scholarly journals c-Fos activates and physically interacts with specific enzymes of the pathway of synthesis of polyphosphoinositides

2011 ◽  
Vol 22 (24) ◽  
pp. 4716-4725 ◽  
Author(s):  
Adolfo R. Alfonso Pecchio ◽  
Andrés M. Cardozo Gizzi ◽  
Marianne L. Renner ◽  
María Molina-Calavita ◽  
Beatriz L. Caputto
Keyword(s):  
Endoplasmic Reticulum ◽  
Transcription Factor ◽  
Biosynthetic Pathway ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Physical Interaction ◽  
Quiescent Cells ◽  
No Activation ◽  
Specific Lipid

The oncoprotein c-Fos is a well-recognized AP-1 transcription factor. In addition, this protein associates with the endoplasmic reticulum and activates the synthesis of phospholipids. However, the mechanism by which c-Fos stimulates the synthesis of phospholipids in general and the specific lipid pathways activated are unknown. Here we show that induction of quiescent cells to reenter growth promotes an increase in the labeling of polyphosphoinositides that depends on the expression of c-Fos. We also investigated whether stimulation by c-Fos of the synthesis of phosphatidylinositol and its phosphorylated derivatives depends on the activation of enzymes of the phosphatidylinositolphosphate biosynthetic pathway. We found that c-Fos activates CDP-diacylglycerol synthase and phosphatidylinositol (PtdIns) 4-kinase II α in vitro, whereas no activation of phosphatidylinositol synthase or of PtdIns 4-kinase II β was observed. Both coimmunoprecipitation and fluorescence resonance energy transfer experiments consistently showed a physical interaction between the N-terminal domain of c-Fos and the enzymes it activates.

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 Subcellular distribution of GABAB receptor homo- and hetero-dimers

2005 ◽  
Vol 388 (1) ◽  
pp. 47-55 ◽  
Author(s):  
Josée-France VILLEMURE ◽  
Lynda ADAM ◽  
Nicola J. BEVAN ◽  
Katy GEARING ◽  
Sébastien CHÉNIER ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Gabab Receptor ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Gabab Receptors ◽  
In Vitro Assays ◽  
Functional Roles ◽  
G Protein Coupled ◽  
Brain And Spinal Cord

GBRs (GABAB receptors; where GABA stands for γ-aminobutyric acid) are G-protein-coupled receptors that mediate slow synaptic inhibition in the brain and spinal cord. In vitro assays have previously demonstrated that these receptors are heterodimers assembled from two homologous subunits, GBR1 and GBR2, neither of which is capable of producing functional GBR on their own. We have used co-immunoprecipitation in combination with bioluminescence and fluorescence resonance energy transfer approaches in living cells to assess directly the interaction between GBR subunits and determine their subcellular localization. The results show that, in addition to forming heterodimers, GBR1 and GBR2 can associate as stable homodimers. Confocal microscopy indicates that, while GBR1/GBR1 homodimers are retained in the endoplasmic reticulum and endoplasmic reticulum–Golgi intermediate compartment, both GBR2/GBR2 homodimers and GBR1/GBR2 heterodimers are present at the plasma membrane. Although these observations shed new light on the assembly of GBR complexes, they raise questions about the potential functional roles of GBR1 and GBR2 homodimers.

scholarly journals In vitro FRET analysis of IRE1 and BiP association and dissociation upon endoplasmic reticulum stress

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Megan C Kopp ◽  
Piotr R Nowak ◽  
Natacha Larburu ◽  
Christopher J Adams ◽  
Maruf MU Ali
Keyword(s):  
Endoplasmic Reticulum ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Misfolded Proteins ◽  
Protein Substrates ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
Substrate Binding Domain

The unfolded protein response (UPR) is a key signaling system that regulates protein homeostasis within the endoplasmic reticulum (ER). The primary step in UPR activation is the detection of misfolded proteins, the mechanism of which is unclear. We have previously suggested an allosteric mechanism for UPR induction (<xref ref-type="bibr" rid="bib3">Carrara et al., 2015</xref>) based on qualitative pull-down assays. Here, we develop an in vitro Förster resonance energy transfer (FRET) UPR induction assay that quantifies IRE1 luminal domain and BiP association and dissociation upon addition of misfolded proteins. Using this technique, we reassess our previous observations and extend mechanistic insight to cover other general ER misfolded protein substrates and their folded native state. Moreover, we evaluate the key BiP substrate-binding domain mutant V461F. The new experimental approach significantly enhances the evidence suggesting an allosteric model for UPR induction upon ER stress.

scholarly journals Interaction of Histone Acetylases and Deacetylases In Vivo

2003 ◽  
Vol 23 (3) ◽  
pp. 1025-1033 ◽  
Author(s):  
Satoshi Yamagoe ◽  
Tomohiko Kanno ◽  
Yuka Kanno ◽  
Shigakazu Sasaki ◽  
Richard M. Siegel ◽  
...  
Keyword(s):  
Dynamic Equilibrium ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Direct Interaction ◽  
Spatial Proximity ◽  
Physical Interaction ◽  
Histone Deacetylase 1 ◽  
Histone Acetylases

