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.

The stoichiometry of the β2-adrenergic receptor (β2AR) was determined using single-molecule fluorescence imaging in living cells. The results showed that β2AR mainly existed as monomers under physiological conditions and exhibited β-arrestin-dependent dimerization upon stimulation with the pharmacological biased ligand carvedilol. The association of β2AR dimerization with biased signalling is revealed.

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 metabolite 4-(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.长期暴露于香烟烟雾中会导致恶性肿瘤和心血管疾病的发病率升高。血栓的形成被认为是吸烟相关心血管疾病发生和死亡的主要原因,但是目前对于吸烟诱发血栓形成的机制仍不清楚。在本工作中,我们利用原子力显微镜单分子力谱技术(AFM-SMFS)研究了香烟中主要成分NNK及其代谢产物NNAL对凝血酶和血栓调节蛋白间相互作用的影响,发现 NNK和NNAL可以降低凝血酶和血栓调节蛋白的结合几率,进一步通过分子动力学模拟的方法验证了NNK和NNAL对凝血酶和血栓调节蛋白结合的抑制及可能的作用机制; 结果表明香烟致癌物可通过抑制凝血酶和血栓调节蛋白的结合来诱发血栓。

Single-particle tracking photoactivated localization microscopy (sptPALM) 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-β type II receptor (TβRII) at high expression level using both traditional single-particle tracking (SPT) and sptPALM. The ligand-induced aggregation of TβRII oligomers was further indicated by sptPALM. Due to the capacity of distinguishing and tracking single molecules within diffraction limit, sptPALM outperforms traditional SPT by providing more accurate biophysical information.用于单粒子示踪的光活化定位显微镜(sptPALM)是研究活细胞中单分子高密度成像和追踪的一种新的有效工具。本文分别利用传统的单粒子追踪方法(SPT)和sptPALM对高表达的 转化生长因子 TGF-β二型受体(TβRII)在细胞膜上的扩散运动进行了表征和比较,同时用sptPALM证实配体刺激下TβRII寡聚体进一步聚集。由于sptPALM可区分并追踪处于衍射极限内的多个单分子,它优于传统的SPT方法,能提供更准确的生物分子动态变化信息。

Endocytosis and intracellular sorting of transforming growth factor-β (TGF-β) receptors play an important regulatory role in TGF-β signaling. Two major endocytic pathways, clathrin- and caveolae-mediated endocytosis, have been reported to independently mediate the internalization of TGF-β receptors. In this study, we demonstrate that the clathrin- and caveolae-mediated endocytic pathways can converge during TGF-β receptor endocytic trafficking. By tracking the intracellular dynamics of fluorescently-labeled TGF-β type I receptor (TβRI), we found that after mediating TβRI internalization, certain clathrin-coated vesicles and caveolar vesicles are fused underneath the plasma membrane, forming a novel type of caveolin-1 and clathrin double-positive vesicles. Under the regulation of Rab5, the fused vesicles are targeted to early endosomes and thus deliver the internalized TβRI to the caveolin-1 and EEA1 double-positive early endosomes (caveolin-1-positive early endosomes). We further showed that the caveolin-1-positive early endosomes are positive for Smad3/SARA, Rab11 and Smad7/Smurf2, and may act as a multifunctional device for TGF-β signaling and TGF-β receptor recycling and degradation. Therefore, these findings uncover a novel scenario of endocytosis, the direct fusion of clathrin-coated and caveolae vesicles during TGF-β receptor endocytic trafficking, which leads to the formation of the multifunctional sorting device, caveolin-1-positive early endosomes, for TGF-β receptors.

The cell wall binding domain (CBD) of bacteriophage lysins can recognize target bacteria with extraordinary specificity through binding to bacterial peptidoglycan, thus it is a promising new probe to identify the corresponding bacterial pathogen. In this work, we used atomic force microscopy (AFM) based single-molecule force spectroscopy to investigate the interaction between the CBD of lysin PlyV12 (PlyV12C) and pathogenic bacterium Staphylococcus aureus (S. aureus). The binding forces of PlyV12C with S. aureus have been measured, and the dissociation process of their binding complex has been characterized. Furthermore, we compared the interactions of PlyV12C–S. aureus and antibody–S. aureus. It is revealed that PlyV12C has a comparable affinity to bacterial peptidoglycans as that of the S. aureus antibody. The results provide new information on the binding properties of lysin CBD with bacterium, and the application of lysin CBD in bacterium detection.

