac1-camps to the C-terminus of PLM. Live cell imaging in PLM-Epac1 and Epac1-camps expressing adult rat ventricular myocytes revealed extensive regulation of NKA/PLM microdomain-associated cAMP levels by β2-adrenoceptors (β2-ARs). Local cAMP pools stimulated by these receptors were tightly controlled by phosphodiesterase (PDE) type 3. In chronic heart failure following myocardial infarction, dramatic reduction of the microdomain-specific β2-AR/cAMP signals and β2-AR dependent PLM phosphorylation was accompanied by a pronounced loss of local PDE3 and an increase in PDE2 effects. Conclusions NKA/PLM complex forms a distinct cAMP microdomain which is directly regulated by β2-ARs and is under predominant control by PDE3. In heart failure, local changes in PDE repertoire result in blunted β2-AR signalling to cAMP in the vicinity of PLM.

scholarly journals Dynamic remodeling of scaffold interactions in dendritic spines controls synaptic excitability

2012 ◽  
Vol 198 (2) ◽  
pp. 251-263 ◽  
Author(s):  
Enora Moutin ◽  
Fabrice Raynaud ◽  
Jonathan Roger ◽  
Emilie Pellegrino ◽  
Vincent Homburger ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Membrane Receptors ◽  
Physical Interaction ◽  
Scaffolding Proteins ◽  
Cellular Functions ◽  
Electrophysiological Recordings ◽  
Living Neurons ◽  
Activity Dependent

Scaffolding proteins interact with membrane receptors to control signaling pathways and cellular functions. However, the dynamics and specific roles of interactions between different components of scaffold complexes are poorly understood because of the dearth of methods available to monitor binding interactions. Using a unique combination of single-cell bioluminescence resonance energy transfer imaging in living neurons and electrophysiological recordings, in this paper, we depict the role of glutamate receptor scaffold complex remodeling in space and time to control synaptic transmission. Despite a broad colocalization of the proteins in neurons, we show that spine-confined assembly/disassembly of this scaffold complex, physiologically triggered by sustained activation of synaptic NMDA (N-methyl-d-aspartate) receptors, induces physical association between ionotropic (NMDA) and metabotropic (mGlu5a) synaptic glutamate receptors. This physical interaction results in an mGlu5a receptor–mediated inhibition of NMDA currents, providing an activity-dependent negative feedback loop on NMDA receptor activity. Such protein scaffold remodeling represents a form of homeostatic control of synaptic excitability.

Abstract 022: Evidence for AT2-receptor-MAS Dimerisation from Fluorescence Resonance Energy Transfer (FRET) Imaging and Fluorescence Cross Correlation Spectroscopy (FCCS)

Hypertension ◽  
2014 ◽  
Vol 64 (suppl_1) ◽  
Author(s):  
Daniel C Villela ◽  
Anke Teichmann ◽  
Sebastian Kirsch ◽  
Maibritt Mardahl ◽  
Lisa M Münter ◽  
...  
Keyword(s):  
Cross Correlation ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Expression Vectors ◽  
Physical Interaction ◽  
At2 Receptor ◽  
Hek 293 ◽  
Fret Efficiency ◽  
In Cells

