Probing Membrane Protein Association Using Concentration‐Dependent Number and Brightness

Paul, Michael; Rainwater, Randall; Zuo, Yi Y.; Gu, Luo; Hristova, Kalina

doi:10.1002/ange.202010049

Cited by 7 publications

(17 citation statements)

References 63 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The molecular brightness, defined as the ratio of the variance of the fluorescence intensity within a membrane region to the mean fluorescence intensity in this region, is known to scale with the oligomer size (24). The molecular brightness distributions of monomeric (Linker for Activation of T-cells, LAT; gray) (25, 26) and dimeric controls (TrkA in the presence of 130 nM nerve growth factor, NGF; black) (27) in 293T cells are shown in Figure 1A, along with the brightness distribution for FGFR1 in 293T cells stably expressing FGFR1-YFP (red). We found that FGFR1, in the absence of ligand (red line), exists in a monomer/dimer equilibrium, as its brightness distribution is between the distributions of LAT (monomer control) and TrkA in the presence of saturating concentration of NGF (dimer control).…”

Section: Resultsmentioning

confidence: 99%

Ligand bias underlies differential signaling of multiple FGFs via FGFR1

Karl

Hristova

Krejčı́

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

FGFR1 signals differently in response to the FGF ligands FGF4, FGF8 and FGF9, but the mechanism behind the differential ligand recognition is poorly understood. Here, we use biophysical tools to quantify multiple aspects of FGFR1 signaling in response to the three FGFs: potency, efficacy, ligand-induced oligomerization and downregulation, and conformation of the active FGFR1 dimers. We show that FGF4, FGF8, and FGF9 are biased ligands, and that bias can explain differences in FGF8 and FGF9-mediated cellular responses. Our data suggest that ligand bias arises due to structural differences in the ligand-bound FGFR1 dimers, which impact the interactions of the FGFR1 transmembrane helices, leading to differential recruitment and activation of the downstream signaling adaptor FRS2. This study expands the mechanistic understanding of FGF signaling during development and brings the poorly understood concept of receptor tyrosine kinase ligand bias into the spotlight.

show abstract

Section: Resultsmentioning

confidence: 99%

Ligand bias underlies differential signaling of multiple FGFs via FGFR1

Karl

Hristova

Krejčı́

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…We thus measured EphA2 oligomer size in EphA2-enriched and depleted regions using Number and Brightness (N&B), which measures fluorescence fluctuations and reports on the oligomer size of a protein complex 34 . Experiments were performed in CHO cells under reversible osmotic stress, as described in 35 , where we imaged the equatorial plane of the membrane. Regions of EphA2-enrichment and EphA2-depletion were analyzed separately, yielding the oligomer size in these regions.…”

Section: Resultsmentioning

confidence: 99%

“…Data for LAT has been previously published. 35 To further investigate EphA2 interactions in EphA2-enriched and -depleted regions, we performed Förster Resonance Energy Transfer (FRET) experiments in HEK293T cells, using the Fully Quantified Spectral Imaging (FSI)-FRET method, with EphA2-mTurquoise and EphA2-eYFP as a FRET pair. 30 The cells were activated with ephrinA1-Fc and imaged with a 2-photon microscope to acquire full fluorescence emission spectra with pixel-level resolution.…”

Section: The Effect Of Lateral Epha2 Segregation On Epha2 Signalingmentioning

confidence: 99%

“…Number and Brightness (N&B) data were acquired and analyzed as described previously 35 . N&B measures the ratio of the variance due to the fluorescence intensity fluctuations over time to the average intensity, which is different for different types of oligomers.…”

Section: Cell Culture and Transfectionmentioning

confidence: 99%

“…Number and Brightness (N&B) measurements of EphA2 oligomer size in EphA2-enriched and -depleted regions in CHO cells.The membrane protein LAT is known to be monomeric and serves as a control. Data for LAT has been previously published 35.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Direct quantification of ligand-induced lipid and protein microdomains with distinctive signaling properties

Wirth

Paul

Pasquale

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

Lipid rafts are known as highly ordered lipid domains that are enriched in saturated lipids such as the ganglioside GM1. While lipid rafts are believed to exist in cells and to serve as signaling platforms through their enrichment in signaling components, they have never been directly observed in the plasma membrane without treatments that artificially cluster GM1 into large lattices. Here we report that microscopic GM1-enriched domains can form, without lipid crosslinking, in the plasma membrane of live mammalian cells expressing the EphA2 receptor tyrosine kinase in response to its ligand ephrinA1-Fc. The GM1-enriched microdomains form concomitantly with EphA2-enriched microdomains, but only partially co-localize with them. To gain insight into how plasma membrane heterogeneity controls signaling, we quantify the degree of EphA2 segregation and study initial EphA2 signaling steps in both EphA2-enriched and EphA2-depleted domains. By measuring dissociation constants, we demonstrate that EphA2 oligomerization is the same in EphA2-enriched and -depleted domains. However, EphA2 interacts preferentially with its downstream effector SRC in EphA2-depleted domains. The ability to induce microscopic GM1-enriched domains in live cells using a ligand for a transmembrane receptor will give us unprecedented opportunities to study the biology of lipid rafts.

show abstract

Direct Quantification of Ligand‐Induced Lipid and Protein Microdomains with Distinctive Signaling Properties**

et al. 2022

Self Cite

View full text Add to dashboard Cite

Lipid rafts are ordered lipid domains that are enriched in saturated lipids, such as the ganglioside GM1. While lipid rafts are believed to exist in cells and to serve as signaling platforms through their enrichment in signaling components, they have not been directly observed in the plasma membrane without treatments that artificially cluster GM1 into large lattices. Here, we report that microscopic GM1-enriched domains can form in the plasma membrane of live mammalian cells expressing the EphA2 receptor tyrosine kinase in response to its ligand ephrinA1-Fc. The GM1-enriched microdomains form concomitantly with EphA2-enriched microdomains. To gain insight into how plasma membrane heterogeneity controls signaling, we quantify the degree of EphA2 segregation and study initial EphA2 signaling steps in both EphA2-enriched and EphA2depleted domains. By measuring dissociation constants, we demonstrate that the propensity of EphA2 to oligomerize is similar in EphA2-enriched and -depleted domains. However, surprisingly, EphA2 interacts preferentially with its downstream effector SRC in EphA2-depleted domains. The ability to induce microscopic GM1-enriched domains in live cells using a ligand for a transmembrane receptor will give us unprecedented opportunities to study the biophysical chemistry of lipid rafts.

show abstract

Probing Membrane Protein Association Using Concentration‐Dependent Number and Brightness

Cited by 7 publications

References 63 publications

Ligand bias underlies differential signaling of multiple FGFs via FGFR1

Ligand bias underlies differential signaling of multiple FGFs via FGFR1

Direct quantification of ligand-induced lipid and protein microdomains with distinctive signaling properties

Direct Quantification of Ligand‐Induced Lipid and Protein Microdomains with Distinctive Signaling Properties**

Contact Info

Product

Resources

About