A strategy of using a gold nanorod (GNR)-loaded electrospun membrane as a photothermal therapy platform of cancer is reported. The strategy takes both the advantages of the excellent photothermal properties of GNRs to selectively kill the cancerous cells, and the widely used biodegradable electrospun membrane to serve as GNR-carrier and surgical recovery material. PEG modified GNRs were embedded into the electrospun fibrous membrane which was composed of PLGA and PLA-b-PEG with an 85:15 ratio. After incubation with the cells in the cell culture medium, the PEG-GNRs were released from the membrane and taken up by cancer cells, allowing the generation of heat upon NIR irradiation to induce cancer cell death. We have demonstrated that the use of PEG-GNR-embedded membrane selectively killed the cancerous cells and effectively inhibited cancer cell proliferation though in vitro experiments. The PEG-GNRs-loaded membrane is a promising material for postsurgical recovery of cancer.Keywords: biodegradable membrane; cancer; electrospun; PEGylated gold nanorods; photothermal therapy;

We achieved single-molecule imaging and tracking of the transforming growth factor type II receptor (TβRII) that was labeled by an organic dye via a genetically encoded unnatural amino acid (UAA) and the copper-free click chemistry. The stoichiometry, mobility and dimerization kinetics of individual TβRII molecules were determined.

Satellite muscle cells play an important role in regeneration of skeletal muscle. However, they are particularly vulnerable to oxidative stress. Herein, we address our efforts on the cytoprotective activities of carboxyfullerenes with different cage size (C60 vs C70) and adduct number on postmitotic muscle cell (C2C12 cell). The correlation of the structural effect on the cytoprotective capability of carboxyfullerenes was evaluated. We find that quadri-malonic acid C70 fullerene (QF70) exhibits higher capability on protecting cells from oxidative-induced stress among these tested carboxyfullerenes. The accumulation of intracellular superoxide dismutase (SOD) is proposed to play an important role in their diverse antioxidative ability. Moreover, the pretreatment of QF70 could also obviously enhance the viability of myotubes originated from oxidative-stressed C2C12 cells, which facilitates the future application of carboxyfullerenes in tissue engineering and nanomedicine.Keywords: C2C12 cell; carboxyfullerene; chemical structure; cytoprotective capability; oxidative stress;

In this work, we have investigated the change of cell wall for the tobacco plant cell (Nicotiana tobacum L. cv. Bright Yellow) under the repression of water-soluble carboxyfullerenes (C70(C(COOH)2)2–4). The adsorption of C70(C(COOH)2)2–4 on cell wall led to the disruption of cell wall and membrane, and consequently, cell growth inhibition. Results from atomic force microscopy (AFM) force measurement and confocal imaging revealed an increase of the glycosyl residue on the cell wall of carboxyfullerene-treated cells, with a time- and dose-dependent manner, and accompanied by the elevated reactive oxygen species (ROS). Moreover, the stimulation-sensitive alteration of glycosyl residue and ROS was demonstrated, which suggested a possible protection strategy for the plant cells under fullerene repression. This study provides the first direct evidence on the change of plant cell wall composition under the repression of fullerene and is the first successful application of AFM ligand-receptor binding force measurement to the living plant cell. The new information present here would help to a better understanding and assessment of the biological effect of fullerenes on plant.

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 of 42 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.

Monitoring single molecules in living cells is becoming a powerful tool for study of the location, dynamics, and kinetics of individual biomolecules in real time. In recent decades, several optical imaging techniques, for example epi-fluorescence microscopy, total internal reflection fluorescence microscopy (TIRFM), confocal microscopy, quasi-TIRFM, and single-point edge excitation subdiffraction microscopy (SPEED), have been developed, and their capability of capturing single-molecule dynamics in living cells has been demonstrated. In this review, we briefly summarize recent advances in the use of these imaging techniques for monitoring single-molecules in living cells for a better understanding of important biological processes, and discuss future developments.