The angiotensin AT2-receptor (AT2R) and the receptor MAS share a strinkingly similar spectrum of signaling mechanisms and protective, physiological actions. Furthermore, cross-inhibition by the respective receptor antagonists has been observed. Therefore we hypothesised that a physical interaction between these two receptors may exist. HEK-293 cells were transfected with vectors encoding MAS or AT2R fused in the C-terminus with the fluorophores CFP or YFP for FRET and GFP or mCherry for FCCS. FRET with photobleaching was used to detect, whether MAS and AT2R are localised in very close proximity (1-10nm) in cell membranes thus indicating dimerisation. FCCS was used to follow simultaneously occurring fluctuations in fluorescence intensity of both labeled molecules. Several controls were applied such as co-transfection of equal amounts of fused and non-fused MAS/AT2R expression vectors for competition, co-tranfection of coding and uncoding pcDNA vectors or co-transfection with an unrelated transmembrane receptor. Experiments were conducted under baseline conditions and in cells treated with AT2R/MAS agonists and antagonists Significant FRET efficiency of 10.8±0.8% was measured for AT2-YFP/MAS-CFP strongly indicating heterodimerisation. FRET efficiency was not altered by AT2R or MAS agonists or antagonists. Non-fluorescent MAS and AT2R competed with fluorescent receptors as indicated by a 50% reduction in FRET efficiency (6.0±0.6%), while empty vectors did not compete (9.6±0.6%). No FRET efficiency was observed with an unrelated transmembrane receptor (0.44±1.44%) indicating specificity of receptor interactions. Both, MAS and AT2R also formed homodimers (7.4±0.8% for MAS, 9.2±0.8% for AT2R). Hetero- and homodimerisations were absent if amino acid C35 of the AT2R was mutated (3,9 ± 1,2%). FCCS corroborated the FRET results and revealed a significantly enhanced cross correlation in cells tranfected with fluorophore-tagged MAS/AT2R when compared to vectors only expressing the fluorophores (8.5±3% vs 11.1±4%; p<0.0001). Our data strongly suggest that MAS and the AT2R form homo- and heterodimers. Studies to investigate the physiological relevance of MAS/AT2R dimerisation are currently being conducted.

AQP2 is a substrate for endogenous PP2B activity within an inner medullary AKAP-signaling complex

2001 ◽  
Vol 281 (5) ◽  
pp. F958-F965 ◽  
Author(s):  
Inho Jo ◽  
Donald T. Ward ◽  
Michelle A. Baum ◽  
John D. Scott ◽  
Vincent M. Coghlan ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Regulatory Subunit ◽  
Relative Molecular Mass ◽  
Signaling Complex ◽  
Anchoring Protein ◽  
Pka Regulatory Subunit

We have demonstrated that inner medullary collecting duct (IMCD) heavy endosomes purified from rat kidney IMCD contain the type II protein kinase A (PKA) regulatory subunit (RII), protein phosphatase (PP)2B, PKCζ, and an RII-binding protein (relative molecular mass ∼90 kDa) representing a putative A kinase anchoring protein (AKAP). Affinity chromatography of detergent-solubilized endosomes on cAMP-agarose permits recovery of a protein complex consisting of the 90-kDa AKAP, RII, PP2B, and PKCζ. With the use of small-particle flow cytometry, RII and PKCζ were localized to an identical population of endosomes, suggesting that these proteins are components of an endosomal multiprotein complex.32P-labeled aquaporin-2 (AQP2) present in these PKA-phosphorylated endosomes was dephosphorylated in vitro by either addition of exogenous PP2B or by an endogenous endosomal phosphatase that was inhibited by the PP2B inhibitors EDTA and the cyclophilin-cyclosporin A complex. We conclude that IMCD heavy endosomes possess an AKAP multiprotein-signaling complex similar to that described previously in hippocampal neurons. This signaling complex potentially mediates the phosphorylation of AQP2 to regulate its trafficking into the IMCD apical membrane. In addition, the PP2B component of the AKAP-signaling complex could also dephosphorylate AQP2 in vivo.

scholarly journals Amplitude of the actomyosin power stroke depends strongly on the isoform of the myosin essential light chain

2015 ◽  
Vol 112 (15) ◽  
pp. 4660-4665 ◽  
Author(s):  
Piyali Guhathakurta ◽  
Ewa Prochniewicz ◽  
David D. Thomas
Keyword(s):  
Light Chain ◽  
Contractile Function ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Catalytic Efficiency ◽  
Power Stroke ◽  
Time Resolved ◽  
Muscle Disorders ◽  
Essential Light Chain ◽  
C Terminus

