scholarly journals Differential mast cell outcomes are sensitive to FcεRI-Syk binding kinetics

2017 ◽  
Vol 28 (23) ◽  
pp. 3397-3414 ◽  
Author(s):  
Samantha L. Schwartz ◽  
Cédric Cleyrat ◽  
Mark J. Olah ◽  
Peter K. Relich ◽  
Genevieve K. Phillips ◽  
...  
Keyword(s):  
Single Molecule ◽  
Calcium Release ◽  
Cytokine Production ◽  
Immunoglobulin E ◽  
Aggregate Size ◽  
Cellular Responses ◽  
Live Cells ◽  
Sh2 Domains ◽  
Downstream Signaling ◽  
Rate Loss

Cross-linking of immunoglobulin E–bound FcεRI triggers multiple cellular responses, including degranulation and cytokine production. Signaling is dependent on recruitment of Syk via docking of its dual SH2 domains to phosphorylated tyrosines within the FcεRI immunoreceptor tyrosine-based activation motifs. Using single-molecule imaging in live cells, we directly visualized and quantified the binding of individual mNeonGreen-tagged Syk molecules as they associated with the plasma membrane after FcεRI activation. We found that Syk colocalizes transiently to FcεRI and that Syk-FcεRI binding dynamics are independent of receptor aggregate size. Substitution of glutamic acid for tyrosine between the Syk SH2 domains (Syk-Y130E) led to an increased Syk-FcεRI off-rate, loss of site-specific Syk autophosphorylation, and impaired downstream signaling. Genome edited cells expressing only Syk-Y130E were deficient in antigen-stimulated calcium release, degranulation, and production of some cytokines (TNF-a, IL-3) but not others (MCP-1, IL-4). We propose that kinetic discrimination along the FcεRI signaling pathway occurs at the level of Syk-FcεRI interactions, with key outcomes dependent upon sufficiently long-lived Syk binding events.

scholarly journals Differential Mast Cell Outcomes Are Sensitive to FcεRI-Syk Binding Kinetics

2017 ◽  
Author(s):  
Samantha L. Schwartz ◽  
Cédric Cleyrat ◽  
Mark Olah ◽  
Peter Relich ◽  
Genevieve Phillips ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Calcium Release ◽  
Aggregate Size ◽  
Live Cells ◽  
Single Molecule Imaging ◽  
Site Specific ◽  
Sh2 Domains ◽  
Downstream Signaling ◽  
Transient Nature

AbstractCrosslinking of IgE-bound FcεRI triggers multiple cellular responses, including degranulation and cytokine production. Signaling is dependent on recruitment of Syk via docking of its dual SH2 domains to phosphorylated tyrosines within the FcεRI immunoreceptor tyrosine-based activation motifs. Using single molecule imaging in live cells, we directly visualized and quantified the binding of individual mNeonGreen-tagged Syk molecules as they associated with the plasma membrane after FcεRI activation. We found that Syk colocalizes transiently to FcεRI and that Syk-FcεRI binding dynamics are independent of receptor aggregate size. Substitution of glutamic acid for tyrosine between the Syk SH2 domains (SykY130E) led to an increased Syk-FcεRI off-rate, loss of site-specific Syk autophosphorylation, and impaired downstream signaling. CRISPR-Cas9 engineered cells expressing only SykY130E were deficient in antigen-stimulated calcium release, degranulation and production of some cytokines (TNF-a, IL-3) but not others (MCP-1, IL-4). We propose that kinetic discrimination along the FcεRI signaling pathway occurs at the level of Syk-FcεRI interactions, with key outcomes dependent upon sufficiently long-lived Syk binding events.SummarySchwartz et al. use single molecule imaging to quantify the transient nature of FcεRI-Syk interactions in live mast cells. A functional mutation that increases Syk off-rate leads to loss of site-specific Syk phosphorylation and impaired signaling, highlighting the importance of finely tuned protein interactions in directing cellular outcomes.

scholarly journals Motional dynamics of single Patched1 molecules in cilia are controlled by Hedgehog and cholesterol

2019 ◽  
Vol 116 (12) ◽  
pp. 5550-5557 ◽  
Author(s):  
Lucien E. Weiss ◽  
Ljiljana Milenkovic ◽  
Joshua Yoon ◽  
Tim Stearns ◽  
W. E. Moerner
Keyword(s):  
Single Molecule ◽  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Transmembrane Protein ◽  
Cellular Level ◽  
Live Cells ◽  
Cholesterol Depletion ◽  
Motional Dynamics ◽  
Directional Transport ◽  
Bulk Behavior