HER2, a member of the epidermal growth factor receptor (ErbB) family, is over-expressed in many cancers. Trastuzumab and Pertuzumab are two monoclonal antibodies targeting different extracellular domains of HER2 for cancer therapy. As Pertuzumab binds to the dimerization arm of HER2, it can block HER2 heterodimerization and in turn ErbB signaling. Whether Trastuzumab has the same function is unclear. In this work, we have applied living-cell single-molecule force spectroscopy (SMFS) by Atomic Force Microscopy (AFM) to investigate the effect of Trastuzumab, as well as Pertuzumab, on HER2-modulated EGF–EGFR interaction. The results demonstrated that EGF bound to EGFR more stably in the cells co-expressing EGFR and HER2, and the binding enhancement in the presence of HER2 was inhibited by either Trastuzumab or Pertuzumab. Trastuzumab is expected to exert a similar inhibition effect on HER2/EGFR dimerization as Pertuzumab, although it does not bind directly to the dimerization arm of HER2.From the Clinical EditorLiving-cell single-molecule force spectroscopy (SMFS) combined by Atomic Force Microscopy (AFM) was used by this team of scientists to investigate the effect of two monoclonal antibodies used in cancer therapy, Trastuzumab and Pertuzumab, on HER2-modulated EGF–EGFR interaction, demonstrating the utility of this technique in characterizing the effects of protein-based therapeutics on membrane receptors.The effect of the HER2 antibodies on HER2 modulated EGF–EGFR interaction was studied by single molecular force spectroscopy using the EGF modified AFM tip and cells expressing GFP tagged EGFR and RFP tagged HER2. It is demonstrated that binding force of EGF–EGFR was higher in the cells co-expressing HER2 and the binding enhancement after recruiting HER2 was inhibited by either Trastuzumab or Pertuzumab. Results from the dynamic force spectroscopy and the derived dissociation kinetic parameters all indicated that Trastuzumab exerted a similar inhibition effect on HER2/EGFR dimerization, although it does not bind directly to HER2 dimerization arm.

Nucleic acid aptamers are single-stranded RNA or DNA molecules that bind to their targets with high specificity and affinity. Although their biomedical applications have been booming, it is still debatable whether an aptamer recognizes its target through “induced fit” or “conformational selection”, a central question in molecular recognition. To address this question, an ATP-binding DNA aptamer was selected as a model system and the conformational properties of this aptamer with and without the presence of ATP were investigated by single-pair Förster resonance energy transfer (spFRET) spectroscopy. The single-molecule results indicate that the aptamer can fold into a double-stranded-like structure, similar to the ligand-bound conformation, even without the presence of ATP. The folded structure is thermally stable at high salt concentrations and becomes rather dynamic at low salt concentrations. Although in the latter condition, the aptamer prefers unfolded structures, it can occasionally migrate to the folded conformation for a short time before being unfolded again. The binding of ATP to the aptamer stabilizes the folded structure, which populates the ligand-bound state of the aptamer, thus shifting the conformational equilibrium. Collectively, our data support that the ATP-binding DNA aptamer recognizes ATP ligand through “conformational selection”.

Cigarette smoking is a well-known risk factor for cardiovascular disease. Smoking can cause vascular endothelial dysfunction and consequently trigger haemostatic activation and thrombosis. However, the mechanism of how smoking promotes thrombosis is not fully understood. Thrombosis is associated with the imbalance of the coagulant system due to endothelial dysfunction. As a vital anticoagulation cofactor, thrombomodulin (TM) located on the endothelial cell surface is able to regulate intravascular coagulation by binding to thrombin, and the binding results in thrombosis inhibition. This work focused on the effects of cigarette smoke extract (CSE) on TM-thrombin binding by atomic force microscopy (AFM) based single-molecule force spectroscopy. The results from both in vitro and live-cell experiments indicated that CSE could notably reduce the binding probability of TM and thrombin. This study provided a new approach and new evidence for studying the mechanism of thrombosis triggered by cigarette smoking.

