scholarly journals Oligomeric Architecture of Mouse Activating Nkrp1 Receptors on Living Cells

2019 ◽  
Vol 20 (8) ◽  
pp. 1884 ◽  
Author(s):  
Ljubina Adámková ◽  
Zuzana Kvíčalová ◽  
Daniel Rozbeský ◽  
Zdeněk Kukačka ◽  
David Adámek ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Lymphoid Cells ◽  
Protein Isoforms ◽  
Major Influence ◽  
Protein Dimerization ◽  
Transmembrane Receptors ◽  
Insight Into

Mouse activating Nkrp1 proteins are commonly described as type II transmembrane receptors with disulfide-linked homodimeric structure. Their function and the manner in which Nkrp1 proteins of mouse strain (C57BL/6) oligomerize are still poorly understood. To assess the oligomerization state of Nkrp1 proteins, mouse activating EGFP-Nkrp1s were expressed in mammalian lymphoid cells and their oligomerization evaluated by Förster resonance energy transfer (FRET). Alternatively, Nkrp1s oligomers were detected by Western blotting to specify the ratio between monomeric and dimeric forms. We also performed structural characterization of recombinant ectodomains of activating Nkrp1 receptors. Nkrp1 isoforms c1, c2 and f were expressed prevalently as homodimers, whereas the Nkrp1a displays larger proportion of monomers on the cell surface. Cysteine-to-serine mutants revealed the importance of all stalk cysteines for protein dimerization in living cells with a major influence of cysteine at position 74 in two Nkrp1 protein isoforms. Our results represent a new insight into the oligomerization of Nkrp1 receptors on lymphoid cells, which will help to determine their function.

scholarly journals Fluorescence resonance energy transfer imaging of PKC signalling in living cells using genetically encoded fluorescent probes

2008 ◽  
Vol 6 (suppl_1) ◽  
Author(s):  
Joachim Goedhart ◽  
Theodorus W.J Gadella
Keyword(s):  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Subcellular Location ◽  
Living Cells ◽  
General Interest ◽  
High Temporal Resolution ◽  
Transmembrane Receptors ◽  
Green Fluorescent ◽  
Single Living Cells

Perception of ligands in the extracellular space by transmembrane receptors initiates signal transduction. The conformation change of the receptor induces changes of intracellular signalling components, including altered cellular concentration, altered subcellular location, altered conformation and altered interacting partners. Biochemical approaches have yielded a lot of information about these processes. However, methods that are compatible with analysis of single living cells are often preferred, since cells are highly organized and their response is usually spatially heterogeneous. In addition, the study of signalling cascades requires high temporal resolution. Fluorescence imaging approaches meet these requirements. Moreover, imaging approaches can be combined with genetically encoded green fluorescent protein-based probes that have a high selectivity and sensitivity for the process/molecule of interest. Nowadays, many genetically encoded probes are available for visualizing signalling in living cells. This review is centred on a key regulator of cellular signalling, protein kinase C (PKC). We will discuss imaging approaches that are used for analysing the molecules involved in activation of PKC, visualizing the dynamics of the location of PKC, measuring the conformation of PKC and quantifying the activity of PKC. These approaches are of general interest since they can be applied to study the dynamics, conformation and activity of any protein in living cells.

scholarly journals A Fluorescence Resonance Energy Transfer-Based Analytical Tool for Nitrate Quantification in Living Cells

ACS Omega ◽  
2020 ◽  
Vol 5 (46) ◽  
pp. 30306-30314
Author(s):  
Urooj Fatima ◽  
Fuad Ameen ◽  
Neha Soleja ◽  
Parvez Khan ◽  
Abobakr Almansob ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Analytical Tool ◽  
Fluorescence Resonance

Use of a-SiC:H multilayer transducers for detection of fluorescence signals from reactive cyan and yellow fluorophores

2011 ◽  
Vol 1321 ◽  
Author(s):  
P. Louro ◽  
M. Vieira ◽  
M. A. Vieira ◽  
J. Costa ◽  
M. Fernandes ◽  
...  
Keyword(s):  
Spectral Response ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Optical Filters ◽  
Electrical Model ◽  
Excitation Light ◽  
Independent Test ◽  
Opposite Behavior ◽  
Blue Background ◽  
Insight Into