The Hedgehog-signaling pathway is an important target in cancer research and regenerative medicine; yet, on the cellular level, many steps are still poorly understood. Extensive studies of the bulk behavior of the key proteins in the pathway established that during signal transduction they dynamically localize in primary cilia, antenna-like solitary organelles present on most cells. The secreted Hedgehog ligand Sonic Hedgehog (SHH) binds to its receptor Patched1 (PTCH1) in primary cilia, causing its inactivation and delocalization from cilia. At the same time, the transmembrane protein Smoothened (SMO) is released of its inhibition by PTCH1 and accumulates in cilia. We used advanced, single molecule-based microscopy to investigate these processes in live cells. As previously observed for SMO, PTCH1 molecules in cilia predominantly move by diffusion and less frequently by directional transport, and spend a fraction of time confined. After treatment with SHH we observed two major changes in the motional dynamics of PTCH1 in cilia. First, PTCH1 molecules spend more time as confined, and less time freely diffusing. This result could be mimicked by a depletion of cholesterol from cells. Second, after treatment with SHH, but not after cholesterol depletion, the molecules that remain in the diffusive state showed a significant increase in the diffusion coefficient. Therefore, PTCH1 inactivation by SHH changes the diffusive motion of PTCH1, possibly by modifying the membrane microenvironment in which PTCH1 resides.

scholarly journals Ionizing Radiation and Translation Control: A Link to Radiation Hormesis?

2020 ◽  
Vol 21 (18) ◽  
pp. 6650
Author(s):  
Usha Kabilan ◽  
Tyson E. Graber ◽  
Tommy Alain ◽  
Dmitry Klokov
Keyword(s):  
Protein Synthesis ◽  
Ionizing Radiation ◽  
Molecular Mechanisms ◽  
Low Dose ◽  
Mrna Translation ◽  
Cellular Responses ◽  
Translation Control ◽  
Cis Acting ◽  
Radiation Hormesis ◽  
Downstream Signaling

Protein synthesis, or mRNA translation, is one of the most energy-consuming functions in cells. Translation of mRNA into proteins is thus highly regulated by and integrated with upstream and downstream signaling pathways, dependent on various transacting proteins and cis-acting elements within the substrate mRNAs. Under conditions of stress, such as exposure to ionizing radiation, regulatory mechanisms reprogram protein synthesis to translate mRNAs encoding proteins that ensure proper cellular responses. Interestingly, beneficial responses to low-dose radiation exposure, known as radiation hormesis, have been described in several models, but the molecular mechanisms behind this phenomenon are largely unknown. In this review, we explore how differences in cellular responses to high- vs. low-dose ionizing radiation are realized through the modulation of molecular pathways with a particular emphasis on the regulation of mRNA translation control.

scholarly journals A Photoactivatable Push−Pull Fluorophore for Single-Molecule Imaging in Live Cells

2008 ◽  
Vol 130 (29) ◽  
pp. 9204-9205 ◽  
Author(s):  
Samuel J. Lord ◽  
Nicholas R. Conley ◽  
Hsiao-lu D. Lee ◽  
Reichel Samuel ◽  
Na Liu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Live Cells ◽  
Single Molecule Imaging

scholarly journals Full characterization of GPCR monomer–dimer dynamic equilibrium by single molecule imaging

2011 ◽  
Vol 192 (3) ◽  
pp. 463-480 ◽  
Author(s):  
Rinshi S. Kasai ◽  
Kenichi G. N. Suzuki ◽  
Eric R. Prossnitz ◽  
Ikuko Koyama-Honda ◽  
Chieko Nakada ◽  
...  
Keyword(s):  
Equilibrium Constant ◽  
Single Molecule ◽  
Dynamic Equilibrium ◽  
Dissociation Rate ◽  
Formyl Peptide Receptor ◽  
Live Cells ◽  
Imaging Method ◽  
Full Characterization ◽  
Before And After ◽  
Dissociation Rate Constants

Receptor dimerization is important for many signaling pathways. However, the monomer–dimer equilibrium has never been fully characterized for any receptor with a 2D equilibrium constant as well as association/dissociation rate constants (termed super-quantification). Here, we determined the dynamic equilibrium for the N-formyl peptide receptor (FPR), a chemoattractant G protein–coupled receptor (GPCR), in live cells at 37°C by developing a single fluorescent-molecule imaging method. Both before and after liganding, the dimer–monomer 2D equilibrium is unchanged, giving an equilibrium constant of 3.6 copies/µm2, with a dissociation and 2D association rate constant of 11.0 s−1 and 3.1 copies/µm2s−1, respectively. At physiological expression levels of ∼2.1 receptor copies/µm2 (∼6,000 copies/cell), monomers continually convert into dimers every 150 ms, dimers dissociate into monomers in 91 ms, and at any moment, 2,500 and 3,500 receptor molecules participate in transient dimers and monomers, respectively. Not only do FPR dimers fall apart rapidly, but FPR monomers also convert into dimers very quickly.

scholarly journals tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

2021 ◽  
Author(s):  
Y. Bousmah ◽  
H. Valenta ◽  
G. Bertolin ◽  
U. Singh ◽  
V. Nicolas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy ◽  
Live Cell ◽  
Live Cells

AbstractYellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow variant derived from the EYFP with a pK1/2 below ∼5.5. Here, we characterize a new yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available, and brighter than most of the monomeric YFPs. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and preserves this property in live cells. As a consequence, tdLanYFP allows the imaging of cellular structures with sub-diffraction resolution with STED nanoscopy. We also demonstrate that the combination of high brightness and strong photostability is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP can be a FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFPa very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging that is also suitable for FRET experiment including at acidic pH.