Aptamers are a new class of molecular probes for protein recognition, detection, and inhibition. Multivalent aptamer–protein binding through aptamer assembly has been currently developed as an effective way to achieve higher protein affinity and selectivity. In this study, the specific interaction between bivalent aptamer Bi-8S and thrombin has been measured directly and quantitatively by atomic force microscopy to investigate the unbinding dynamics and dissociation energy landscape of the multivalent interaction. Bivalent aptamer Bi-8S contains thrombin’s two aptamers, 15apt and 27apt, which are linked by eight spacer phosphoramidites. The results revealed the sequential dissociation of the two aptamers. Moreover, the dynamic force spectroscopy data revealed that the 27apt’s binding to the thrombin remains largely unaffected by the eight-spacer phosphoramidites within Bi-8S. In contrast, the eight-spacer phosphoramidites stabilized the 15apt–thrombin binding.

Some researchers have questioned whether synthetic aptamers bind as robustly as natural antibodies. To address this issue, we used single-molecule atomic force microscopy to measure the rupture force between a protein and both its aptamer and its antibody. The rupture force on live cell membranes between the aptamer and protein was 46 ± 26 pN; the force with the antibody was 68 ± 33 pN, we conclude that the binding forces are about equal.

A water-soluble covalently linked C60-porphyrin compound–C60Por is reported to show enhanced DNA-cleaving activity. The highly enhanced DNA-cleaving activity is expected to be ascribed to the high water-solubility and affinity of C60Por to DNA, as well as to the accessibility to the charge-separated states C60˙−–Por˙+ formed under radiation.

With single-molecule fluorescence imaging and single-molecule force measurement, we have found that the natural compound Naringenin exerts an inhibition effect on TGF-β ligand–receptor interaction, the initial step of TGF-β signaling.

A novel fluorescence aptasensor based on DNA charge transport for sensitive protein detection has been developed. A 15nt DNA aptamer against thrombin was used as a model system. The aptamer was integrated into a double strand DNA (dsDNA) that was labeled with a hole injector, naphthalimide (NI), and a fluorophore, Alexa532, at its two ends. After irradiation by UV light, the fluorescence of Alexa532 was bleached due to the oxidization of Alexa532 by the positive charge transported from naphthalimide through the dsDNA. In the presence of thrombin, the binding of thrombin to the aptamer resulted in the unwinding of the dsDNA into ssDNA, which led to the blocking of charge transfer and the strong fluorescence emission of Alexa532. By monitoring the fluorescence signal change, we were able to detect thrombin in homogeneous solutions with high selectivity and high sensitivity down to 1.2 pM. Moreover, as DNA charge transfer is resistant to interferences from biological contexts, the aptasensor can be used directly in undiluted serum with similar sensitivity as that in buffer. This new sensing strategy is expected to promote the exploitation of aptamer-based biosensors for protein assays in complex biological matrixes.

Influences of substrate stiffness on mechanical properties of cardiac myocytes and fibroblasts were investigated by cell elasticity measurement with atomic force microscopy. The cells were cultured on collagen-coated polyacrylamide substrates with gradient rigidity. While cardiac myocytes showed no evident change in cell elasticity on different substrates, cardiac fibroblasts displayed the non-monotonic dependence on substrate stiffness with a maximum elastic modulus. Moreover, the elasticity change of cardiac fibroblasts with substrates stiffness was found to be regulated by actin filaments. Study of the effect of substrate stiffness on cell elasticity for different cardiac cells provides new information for the better understanding of cardiac physiology and pathology.

Fluorescence imaging of single molecules is becoming a powerful tool to examine biological processes at the molecular level. Using total internal reflection fluorescence microscopy (TIRFM), it has been possible to study the dynamic behavior of single molecules on living cell membranes. Herein, we briefly review the application of TIRFM-based single-molecule imaging in studies of membrane receptors involved in signal transduction. Furthermore, we discuss several examples of our own research on growth factor receptors, including TGF-β receptors, HER2, and EGFR, and speculate possible applications of this technique to investigate other cellular events occurring on or near the plasma-membrane.