ABSTRACTThe transducer consists of a p-i’(a-SiC:H)-n/p-i(a-Si:H)-n heterostructures produced by PECVD and optimized for the detection of the fluorescence resonance energy transfer between fluorophores with excitation in the violet(400 nm) and emissions in the cyan (470 nm) and yellow (588 nm) range of the spectrum. The thickness and the absorption coefficient of the i’- and i- layers were tailored for cyan and yellow optical confinement, respectively in the front and back photodiodes acting both as optical filters. The devices were characterized through transmittance and spectral response measurements and under different electrical.To simulate the FRET pairs and the excitation light a chromatic time dependent combination of violet, cyan and yellow wavelengths was applied to the device. The generated photocurrent was measured under negative and positive bias to readout the combined spectra. The independent test signals were chosen in order to sample all the possible chromatic. Different wavelength backgrounds were also superimposed.Results show that under negative bias the phorocurrent signal presents eight separate levels each one assigned to the different polychromatic mixtures. If a blue background is superimposed the yellow channel is enhanced and the cyan suppressed while under red irradiation the opposite behavior occurs. So under appropriated steady state optical bias the sensor will detect separately the cyan and yellow fluorescence pairs. An electrical model, supported by a numerical simulation, gives insight into the transduction mechanism.

scholarly journals Visualization of growth signal transduction cascades in living cells with genetically encoded probes based on Förster resonance energy transfer

2008 ◽  
Vol 363 (1500) ◽  
pp. 2143-2151 ◽  
Author(s):  
Kazuhiro Aoki ◽  
Etsuko Kiyokawa ◽  
Takeshi Nakamura ◽  
Michiyuki Matsuda
Keyword(s):  
Signal Transduction ◽  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Small Gtpases ◽  
Forster Resonance Energy Transfer ◽  
Fluorescence Probes ◽  
Förster Resonance Energy Transfer ◽  
Förster Resonance

Fluorescence probes based on the principle of Förster resonance energy transfer (FRET) have shed new light on our understanding of signal transduction cascades. Among them, unimolecular FRET probes containing fluorescence proteins are rapidly increasing in number because these genetically encoded probes can be easily loaded into living cells and allow simple acquisition of FRET images. We have developed probes for small GTPases, tyrosine kinases, serine–threonine kinases and phosphoinositides. Images obtained with these probes have revealed that membrane protrusions such as nascent lamellipodia or neurites provide an active signalling platform in the growth factor-stimulated cells.

scholarly journals Elucidation and refinement of synthetic receptor mechanisms

2020 ◽  
Author(s):  
Hailey I. Edelstein ◽  
Patrick S. Donahue ◽  
Joseph J. Muldoon ◽  
Anthony K. Kang ◽  
Taylor B. Dolberg ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Functional Performance ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Performance Characteristics ◽  
Therapeutic Gene ◽  
High Performing ◽  
Synthetic Receptor ◽  
And Performance ◽  
Insight Into

ABSTRACTSynthetic receptors are powerful tools for engineering mammalian cell-based devices. These biosensors enable cell-based therapies to perform complex tasks such as regulating therapeutic gene expression in response to sensing physiological cues. Although multiple synthetic receptor systems now exist, many aspects of receptor performance are poorly understood. In general, it would be useful to understand how receptor design choices influence performance characteristics. In this study, we examined the modular extracellular sensor architecture (MESA) and systematically evaluated previously unexamined design choices, yielding substantially improved receptors. A key finding that might extend to other receptor systems is that the choice of transmembrane domain (TMD) is important for generating high-performing receptors. To provide mechanistic insights, we adopted and employed a Förster resonance energy transfer (FRET)-based assay to elucidate how TMDs affect receptor complex formation and connected these observations to functional performance. To build further insight into these phenomena, we developed a library of new MESA receptors that sense an expanded set of ligands. Based upon these explorations, we conclude that TMDs affect signaling primarily by modulating intracellular domain geometry. Finally, to guide the design of future receptors, we propose general principles for linking design choices to biophysical mechanisms and performance characteristics.

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