ABSTRACT Having opposing enzymatic activities, histone acetylases (HATs) and deacetylases affect chromatin and regulate transcription. The activities of the two enzymes are thought to be balanced in the cell by an unknown mechanism that may involve their direct interaction. Using fluorescence resonance energy transfer analysis, we demonstrated that the acetylase PCAF and histone deacetylase 1 (HDAC1) are in close spatial proximity in living cells, compatible with their physical interaction. In agreement, coimmunoprecipitation assays demonstrated that endogenous HDACs are associated with PCAF and another acetylase, GCN5, in HeLa cells. We found by glycerol gradient sedimentation analysis that HATs are integrated into a large multiprotein HDAC complex that is distinct from the previously described HDAC complexes containing mSin3A, Mi-2/NRD, or CoREST. This HDAC-HAT association is partly accounted for by a direct protein-protein interaction observed in vitro. The HDAC-HAT complex may play a role in establishing a dynamic equilibrium of the two enzymes in vivo.

Quantitative Ratiometric pH Imaging Using a 1,2-Dioxetane Chemiluminescence Resonance Energy Transfer Sensor

2020 ◽  
Author(s):  
Lucas S. Ryan ◽  
Jeni Gerberich ◽  
Uroob Haris ◽  
ralph mason ◽  
Alexander Lippert
Keyword(s):  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Whole Body ◽  
Photon Flux ◽  
Ph Homeostasis ◽  
Ph Imaging ◽  
Confounding Variables ◽  
Significant Difference

<p>Regulation of physiological pH is integral for proper whole-body and cellular function, and disruptions in pH homeostasis can be both a cause and effect of disease. In light of this, many methods have been developed to monitor pH in cells and animals. In this study, we report a chemiluminescence resonance energy transfer (CRET) probe Ratio-pHCL-1, comprised of an acrylamide 1,2-dioxetane chemiluminescent scaffold with an appended pH-sensitive carbofluorescein fluorophore. The probe provides an accurate measurement of pH between 6.8-8.4, making it viable tool for measuring pH in biological systems. Further, its ratiometric output is independent of confounding variables. Quantification of pH can be accomplished both using common fluorimetry and advanced optical imaging methods. Using an IVIS Spectrum, pH can be quantified through tissue with Ratio-pHCL-1, which has been shown in vitro and precisely calibrated in sacrificed mouse models. Initial studies showed that intraperitoneal injections of Ratio-pHCL-1 into sacrificed mice produce a photon flux of more than 10^10 photons per second, and showed a significant difference in ratio of emission intensities between pH 6.0, 7.0, and 8.0.</p> <b></b><i></i><u></u><sub></sub><sup></sup><br>

Programmable in vitro Co-Encapsidation of Guest Proteins Inside Protein Nanocages

2018 ◽  
Author(s):  
Noor H. Dashti ◽  
Rufika S. Abidin ◽  
Frank Sainsbury
Keyword(s):  
Self Assembly ◽  
Mammalian Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Super Resolution ◽  
Cell Engineering ◽  
Particle Analysis ◽  
Protein Cages

Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages have been developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both <i>in vitro</i> and <i>in vivo</i> cell engineering. However, there is a lack of platforms in bionanotechnology that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for <i>in vivo</i> self-sorting of cargo-linked capsomeres of the Murine polyomavirus (MPyV) major coat protein that enables controlled encapsidation of guest proteins by <i>in vitro</i> self-assembly. Using Förster resonance energy transfer (FRET) we demonstrate the flexibility in this system to support co-encapsidation of multiple proteins. Complementing these ensemble measurements with single particle analysis by super-resolution microscopy shows that the stochastic nature of co-encapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable co-encapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.

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 Biological nanoscale fluorescent probes: From structure and performance to bioimaging

2020 ◽  
Vol 39 (1) ◽  
pp. 209-221
Author(s):  
Jiafeng Wan ◽  
Xiaoyuan Zhang ◽  
Kai Zhang ◽  
Zhiqiang Su
Keyword(s):  
Fluorescent Probes ◽  
Organic Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
High Sensitivity ◽  
Biological Imaging ◽  
Fluorescent Materials ◽  
And Performance

Abstract In recent years, nanomaterials have attracted lots of attention from researchers due to their unique properties. Nanometer fluorescent materials, such as organic dyes, semiconductor quantum dots (QDs), metal nano-clusters (MNCs), carbon dots (CDs), etc., are widely used in biological imaging due to their high sensitivity, short response time, and excellent accuracy. Nanometer fluorescent probes can not only perform in vitro imaging of organisms but also achieve in vivo imaging. This provides medical staff with great convenience in cancer treatment. Combined with contemporary medical methods, faster and more effective treatment of cancer is achievable. This article explains the response mechanism of three-nanometer fluorescent probes: the principle of induced electron transfer (PET), the principle of fluorescence resonance energy transfer (FRET), and the principle of intramolecular charge transfer (ICT), showing the semiconductor QDs, precious MNCs, and CDs. The excellent performance of the three kinds of nano fluorescent materials in biological imaging is highlighted, and the application of these three kinds of nano fluorescent probes in targeted biological imaging is also introduced. Nanometer fluorescent materials will show their significance in the field of biomedicine.

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