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

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.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e17-06-0350 ◽

2017 ◽

Vol 28 (23) ◽

pp. 3397-3414 ◽

Cited By ~ 10

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.

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Proximity-assisted photoactivation (PAPA): Detecting molecular interactions in live-cell single-molecule imaging

10.1101/2021.12.13.472508 ◽

2021 ◽

Author(s):

Thomas George Wade Graham ◽

John Joseph Ferrie ◽

Gina M. Dailey ◽

Robert Tjian ◽

Xavier Darzacq

Keyword(s):

Single Molecule ◽

Molecular Interactions ◽

Protein Interactions ◽

Protein Complexes ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Live Cells ◽

Single Molecule Imaging ◽

Protein Protein Interactions ◽

Self Association

Single-molecule imaging provides a powerful way to study biochemical processes in live cells, yet it remains challenging to track single molecules while simultaneously detecting their interactions. Here we describe a novel property of rhodamine dyes, proximity-assisted photoactivation (PAPA), in which one fluorophore (the "sender") can reactivate a second fluorophore (the "receiver") from a dark state. PAPA requires proximity between the two fluorophores, yet it operates at a longer average intermolecular distance than Forster resonance energy transfer (FRET). We show that PAPA can be used in live cells both to detect protein-protein interactions and to highlight a sub-population of labeled protein complexes in which two different labels are in proximity. In proof-of-concept experiments, PAPA detected the expected correlation between androgen receptor self-association and chromatin binding at the single-cell level. These results establish a new way in which a photophysical property of fluorophores can be harnessed to study molecular interactions in single-molecule imaging of live cells.

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Faculty Opinions recommendation of Single-molecule imaging reveals receptor-G protein interactions at cell surface hot spots.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.732005651.793538451 ◽

2017 ◽

Author(s):

Arun Shukla

Keyword(s):

Cell Surface ◽

G Protein ◽

Single Molecule ◽

Protein Interactions ◽

Hot Spots ◽

Single Molecule Imaging

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Faculty Opinions recommendation of Single-molecule imaging reveals receptor-G protein interactions at cell surface hot spots.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.732005651.793541682 ◽

2018 ◽

Author(s):

Marcus Thelen

Keyword(s):

Cell Surface ◽

G Protein ◽

Single Molecule ◽

Protein Interactions ◽

Hot Spots ◽

Single Molecule Imaging

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

Journal of the American Chemical Society ◽

10.1021/ja802883k ◽

2008 ◽

Vol 130 (29) ◽

pp. 9204-9205 ◽

Cited By ~ 146

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

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Kappa but not delta or mu opioid receptors form homodimers at low membrane densities

Cellular and Molecular Life Sciences ◽

10.1007/s00018-021-03963-y ◽

2021 ◽

Author(s):

Kristina Cechova ◽

Chenyang Lan ◽

Matus Macik ◽

Nicolas P. F. Barthes ◽

Manfred Jung ◽

...

Keyword(s):

Opioid Receptors ◽

Single Molecule ◽

Protein Interactions ◽

Live Cell Imaging ◽

Specific Effect ◽

Single Molecule Imaging ◽

Physiological Conditions ◽

Biochemical Assays ◽

A Cell ◽

Membrane Density

AbstractOpioid receptors (ORs) have been observed as homo- and heterodimers, but it is unclear if the dimers are stable under physiological conditions, and whether monomers or dimers comprise the predominant fraction in a cell. Here, we use three live-cell imaging approaches to assess dimerization of ORs at expression levels that are 10–100 × smaller than in classical biochemical assays. At membrane densities around 25/µm2, a split-GFP assay reveals that κOR dimerizes, while µOR and δOR stay monomeric. At receptor densities < 5/µm2, single-molecule imaging showed no κOR dimers, supporting the concept that dimer formation depends on receptor membrane density. To directly observe the transition from monomers to dimers, we used a single-molecule assay to assess membrane protein interactions at densities up to 100 × higher than conventional single-molecule imaging. We observe that κOR is monomeric at densities < 10/µm2 and forms dimers at densities that are considered physiological. In contrast, µOR and δOR stay monomeric even at the highest densities covered by our approach. The observation of long-lasting co-localization of red and green κOR spots suggests that it is a specific effect based on OR dimerization and not an artefact of coincidental encounters.

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