scholarly journals A method for imaging single molecules at the plasma membrane of live cells within tissue slices

2020 ◽  
Vol 153 (1) ◽  
Author(s):  
Gregory I. Mashanov ◽  
Tatiana A. Nenasheva ◽  
Tatiana Mashanova ◽  
Catherine Maclachlan ◽  
Nigel J.M. Birdsall ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Lines ◽  
Single Molecule ◽  
Cardiac Tissue ◽  
Single Molecules ◽  
Live Cells ◽  
Biological Macromolecules ◽  
Tissue Slices ◽  
Tissue Samples ◽  
Mammalian Cell Lines

Recent advances in light microscopy allow individual biological macromolecules to be visualized in the plasma membrane and cytosol of live cells with nanometer precision and ∼10-ms time resolution. This allows new discoveries to be made because the location and kinetics of molecular interactions can be directly observed in situ without the inherent averaging of bulk measurements. To date, the majority of single-molecule imaging studies have been performed in either unicellular organisms or cultured, and often chemically fixed, mammalian cell lines. However, primary cell cultures and cell lines derived from multi-cellular organisms might exhibit different properties from cells in their native tissue environment, in particular regarding the structure and organization of the plasma membrane. Here, we describe a simple approach to image, localize, and track single fluorescently tagged membrane proteins in freshly prepared live tissue slices and demonstrate how this method can give information about the movement and localization of a G protein–coupled receptor in cardiac tissue slices. In principle, this experimental approach can be used to image the dynamics of single molecules at the plasma membrane of many different soft tissue samples and may be combined with other experimental techniques.

scholarly journals On demand MyD88 oligomerization is controlled by IRAK4 during Myddosome signaling

2020 ◽  
Author(s):  
Rafael Deliz-Aguirre ◽  
Fakun Cao ◽  
Fenja H. U. Gerpott ◽  
Nichanok Auevechanichkul ◽  
Mariam Chupanova ◽  
...  
Keyword(s):  
Self Assembly ◽  
Intracellular Signaling ◽  
Innate Immune ◽  
Live Cells ◽  
Signaling Proteins ◽  
Protein Oligomerization ◽  
Downstream Signaling ◽  
Oligomeric Complex ◽  
Mechanistic Basis ◽  
Cell Signaling Pathways

AbstractA recurring feature of innate immune receptor signaling is the self-assembly of signaling proteins into oligomeric complexes. The Myddosome is an oligomeric complex that is required to transmit inflammatory signals from TLR/IL1Rs and consists of MyD88 and IRAK family kinases. However, the molecular basis for how Myddosome proteins self-assemble and regulate intracellular signaling remains poorly understood. Here, we developed a novel assay to analyze the spatiotemporal dynamics of IL1R and Myddosome signaling in live cells. We found that MyD88 oligomerization is inducible and initially reversible. Moreover, the formation of larger, stable oligomers consisting of more than 4 MyD88s triggers the sequential recruitment of IRAK4 and IRAK1. Notably, genetic knockout of IRAK4 enhanced MyD88 oligomerization, indicating that IRAK4 controls MyD88 oligomer size and growth. MyD88 oligomer size thus functions as a physical threshold to trigger downstream signaling. These results provide a mechanistic basis for how protein oligomerization might function in cell signaling pathways.

scholarly journals Live cell micropatterning reveals the dynamics of signaling complexes at the plasma membrane

2014 ◽  
Vol 207 (3) ◽  
pp. 407-418 ◽  
Author(s):  
Sara Löchte ◽  
Sharon Waichman ◽  
Oliver Beutel ◽  
Changjiang You ◽  
Jacob Piehler
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Spatial Organization ◽  
Polymer Brush ◽  
Effector Proteins ◽  
Live Cells ◽  
Type I ◽  
Rgd Peptides ◽  
Single Molecule Level ◽  
Cell Micropatterning

Interactions of proteins in the plasma membrane are notoriously challenging to study under physiological conditions. We report in this paper a generic approach for spatial organization of plasma membrane proteins into micropatterns as a tool for visualizing and quantifying interactions with extracellular, intracellular, and transmembrane proteins in live cells. Based on a protein-repellent poly(ethylene glycol) polymer brush, micropatterned surface functionalization with the HaloTag ligand for capturing HaloTag fusion proteins and RGD peptides promoting cell adhesion was devised. Efficient micropatterning of the type I interferon (IFN) receptor subunit IFNAR2 fused to the HaloTag was achieved, and highly specific IFN binding to the receptor was detected. The dynamics of this interaction could be quantified on the single molecule level, and IFN-induced receptor dimerization in micropatterns could be monitored. Assembly of active signaling complexes was confirmed by immunostaining of phosphorylated Janus family kinases, and the interaction dynamics of cytosolic effector proteins recruited to the receptor complex were unambiguously quantified by fluorescence recovery after photobleaching.

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