We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to determine the role of myosin essential light chains (ELCs) in structural transitions within the actomyosin complex. Skeletal muscle myosins have two ELC isoforms, A1 and A2, which differ by an additional 40–45 residues at the N terminus of A1, and subfragment 1 (S1) containing A1 (S1A1) has higher catalytic efficiency and higher affinity for actin than S1A2. ELC’s location at the junction between the catalytic and light-chain domains gives it the potential to play a central role in the force-generating power stroke. Therefore, we measured site-directed TR-FRET between a donor on actin and an acceptor near the C terminus of ELC, detecting directly the rotation of the light-chain domain (lever arm) relative to actin (power stroke), induced by the interaction of ATP-bound myosin with actin. TR-FRET resolved the weakly bound (W) and strongly bound (S) states of actomyosin during the W-to-S transition (power stroke). We found that the W states are essentially the same for the two isoenzymes, but the S states are quite different, indicating a much larger movement of S1A1. FRET from actin to a probe on the N-terminal extension of A1 showed close proximity to actin. We conclude that the N-terminal extension of A1-ELC modulates the W-to-S structural transition of acto-S1, so that the light-chain domain undergoes a much larger power stroke in S1A1 than in S1A2. These results have profound implications for understanding the contractile function of actomyosin, as needed in therapeutic design for muscle disorders.

scholarly journals Association with Coregulators Is the Major Determinant Governing Peroxisome Proliferator-activated Receptor Mobility in Living Cells

2006 ◽  
Vol 282 (7) ◽  
pp. 4417-4426 ◽  
Author(s):  
Cicerone Tudor ◽  
Jérôme N. Feige ◽  
Harikishore Pingali ◽  
Vidya Bhushan Lohray ◽  
Walter Wahli ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Molecular Mechanisms ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Peroxisome Proliferator Activated Receptors

The nucleus is an extremely dynamic compartment, and protein mobility represents a key factor in transcriptional regulation. We showed in a previous study that the diffusion of peroxisome proliferator-activated receptors (PPARs), a family of nuclear receptors regulating major cellular and metabolic functions, is modulated by ligand binding. In this study, we combine fluorescence correlation spectroscopy, dual color fluorescence cross-correlation microscopy, and fluorescence resonance energy transfer to dissect the molecular mechanisms controlling PPAR mobility and transcriptional activity in living cells. First, we bring new evidence that in vivo a high percentage of PPARs and retinoid X receptors is associated even in the absence of ligand. Second, we demonstrate that coregulator recruitment (and not DNA binding) plays a crucial role in receptor mobility, suggesting that transcriptional complexes are formed prior to promoter binding. In addition, association with coactivators in the absence of a ligand in living cells, both through the N-terminal AB domain and the AF-2 function of the ligand binding domain, provides a molecular basis to explain PPAR constitutive activity.

scholarly journals Affinity of Skp to OmpC revealed by single-molecule detection

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Sichen Pan ◽  
Chen Yang ◽  
Xin Sheng Zhao
Keyword(s):  
Single Molecule ◽  
Molecular Chaperones ◽  
Outer Membrane Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Hill Coefficient ◽  
Stoichiometric Ratio ◽  
Single Molecule Level

Abstract Outer membrane proteins (OMPs) are essential to gram-negative bacteria, and molecular chaperones prevent the OMPs from aggregation in the periplasm during the OMPs biogenesis. Skp is one of the molecular chaperones for this purpose. Here, we combined single-molecule fluorescence resonance energy transfer and fluorescence correlation spectroscopy to study the affinity and stoichiometric ratio of Skp in its binding with OmpC at the single-molecule level. The half concentration of the Skp self-trimerization (C1/2) was measured to be (2.5 ± 0.7) × 102 nM. Under an Skp concentration far below the C1/2, OmpC could recruit Skp monomers to form OmpC·Skp3. The affinity to form the OmpC·Skp3 complex was determined to be (5.5 ± 0.4) × 102 pM with a Hill coefficient of 1.6 ± 0.2. Under the micromolar concentrations of Skp, the formation of OmpC·(Skp3)2 was confirmed, and the dissociation constant of OmpC·(Skp3)2 was determined to be 1.2 ± 0.4 μM. The precise information will help us to quantitatively depict the role of Skp in the biogenesis of OMPs.

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