Single-molecule imaging reveals the stoichiometry change of epidermal growth factor receptor during transactivation by β2-adrenergic receptor

Zhang, Mingliang; He, Kangmin; Wu, Jimin; Li, Nan; Yuan, Jinghe; Zhou, Wei; Ye, Zi; Li, Zijian; Xiao, Han; Lv, Zhizhen; Zhang, Youyi; Fang, Xiaohong

doi:10.1007/s11426-017-9072-5

Cited by 10 publications

(12 citation statements)

References 43 publications

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“…Single-molecule imaging. Single-molecule fluorescence imaging was performed by a home-built TIRF microscope using an inverted Olympus IX71 microscope equipped with a total internal reflective fluorescence illuminator, a 100×/1.45 NA Plan Apochromatic TIRF objective and an electron-multiplying charge-coupled device (EMCCD) camera (Andor iXon DU-897D BV) 46,50 . EGFP molecules were excited by a 488 nm laser at 1 mW (60 W/cm2) (Melles Griot, Carlsbad, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

Analyzing protein dynamics from fluorescence intensity traces using unsupervised deep learning network

Yuan

Zhao

et al. 2020

Commun Biol

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We propose an unsupervised deep learning network to analyze the dynamics of membrane proteins from the fluorescence intensity traces. This system was trained in an unsupervised manner with the raw experimental time traces and synthesized ones, so neither predefined state number nor pre-labelling were required. With the bidirectional Long Short-Term Memory (biLSTM) networks as the hidden layers, both the past and future context can be used fully to improve the prediction results and can even extract information from the noise distribution. The method was validated with the synthetic dataset and the experimental dataset of monomeric fluorophore Cy5, and then applied to extract the membrane protein interaction dynamics from experimental data successfully.

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Section: Methodsmentioning

confidence: 99%

Analyzing protein dynamics from fluorescence intensity traces using unsupervised deep learning network

Yuan

Zhao

et al. 2020

Commun Biol

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“…Again, the use of SPT has proved to be useful in shedding further light on the interactions of EGFR. A recent study used SPT to track both EGFR and the GPCR beta-2 adrenergic receptor (β2-AR) in live HeLa cells [136], directly observing the stoichiometry and dynamics of EGFR transactivation. They used isoprotenerol (ISO) to activate β2-AR, and found that this induced EGFR dimerization and slower diffusion.…”

Section: Interactions With Other Receptors the Endocytic Pathwaymentioning

confidence: 99%

A Brief History of Single-Particle Tracking of the Epidermal Growth Factor Receptor

Clarke

Martin-Fernandez

2019

MPs

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Single-particle tracking (SPT) has been used and developed over the last 25 years as a method to investigate molecular dynamics, structure, interactions, and function in the cellular context. SPT is able to show how fast and how far individual molecules move, identify different dynamic populations, measure the duration and strength of intermolecular interactions, and map out structures on the nanoscale in cells. In combination with other techniques such as macromolecular crystallography and molecular dynamics simulation, it allows us to build models of complex structures, and develop and test hypotheses of how these complexes perform their biological roles in health as well as in disease states. Here, we use the example of the epidermal growth factor receptor (EGFR), which has been studied extensively by SPT, demonstrating how the method has been used to increase our understanding of the receptor’s organization and function, including its interaction with the plasma membrane, its activation, clustering, and oligomerization, and the role of other receptors and endocytosis. The examples shown demonstrate how SPT might be employed in the investigation of other biomolecules and systems.

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“…In the traditional view of TβR signaling, the receptors are believed to exist as dimers or oligomers in the resting cells, and ligand binding induces the formation of heteromeric TGF-β receptor complexes each containing a dimer of type II TβR and a dimer of type I TβR, which initiates signaling. Our previous research discovered that at a low expression level, TGFβ receptors mainly exist as monomers on the cell membrane, and they also follow the general rule of growth-factor-receptor activation, in which ligands induce activation through monomer dimerization. , This activation model we proposed has been successfully applied to the study of the signaling capability of a pair of type II TβR and type I TβR molecules as well as to the study of the mechanism of several inhibitors of TβR-signaling pathways. , Here, by our FRAP-SMI method, we found that TβRII is mainly transported to the cell membrane in its monomeric form, independent of TGF-β1 stimulation. This result provided additional evidence supporting our previous finding of monomeric TβRs on cell membranes.…”

Section: Results and Discussionmentioning

confidence: 88%

“…Our previous research discovered that at a low expression level, TGFβ receptors mainly exist as monomers on the cell membrane, and they also follow the general rule of growth-factor-receptor activation, in which ligands induce activation through monomer dimerization. 2,31 This activation model we proposed has been successfully applied to the study of the signaling capability of a pair of type II TβR and type I TβR molecules as well as to the study of the mechanism of several inhibitors of TβR-signaling pathways. 15,25 Here, by our FRAP-SMI method, we found that TβRII is mainly transported to the cell membrane in its monomeric form, independent of TGF-β1 stimulation.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Quantitative Characterization of the Membrane Dynamics of Newly Delivered TGF-β Receptors by Single-Molecule Imaging

Zhang

et al. 2018

Anal. Chem.

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The dynamics and stoichiometry of receptors newly delivered on the plasma membrane play a vital role in cell signal transduction, yet knowledge of this process is limited because of the lack of suitable analytical methods. Here we developed a new strategy that combines single-molecule imaging (SMI) and fluorescence recovery after photobleaching (FRAP), named FRAP-SMI, to monitor and quantify individual newly delivered and inserted transmembrane receptors on plasma membranes of living cells. Transforming-growth-factor-β type II receptor (TβRII), a typical serine/threoninekinase receptor, was studied with this method. We first eliminated the fluorescence signals from the pre-existing EGFP-labeled TβRII molecules on the plasma membrane, and then we recorded the individual newly appeared TβRII-GFP by total-internal-reflection fluorescence imaging. The fluorescence-intensity distributions, photobleaching steps, and diffusion rates of the single TβRII-GFP molecules were analyzed. We reported, for the first time, that TβRII was transported to the plasma membrane mainly in the monomeric form in both resting and TGF-β1stimulated cells. This strongly supported our former discovery that TβRII could exist as a monomer on the cell membrane. We also found that ligand stimulation resulted in enhanced delivery rates and prolonged membrane-association times for the TβRII molecules. On the basis of these observations, we proposed a mechanism of TGF-β1-induced TβRII dimerization for receptor activation. Our method provides a useful tool for the real-time quantification of the spatial arrangement, mobility, and oligomerization of cell-surface proteins in living cells, thus providing a better understanding of cell signaling.

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Single-molecule imaging reveals the stoichiometry change of epidermal growth factor receptor during transactivation by β2-adrenergic receptor

Cited by 10 publications

References 43 publications

Analyzing protein dynamics from fluorescence intensity traces using unsupervised deep learning network

Analyzing protein dynamics from fluorescence intensity traces using unsupervised deep learning network

A Brief History of Single-Particle Tracking of the Epidermal Growth Factor Receptor

Quantitative Characterization of the Membrane Dynamics of Newly Delivered TGF-β Receptors by Single-Molecule Imaging

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