Biomolecule monitoring and nanobiology

Brief Introduction

Real time detection and characterization of biomolecules in live cells enables the investigation of their physicochemical parameters and biological behaviors, which will greatly enrich our knowledge on the molecular mechanisms of cellular processes. This research aims at the development of ultrasensitive methods for the real time biomolecule monitoring at the single molecule level in live cells, to reveal the role and function of some key proteins involved in the signal transduction of major pathways. The research mainly includes following activities:

1.         Developing single-molecule detection techniques, which includes single-molecule fluorescence imaging, super-resolution fluorescence imaging, AFM based single-molecule force spectroscopy and their integrated methods.

2.         Imaging and tracking of cell signal transduction proteins to study the dynamic process of their activation and transduction at the single molecule level.

3.         Single molecule force spectrum and dynamic force spectra measurements for the study of ligand-receptor interaction, and for the investigation of the stability of signaling complexes as well as the mechanism of drugs.

4.         Investigating nanoparticle-cell interaction and biological effect of nanomaterials based on the real time detection techniques.

5.         Developing highly sensitive protein sensors based on the aptamers as novel molecular probes.

Representative Research Work

Single-molecule imaging reveals transforming growthfactor-β-induced type II receptordimerization

Wei Zhang, Yaxin Jiang, Qiang Wang, Xinyong Ma, Zeyu Xiao, Wei Zuo, Xiaohong Fang*, and Ye-Gang Chen*. PNAS, 2009, 106, 15679-15683.

Transforming growth factor-β(TGF-β) elicits its signals throughtwo transmembrane serine/threonine kinase receptors, type II(TβRII) and type I receptors. It is generally believed that the initialreceptor dimerization is an essential event for receptor activation.However, previous studies suggested that TGF-βsignals by bindingto the preexisting TβRII homodimer. Here, using single moleculemicroscopy to image green fluorescent protein (GFP)-labeled TβRIIon the living cell surface, we demonstrated that the receptor couldexist as monomers at the low expression level in resting cells and dimerize upon TGF-β stimulation. This work reveals a model inwhich the activation of serine-threonine kinase receptors is alsoaccomplished via dimerization of monomers, suggesting that receptordimerization is a general mechanism for ligand-inducedreceptor activation.

Analysis of bleaching steps of single TβRII-GFP molecules imaged withthe fixed HeLa cells.

A single-molecule study of the inhibition effect of Naringenin ontransforming growth factor-βligand-receptor binding

Yong Yang, YongchunXu, Tie Xia, Fangjin Chen, Chunling Zhang, Wei Liang, Luhua Lai and Xiaohong Fang*. Chem Comm., 2011, 47, 5440-5442.

With single-molecule fluorescence imaging and single-moleculeforce measurement, we have found that the natural compound Naringenin exerts an inhibition effect on TGF-b ligand-receptorinteraction, the initial step of TGF-βsignaling.

Frequency of one- and two-step bleaching events under various conditions.

Study of inhibition effect of herceptin on interaction betweenheregulin and ErbB receptors HER3/HER2 by single-moleculeforce spectroscopy

Xiaoli Shi, Li Xu, Junping Yu, Xiaohong Fang*. Exp Cell Res. 2009, 315, 2847-2855.

Herceptin is a monoclonal antibody against HER2, which is a member of the epidermal growth factor receptor (ErbB) family and is overexpressed in many cancers. In this work, we have applied single-molecule force spectroscopy to study the effect of Herceptin on HER2 modulated ligand-receptor interaction for ErbB signaling in living cells. Heregulin beta1 (HRG), the specific ligand of HER3, was used for HER2 activation as HER3 is the preferable dimerization partner of HER2 and HER3/HER2 is the most representative heterodimer found in cancer. Our results demonstrated a more stable binding of HRG to the cells co-expressing HER3 and HER2 than those expressing HER3 alone. Moreover, the binding force of Herceptin and HER2 is as strong as that of HRG and HER3/HER2. With the addition of Herceptin, the binding strength of HRG to the cells co-expressing HER3 and HER2 decreased. The presence of Herceptin changed the dynamic force spectrum of HRG-HER3/HER2 to that similar to HRG-HER3. Therefore, the enhancement in HRG-HER3 binding after recruiting HER2 was inhibited by Herceptin. The method offers a new approach to study the molecular mechanism of Herceptin anti-cancer effect.

Forces measured at differentloading rates for HRG-modified tips

Long-distance intercellular connectivity between cardiomyocytes and cardiofibroblasts mediatedby membrane nanotubes

Kangmin He, Xiaoli Shi, Xuejie Zhang, Song Dang, Xiaowei Ma, Fei Liu, Ming Xu, ZhizhenLv, Dong Han, Xiaohong Fang*, and Youyi Zhang*. Cardiovascular Res., 2011, 92, 39-47.

By membrane dye staining, we observed long, thin membrane nanotubular structures containing actin and microtubulesthat connected neonatal rat ventricular CMs and FBs. By single-particle tracking, we observed vehicles movingbetween CMs and FBs within the membrane nanotubes. By dual colour staining, confocal imaging and flow cytometry, we observed mitochondria exchange between CMs and FBs in a coculture system. By combined atomic forcemicroscopy (AFM) and confocal microscopy, we observed calcium signal propagation from AFM-stimulated CM(or FB) to unstimulated FB (or CM) via membrane nanotubes. By membrane and cytoskeleton staining, we observed similar nanotubular structures in adult mouse heart tissue, which suggests their physiological relevance.

Calcium signal propagation between two membrane nanotube-connected cells but not the adjacent unconnected cell

Carbon nanotubes as molecular transporters for walled plant cells

Qiaoling Liu, Bo Chen, Qinli Wang, Xiaoli Shi, Zeyu Xiao, Jinxin Lin, and Xiaohong Fang*. Nano Lett., 2009,9, 1007-1010.

We have investigated the capability of single-walled carbon nanotubes (SWNTs) to penetrate the cell wall and cell membrane of intact plantcells. 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 SWNTscould deliver different cargoes into different plant cell organelles.

Confocal microscopy images of BY-2 incubated withSWNT

Single-Molecule Detection of Proteins Using Aptamer Functionalized Molecular Electronic Devices

Song Liu, Xinyue Zhang, WangxiLuo, Zhenxing Wang, XuefengGuo*, Michael L. Steigerwald, and Xiaohong Fang*. Angew. Chem. Int. Ed. 2011, 50, 2496 –2502

We detail here a practical yet reliableapproach in which molecular electronics are interfaced withbiological systems to realize the label-free, real-time, reversible electrical detection of DNA and/or protein activities.This method uses functional single-molecule devices toachieve ultrahigh selectivity and sensitivity.

Schematic representation of the sensing mechanism showing how single-molecule devicescan detect proteins at the single-molecule level