Molecular medicine is an emerging field focused on understanding the molecular basis of diseases and translating this information into strategies for diagnosis and therapy. This approach could lead to personalized medical treatments. Currently, our ability to understand human diseases at the molecular level is limited by the lack of molecular tools to identify and characterize the distinct molecular features of the disease state, especially for diseases such as cancer. Among the new tools being developed by researchers including chemists, engineers, and other scientists is a new class of nucleic acid probes called aptamers, which are ssDNA/RNA molecules selected to target a wide range of molecules and even cells. In this Account, we will focus on the use of aptamers, generated from cell-based selections, as a novel molecular tool for cancer research. Cancers originate from mutations of human genes. These genetic alterations result in molecular changes to diseased cells, which, in turn, lead to changes in cell morphology and physiology. For decades, clinicians have diagnosed cancers primarily based on the morphology of tumor cells or tissues. However, this method does not always give an accurate diagnosis and does not allow clinicians to effectively assess the complex molecular alterations that are predictive of cancer progression. As genomics and proteomics do not yet allow a full access to this molecular knowledge, aptamer probes represent one effective and practical avenue toward this goal. One special feature of aptamers is that we can isolate them by selection against cancer cells without prior knowledge of the number and arrangement of proteins on the cellular surface. These probes can identify molecular differences between normal and tumor cells and can discriminate among tumor cells of different classifications, at different disease stages, or from different patients. This Account summarizes our recent efforts to develop aptamers through cell-SELEX for the study of cancer and apply those aptamers in cancer diagnosis and therapy. We first discuss how we select aptamers against live cancer cells. We then describe uses of these aptamers. Aptamers can serve as agents for molecular profiling of specific cancer types. They can also be used to modify therapeutic reagents to develop targeted cancer therapies. Aptamers are also aiding the discovery of new cancer biomarkers through the recognition of membrane protein targets. Importantly, we demonstrate how molecular assemblies can integrate the properties of aptamers and, for example, nanoparticles or microfluidic devices, to improve cancer cell enrichment, detection and therapy.

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.

The interaction between the cell adhesion molecule CD11b and its ligand ICAM-1 plays an important role in inflammatory responses in the disease of atherosclerosis. Atorvastatin is a commonly prescribed statin drug which has been considered as one of the most potent therapeutic agents for atherosclerosis due to its lipid-lowering effect. Recently, there is a growing body of evidence that atorvastatin has anti-inflammatory effect. We have applied the advanced method of live-cell single-molecule force spectroscopy to investigate the effect of atorvastatin on adhesion force between ICAM-1 and CD11b. Our result showed that single-molecule binding force of ICAM-1 and CD11b detected by AFM in the living cells was about 40 pN, and atorvastatin did not affect this force by blocking ICAM-1 or CD11b. This was different from the ICAM-1 monoclonal antibody, which could directly reduce the binding force of ICAM-1 and CD11b. Flow cytometry results revealed that atorvastatin pretreatment decreased the ICAM-1 expression in TNF-α activated HUVECs, which may contribute to its anti-inflammatory effect. The study provides a new approach to study anti-inflammatory mechanism for clinic drugs.

The effect of water-soluble fullerene C70(C(COOH)2)4−8 on plant growth was investigated, using the transgenic seedling lines expressing fluorescent makers. The retarded roots with shortened length and loss of root gravitropism were observed for seedlings grown in the fullerene-containing medium. Fluorescence imaging revealed the abnormalities of root tips in hormone distribution, cell division, microtubule organization, and mitochondrial activity. The study of the inhibitory effects at the cellular level provides new information on the phytotoxicity mechanism of fullerene.Keywords: fluorescence imaging; fullerenes; green fluorescence protein; phytotoxicity; transgenic plants

We have investigated the capability of single-walled carbon nanotubes (SWNTs) to penetrate the cell wall and cell membrane of intact plant cells. Confocal fluorescence images revealed the cellular uptake of both SWNT/fluorescein isothiocyanate and SWNT/DNA conjugates, demonstrating that SWNTs also hold great promise as nanotransporters for walled plant cells. Moreover, the result suggested that SWNTs could deliver different cargoes into different plant cell organelles.

