We have built an integrated imaging system by combining stimulated emission depletion(STED)microscope and atomic force microscope(AFM).The STED microscope was constructed based on the supercontinuum fiber laser and a super lateral resolution of42 nm was achieved.With this integrated imaging system,morphological features,mechanical parameters and fluorescence super resolution imaging were obtained simultaneously for both nanobeads and fixed cell samples.This new integrated imaging system is expected to obtain comprehensive information at the nanoscale for studies in nanobiology and nanomedicine.
The many kinds of cell structures involved in cell-cell communication include tight junction,adherens junction and gap junction,but almost all are between adjacent cells.Recently,a general and dynamic membrane tether,termed tunneling nanotubes or membrane nanotubes(MNTs),was discovered to be involved in communication between distant cells.By facilitating intercellular communication,MNTs contribute to many biological functions and pathologic changes in cells.Many works have revealed the structure,formation and functional properties of MNTs.However,as novel structures,further research is needed.
Single-particle tracking photoactivated localization microscopy(spt PALM) has recently emerged as a powerful tool for high-density imaging and tracking of individual molecules in living cells. In this work, we have monitored and compared the diffusion dynamics of TGF-b type II receptor(Tb RII) at high expression level using both traditional single-particle tracking(SPT) and spt PALM.The ligand-induced aggregation of Tb RII oligomers was further indicated by spt PALM. Due to the capacity of distinguishing and tracking single molecules within diffraction limit, spt PALM outperforms traditional SPT by providing more accurate biophysical information.
Zi YeNan LiLibo ZhaoYahong SunHefei RuanMingliang ZhangJinghe YuanXiaohong Fang
Exposure to cigarette smoke is a major risk factor for cancer and cardiovascular disease. Thrombosis is regarded as the main reason for smoking-related cardiovascular disease. However, the detail mechanism of how smoking promotes thrombosis is not fully understood. In this work, we investigated the impacts of one major cigarette carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK) as well as its metabolite4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol(NNAL)on a key process in thrombosis regulation: thrombin–thrombomodulin(TM) binding. Atomic force microscopy based single-molecule force spectroscopy was applied to measure both in vitro and in vivo binding force of thrombin to TM in the absence and presence of NNK and NNAL respectively. The results revealed that NNK and NNAL can reduce the binding probability of TM and thrombin. The inhibition effect and underlying mechanism was further studied by molecular simulation. As indicated by our results, the cigarette carcinogens could cause a higher risk of thrombosis through the disruption of TM–thrombin interaction.
As a prototype of the TGF-βsuperfamily cytokines,TGF-βis well known for its diverse roles in embryogenesis and adult tissue homeostasis.TGF-βevokes cellular responses by signaling mainly through cell membrane receptors and transcription factor R-Smads and Co-Smad(Smad4),while an inhibitory Smad,Smad7,acts as a critical negative regulator of TGF-βsignaling.Smad7 antagonizes TGF-βsignaling by regulating the stability or activity of the receptors or blocking the DNA binding of the functional R-Smad-Smad4 complex in the nucleus.However,the function of Smad7 in the nucleus is not fully understood.Yin Yang 1(YY1)is a ubiquitously expressed transcription factor with multiple functions.It has been reported that YY1 can inhibit Smad-dependent transcriptional responses and TGF-β/BMP-induced cell differentiation independently of its DNA binding ability.In this study,we found that Smad7 interacts with YY1 and the interaction is attenuated by TGF-βsignaling.Reporter assays and target gene expression analyses revealed that Smad7 and YY1 act in concert to inhibit TGF-β-induced transcription in the nucleus.Furthermore,Smad7 could enhance the interaction of YY1 with the histone deacetylase HDAC1.Consistently,YY1 and HDAC1 augmented the transcription repression activity of Smad7 in Gal4-luciferase reporter analysis.Therefore,our findings define a novel mechanism of Smad7 and YY1 to antagonize TGF-βsignaling.
YAN XiaoHuaPAN JunXIONG WanWanCHENG MinZhangSUN YingYuanZHANG SuPingCHEN YeGuang
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 thatβ2-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.
