Jinghe Yuan

Monomeric type I and type III transforming growth factor-β receptors and their dimerization revealed by single-molecule imaging

Transforming growth factor-β (TGF-β) binds with two transmembrane serine/threonine kinase receptors, type II (TβRII) and type I receptors (TβRI), and one accessory receptor, type III receptor (TβRIII), to transduce signals across cell membranes. Previous biochemical studies suggested that TβRI and TβRIII are preexisted homo-dimers. Using single-molecule microscopy to image green fluorescent protein-labeled membrane proteins, for the first time we have demonstrated that TβRI and TβRIII could exist as monomers at a low expression level. Upon TGF-β1 stimulation, TβRI follows the general ligand-induced receptor dimerization model for activation, but this process is TβRII-dependent. The monomeric status of the non-kinase receptor TβRIII is unchanged in the presence of TGF-β1. With the increase of receptor expression, both TβRI and TβRIII can be assembled into dimers on cell surfaces.

Single-molecule imaging revealed enhanced dimerization of transforming growth factor β type II receptors in hypertrophic cardiomyocytes.

Transforming growth factor β (TGF-β) signaling plays an important role in the pathogenesis of cardiac hypertrophy. However, the molecular mechanism of TGF-β signaling during the process of cardiac remodeling remains poorly understood. In the present study, by employing single-molecule fluorescence imaging approach, we demonstrated that in neonatal rat cardiomyocytes, TGF-β type II receptors (TβRII) existed as monomers at the low expression level, and dimerized upon TGF-β1 stimulation. Importantly, for the first time, we found the increased dimerization of TβRII in hypertrophic cardiomyocytes comparing to the normal cardiomyocytes. The enhanced TβRII dimerization was correlated with the enhanced Smad3 phosphorylation levels. These results provide new information on the mechanism of TGF-β signaling in cardiac remodeling.

Analysis of the Steps in Single‐Molecule Photobleaching Traces by Using the Hidden Markov Model and Maximum‐Likelihood Clustering

The step analysis of single-molecule photobleaching data offers a new approach for studying protein stoichiometry under physiological conditions. As such, it is important to develop suitable algorithms that can accurately extract the step events from the noisy single-molecule data. Herein, we report a HMM method that combines maximum-likelihood clustering for initializing the emission-probability distribution of the HMMs with an extended silhouette clustering criterion for estimating the state number of single molecules. In this way, the limitations of standard HMM in terms of processing typical single-molecule data with a short sequence are overcome. By using this method, the number and time points of the step events are automatically determined, without the introduction of any subjectivity. Simulation experiments on the experimental photobleaching data indicate that our method is very effective and robust in the analysis of single-molecule fluorescence photobleaching curves if the signal/noise ratio is larger than 2:1. This method was employed for processing photobleaching data that were obtained from single-molecule fluorescence imaging of transforming growth factor typeII receptors on a cell surface. This method is also expected to be applicable to the analysis of other stepwise events.

Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane

Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction.

Nanoscale Distribution of Transforming Growth Factor Receptor on Post-Golgi Vesicle Revealed by Super-resolution Microscopy.

Transforming growth factor-β (TGF-β) type II receptor (TβRII) plays a critical role in the initiation of TGF-β signaling pathway; therefore, the study of its synthesis and transport processes is of great important. In this work, we achieved super-resolution imaging of a new type of TβRII-containing post-Golgi vesicle by our home-built stimulated emission depletion (STED) microscope. We visualized the ring-shaped structure of these vesicles containing newly synthesized TβRII in the cytoplasm and characterized their size distribution from 300 to 1000 nm. These vesicles could be swollen by chloroquine treatment. Further investigation revealed that TβRII formed clusters on the outer ring of the post-Golgi vesicles. This study offers new information on the intracellular transportation of TGF-β receptors for better understanding its signaling process.

Single-molecule imaging reveals the stoichiometry change of epidermal growth factor receptor during transactivation by β 2 -adrenergic receptor

Stimulation of G protein-coupled receptors (GPCRs) can lead to the transactivation of the epidermal growth factor receptors (EGFR). The cross-communication between the two signaling pathways regulates several important physiological or pathological processes. However, the molecule mechanism underlying EGFR transactivation remains poorly understood. Here, we aim to study the GPCR-mediated EGFR transactivation process using the single-molecule fluorescence imaging and tracking approach. We found that although EGFR existed as monomers at the plasma membrane of resting cells, they became dimers and thus diffused slower following the activation of β2-adrenergic receptor (β2-AR) by isoproterenol (ISO). We further proved that β2-AR-mediated changes of EGFR in stoichiometry and dynamics were mediated by Src kinase. Thus, the observations obtained via the single-molecule imaging and tracking methods shed new insights into the molecular mechanism of EGFR transactivation at single molecule level.

Mammalian actin-binding protein 1/HIP-55 is essential for the scission of clathrin-coated pits by regulating dynamin-actin interaction

Actin and dynamin work cooperatively to drive the invagination and scission of clathrin‐coated pits (CCPs). However, little is known about the mechanism that orchestrates the spatiotemporal recruitment of dynamin and actin. Here, we have identified the mammalian actin‐binding protein 1 (mAbp1; also called HIP‐55 or SH3P7), which could bind to clathrin, actin, as well as dynamin, as an adaptor that links the dynamic recruitment of dynamin and actin for the scission of CCPs. Live‐cell imaging reveals that mAbp1 is specifically recruited at a late stage of the long‐lived CCPs. mAbp1 knockdown impaired CCP scission by reducing dynamin recruitment at the plasma membrane. However, actin disruption remarkably eliminates mAbp1 recruitment and thus dynamin recruitment. These data suggest that by binding to both clathrin and F‐actin, mAbp1 is specifically recruited at a late stage of CCP formation, which subsequently recruits dynamin to CCPs. He, K., Xing, R., Yan, X., Tian, A., Zhang, M., Yuan, J., Lv, Z., Fang, X., Li, Z., and Zhang, Y.—Mammalian actin‐binding protein 1/HIP‐55 is essential for the scission of clathrin‐coated pits by regulating dynamin‐actin interaction. FASEB J. 29, 2495‐2503 (2015). www.fasebj.org

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

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.