In this work, we have developed new aptamer probes for non-small cell lung cancer (NSCLC) by directing the aptamer selection process against the living cells of adenocarcinoma, the most common subtype of NSCLC. A panel of single-stranded DNA (ssDNA) aptamers were generated and evaluated for adenocarcinoma cell recognition. The aptamers bound to the adenocarcinoma cells with dissociation constants in the nanomolar range and the binding of the selected aptamers to the adenocarcinoma cells were significantly stronger than the other cancerous lung cells as well as other types of cancer cells. Moreover, the application of the aptamers to the clinical tissue section samples showed the differentiation of adenocarcinoma from normal lung tissue and other subtypes of lung cancer. The aptamers are expected to be new molecular probes for the investigation of the molecular bases of different NSCLC subtypes and their biological heterogeneity, which is valuable for advancing NSCLC diagnosis and treatment.

Transforming growth factor-β (TGF-β) elicits its signals through two transmembrane serine/threonine kinase receptors, type II (TβRII) and type I receptors. It is generally believed that the initial receptor dimerization is an essential event for receptor activation. However, previous studies suggested that TGF-β signals by binding to the preexisting TβRII homodimer. Here, using single molecule microscopy to image green fluorescent protein (GFP)-labeled TβRII on the living cell surface, we demonstrated that the receptor could exist as monomers at the low expression level in resting cells and dimerize upon TGF-β stimulation. This work reveals a model in which the activation of serine-threonine kinase receptors is also accomplished via dimerization of monomers, suggesting that receptor dimerization is a general mechanism for ligand-induced receptor activation.

Water-soluble gadofullerides exhibited high efficiency as magnetic resonance imaging (MRI) contrast agents. In this paper, we report the conjugation of the newly synthesized gadofulleride, Gd@C82O6(OH)16(NHCH2CH2COOH)8, with the antibody of green fluorescence protein (anti-GFP), as a model for “tumor targeted” imaging agents based on endohedral metallofullerenes. In this model system, the activity of the anti-GFP conjugate can be conveniently detected by green fluorescence protein (GFP), leading to in vitro experiments more direct and facile than those of tumor antibodies. Objective-type total internal reflection fluorescence microscopy revealed that each gadofulleride aggregate conjugated on average five anti-GFPs, and the activity of anti-GFPs was preserved after conjugation. In addition, the gadofulleride/antibody conjugate exhibited higher water proton relaxivity (12.0 mM−1 s−1) than the parent gadofulleride aggregate (8.1 mM−1 s−1) in phosphate buffered saline at 0.35 T, as also confirmed by T1-weighted images of phantoms. These observations clearly indicate that the synthesized gadofulleride/antibody conjugate not only has targeting potential, but also exhibits higher efficiency as an MRI contrast agent.

Conjugation of the cell-penetrating peptide derived from the human immunodeficiency virus-1 transactivator protein (TAT) to semiconductor quantum dots (QDs) is an effective way to enhance transmembrane delivery of QDs for intracellular and molecular imaging. In this work, the internalization pathway of TAT-QDs was studied systematically in living cells. Cellular uptake of TAT-QDs, under different endocytosis-inhibiting conditions, was compared by fluorescence imaging and flow cytometry. The results suggest TAT-QDs internalize through lipid-raft-dependent macropinocytosis, which is different from that of FITC-labeled TAT. They also provide new information for better understanding of the TAT-mediated cell uptake mechanism.

The adsorption of plasma protein fibrinogen on the self-assembled monolayers (SAMs) of n-hexadecyl mercaptan and citrate was investigated with atomic force microscopy (AFM). On the basis of the preparation of these two flat SAMs on Au(1 1 1), high-resolution AFM images of bovine fibrinogen were obtained with different protein concentrations. The results indicated that the surface chemical composition significantly affected the adsorption behavior of fibrinogen. Since fibrinogen plays a key role in the regulation of both haemostasis and thrombosis, high-resolution AFM imaging on SAMs is expected to be an effective approach to study the haemocompatibility of materials with different surface chemistry.

