scholarly journals Ca2+-Dependent Protein Kinase 6 Enhances KAT2 Shaker Channel Activity in Arabidopsis thaliana

2021 ◽  
Vol 22 (4) ◽  
pp. 1596
Author(s):  
Elsa Ronzier ◽  
Claire Corratgé-Faillie ◽  
Frédéric Sanchez ◽  
Christian Brière ◽  
Tou Cheu Xiong
Keyword(s):  
Arabidopsis Thaliana ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Physical Interaction ◽  
Fluorescence Lifetime Imaging ◽  
Dependent Manner ◽  
In Planta ◽  
Knock Out ◽  
Calcium Dependent ◽  
Dependent Protein

Post-translational regulations of Shaker-like voltage-gated K+ channels were reported to be essential for rapid responses to environmental stresses in plants. In particular, it has been shown that calcium-dependent protein kinases (CPKs) regulate Shaker channels in plants. Here, the focus was on KAT2, a Shaker channel cloned in the model plant Arabidopsis thaliana, where is it expressed namely in the vascular tissues of leaves. After co-expression of KAT2 with AtCPK6 in Xenopuslaevis oocytes, voltage-clamp recordings demonstrated that AtCPK6 stimulates the activity of KAT2 in a calcium-dependent manner. A physical interaction between these two proteins has also been shown by Förster resonance energy transfer by fluorescence lifetime imaging (FRET-FLIM). Peptide array assays support that AtCPK6 phosphorylates KAT2 at several positions, also in a calcium-dependent manner. Finally, K+ fluorescence imaging in planta suggests that K+ distribution is impaired in kat2 knock-out mutant leaves. We propose that the AtCPK6/KAT2 couple plays a role in the homeostasis of K+ distribution in leaves.

scholarly journals The 5-HT4 receptor interacts with adhesion molecule L1 to modulate morphogenic signaling in neurons

2021 ◽  
Vol 134 (4) ◽  
pp. jcs249193
Author(s):  
Simon Bennet Sonnenberg ◽  
Jonah Rauer ◽  
Christoph Göhr ◽  
Nataliya Gorinski ◽  
Sophie Kristin Schade ◽  
...  
Keyword(s):  
Adhesion Molecule ◽  
Dendritic Spines ◽  
Hippocampal Neurons ◽  
Serotonin Receptor ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Mitogen Activated Protein Kinase ◽  
Physical Interaction ◽  
Dependent Manner

ABSTRACTMorphological remodeling of dendritic spines is critically involved in memory formation and depends on adhesion molecules. Serotonin receptors are also implicated in this remodeling, though the underlying mechanisms remain enigmatic. Here, we uncovered a signaling pathway involving the adhesion molecule L1CAM (L1) and serotonin receptor 5-HT4 (5-HT4R, encoded by HTR4). Using Förster resonance energy transfer (FRET) imaging, we demonstrated a physical interaction between 5-HT4R and L1, and found that 5-HT4R–L1 heterodimerization facilitates mitogen-activated protein kinase activation in a Gs-dependent manner. We also found that 5-HT4R–L1-mediated signaling is involved in G13-dependent modulation of cofilin-1 activity. In hippocampal neurons in vitro, the 5-HT4R–L1 pathway triggers maturation of dendritic spines. Thus, the 5-HT4R–L1 signaling module represents a previously unknown molecular pathway regulating synaptic remodeling.

scholarly journals Discs large 1 controls daughter-cell polarity after cytokinesis in vertebrate morphogenesis

2018 ◽  
Vol 115 (46) ◽  
pp. E10859-E10868 ◽  
Author(s):  
Yuwei Li ◽  
Jason A. Junge ◽  
Cosimo Arnesano ◽  
Garrett G. Gross ◽  
Jeffrey H. Miner ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Protein Interactions ◽  
Protein Function ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Explant Culture ◽  
Scaffolding Protein ◽  
Fluorescence Lifetime Imaging ◽  
Discs Large

Vertebrate embryogenesis and organogenesis are driven by cell biological processes, ranging from mitosis and migration to changes in cell size and polarity, but their control and causal relationships are not fully defined. Here, we use the developing limb skeleton to better define the relationships between mitosis and cell polarity. We combine protein-tagging and -perturbation reagents with advanced in vivo imaging to assess the role of Discs large 1 (Dlg1), a membrane-associated scaffolding protein, in mediating the spatiotemporal relationship between cytokinesis and cell polarity. Our results reveal that Dlg1 is enriched at the midbody during cytokinesis and that its multimerization is essential for the normal polarity of daughter cells. Defects in this process alter tissue dimensions without impacting other cellular processes. Our results extend the conventional view that division orientation is established at metaphase and anaphase and suggest that multiple mechanisms act at distinct phases of the cell cycle to transmit cell polarity. The approach employed can be used in other systems, as it offers a robust means to follow and to eliminate protein function and extends the Phasor approach for studying in vivo protein interactions by frequency-domain fluorescence lifetime imaging microscopy of Förster resonance energy transfer (FLIM-FRET) to organotypic explant culture.

Automated multiwell fluorescence lifetime imaging for Förster resonance energy transfer assays and high content analysis

2015 ◽  
Vol 7 (10) ◽  
pp. 4071-4089 ◽  
Author(s):  
Douglas J. Kelly ◽  
Sean C. Warren ◽  
Dominic Alibhai ◽  
Sunil Kumar ◽  
Yuriy Alexandrov ◽  
...  
Keyword(s):  
Content Analysis ◽  
Energy Transfer ◽  
Fluorescence Lifetime ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Förster Resonance Energy Transfer ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
High Content Analysis ◽  
Förster Resonance

An HCA-FLIM instrument is presented alongside exemplar oligomerisation, intermolecular and intramolecular FRET assays that require robust measurement of small lifetime changes.

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 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.

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