Atomic force microscopy (AFM) and scanning tunneling microscopy (STM) have been employed in situ and ex situ to directly study the aggregation of β-amyloid(1-42) (Aβ42) peptide on hydrophobic graphite.From in situ AFM images, Aβ42 peptides were seen to aggregate into the sheets that preferred to three orientations with characteristic 3-fold symmetry (Proc. Natl. Acad. Sci. USA 96 (1999) 3688). The sheets were formed by parallel narrow lines with a height of 0.8–1.0 nm and a width of 12–14 nm. The narrow lines looked like beaded chains and have a right-handed axial periodicity.The high-resolution ex situ AFM and STM images showed that some fibrils of β-amyloid had a characteristic domain texture, indicating they were formed through the association of protofibrils and monomers. The fibril containing lateral associated filaments that exhibited right-handed twist was clearly observed in the STM image.These results provide important clues to study the detailed structure of β-amyloid aggregates and the mechanism of the Aβ fibrils formation on hydrophobic surface.

Single-molecule fluorescence imaging with living cells offers a new approach to visualize and monitor individual proteins during their cellular activities. It facilities the study of cell signaling proteins whose structures and interactions are highly temporal and spatial regulated. In this review, we will mainly present our recent work on probing the dynamics of two types of important transmembrane growth factor receptors, transforming growth factor β receptors and epidermal growth factor receptors, by single-molecule fluorescence microscopy. This includes the characterization of receptor stoichiometry, monomer–dimer interconversion kinetics, effects of microenvironment on receptor membrane diffusion, and intracellular transportation under different signaling conditions. Related studies on these receptors from other groups, as well as the method developments, will be also discussed. Single-molecule study helps to achieve a better understanding of the molecular mechanism of receptor activation, endocytosis and other molecular events in transmembrane signaling.

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 metabolite 4-(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.

Single-particle tracking photoactivated localization microscopy (sptPALM) 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-β type II receptor (TβRII) at high expression level using both traditional single-particle tracking (SPT) and sptPALM. The ligand-induced aggregation of TβRII oligomers was further indicated by sptPALM. Due to the capacity of distinguishing and tracking single molecules within diffraction limit, sptPALM outperforms traditional SPT by providing more accurate biophysical information.

The stoichiometry of the β2-adrenergic receptor (β2AR) was determined using single-molecule fluorescence imaging in living cells. The results showed that β2AR mainly existed as monomers under physiological conditions and exhibited β-arrestin-dependent dimerization upon stimulation with the pharmacological biased ligand carvedilol. The association of β2AR dimerization with biased signalling is revealed.

We achieved single-molecule imaging and tracking of the transforming growth factor type II receptor (TβRII) that was labeled by an organic dye via a genetically encoded unnatural amino acid (UAA) and the copper-free click chemistry. The stoichiometry, mobility and dimerization kinetics of individual TβRII molecules were determined.

With single-molecule fluorescence imaging and single-molecule force measurement, we have found that the natural compound Naringenin exerts an inhibition effect on TGF-β ligand–receptor interaction, the initial step of TGF-β signaling.

Influences of substrate stiffness on mechanical properties of cardiac myocytes and fibroblasts were investigated by cell elasticity measurement with atomic force microscopy. The cells were cultured on collagen-coated polyacrylamide substrates with gradient rigidity. While cardiac myocytes showed no evident change in cell elasticity on different substrates, cardiac fibroblasts displayed the non-monotonic dependence on substrate stiffness with a maximum elastic modulus. Moreover, the elasticity change of cardiac fibroblasts with substrates stiffness was found to be regulated by actin filaments. Study of the effect of substrate stiffness on cell elasticity for different cardiac cells provides new information for the better understanding of cardiac physiology and pathology.

A water-soluble covalently linked C60-porphyrin compound–C60Por is reported to show enhanced DNA-cleaving activity. The highly enhanced DNA-cleaving activity is expected to be ascribed to the high water-solubility and affinity of C60Por to DNA, as well as to the accessibility to the charge-separated states C60˙−–Por˙+ formed under radiation.