Jun Chu

A novel far-red bimolecular fluorescence complementation system that allows for efficient visualization of protein interactions under physiological conditions

Fluorescent protein (FP) has enabled the analysis of biomolecular interactions in living cells, and bimolecular fluorescence complementation (BiFC) represents one of the newly developed imaging technologies to directly visualize protein-protein interactions in living cells. Although 10 different FPs that cover a broad range of spectra have been demonstrated to support BiFC, only Cerulean (cyan FP variant), Citrine and Venus (yellow FP variants)-based BiFC systems can be used under 37 degrees C physiological temperature. The sensitivity of two mRFP-based red BiFC systems to higher temperatures (i.e., 37 degrees C) limits their applications in most mammalian cell-based studies. Here we report that mLumin, a newly isolated far-red fluorescent protein variant of mKate with an emission maximum of 621 nm, enables BiFC analysis of protein-protein interactions at 37 degrees C in living mammalian cells. Furthermore, the combination of mLumin with Cerulean- and Venus-based BiFC systems allows for simultaneous visualization of three pairs of protein-protein interactions in the same cell. The mLumin-based BiFC system will facilitate simultaneous visualization of multiple protein-protein interactions in living cells and offer the potential to visualize protein-protein interactions in living animals.

Visualization of β-secretase cleavage in living cells using a genetically encoded surface-displayed FRET probe

The human beta-secretase, BACE, plays a key role in the generation of pathogenic amyloid beta-peptide (Abeta) in Alzheimers disease and has been identified as an ideal target for therapy. Previous studies reported the monitoring of BACE activity in vitro utilizing chemical synthesized sensors. Here we describe the first genetically encoded FRET probe that can detect BACE activity in vivo. The FRET probe was constructed with the BACE substrate site (BSS) and two mutated green fluorescent proteins. In living cell, the FRET probe was directed to the secretory pathway and anchored on the cell surface to measure BACE enzymatic activity. The results show that the FRET probe can be cleaved by BACE effectively in vivo, suggesting that the probe can be used for real-time monitoring of BACE activity. This assay provides a novel platform for BACE inhibitor screening in vivo.

A new red bimolecular fluorescence complementation based on TagRFP

Fluorescent proteins have become extremely popular tools for in vivo imaging as well as the study of localization, motility and interaction of proteins in living cells. Bimolecular fluorescence complementation (BiFC) analysis based on fluorescent proteins enables direct and high throughput visualization of protein-protein interactions in living cells. Two red Bimolecular Fluorescent Complementation (BiFC) systems based on mRFP variants have been reported. However, some physical-chemical characteristics of mRFP limited their applications, such as low pH-stability, relative low brightness and low maturation rate. We have developed a new red BiFC system based on TagRFP, a novel monomeric red fluorescent protein with high brightness, complete chromophore maturation, prolonged fluorescence lifetime and high pH-stability. In this study, bFos and bJun were used as the positive protein-protein interaction pair, a mutant of bFos (bFos) and bJun were used as the negative protein-protein interaction pair. bFos/ΔbFos was fused to N-terminal fragment of TagRFP, and bJun was fused to C-terminal fragment of TagRFP. The BiFC systems based on TagRFP was confirmed in living mammalian cells. Furthermore, the BiFC based on TagRFP allow analyzing multi-protein interactions when combined with other BiFC systems. Thus, the BiFC based on TagRFP is very useful for investigating the complicated and significant molecular mechanisms of multi-protein complex in living cells.

Simultaneous imaging of two initiator caspases during cisplatin-induced HeLa apoptosis

Caspase-2 is an initiating caspase required for stress-induced apoptosis in various human cancer cells. Activation of caspase-9, a key event in stress-mediated apoptosis, also has been shown to be an initiator caspase. However, the timing or activation sequence of these initiator caspases, which trigger apoptotic pathway, is unclear. Here we report caspase-2 and caspase-9 dynamics during cisplatin-induced HeLa apoptosis using Double Fluorescence Resonance Energy Transfer (FRET) technique. Two FRET probes were constructed that each encoded a CRS (caspase-2 or caspase-9 recognition Site) fused with a cyan/yellow fluorescent protein (CFP/YFP) and a red fluorescent protein (DsRed) (CFP/YFP-CRS-DsRed). By using two probes, CFP-C2-DsRed and YFP-C9-DsRed, we carried out simultaneous double-FRET analysis and revealed that activation of caspase-2 had the same time course with caspase-9. These data suggest parallel activation of initiator caspase-2 and caspase-9 in cisplatin-induced cell death.

A feasible method for comparing the power dependent photostability of fluorescent proteins

A feasible method of combining the concept of fluorescence half-life and the power dependent photobleaching rate for characterizing the practical photostability of fluorescent proteins (FPs) was introduced. Furthermore, by using a fluorescent photostability standard, a relative comparison of the photostabilty of FPs from different research groups was proposed, which would be of great benefit for developing novel FPs with optimized emission wavelength, better brightness, and improved photostability. We used rhodamine B as an example to verify this method and evaluate the practical photostability of a far-red FP, mKate-S158C. Experimental results indicated good potential of this method for further study.

In vivo optical imaging of bacterial infection and antibiotic response in intact nude mice

We describe imaging the luminance of red fluorescent protein (DsRed2)-expressing bacteria from outside intact infected animals. This simple, nonintrusive technique can show in great detail the temporal behavior of the infectious process. Fluorescence stereo microscope, laser and cooled CCD are expensive to many institutes, we set up an inexpensive compact whole-body fluorescent imaging tool, which consisted of a digital camera, fluorescence filters and a mercury 50-W lamp power supply as excitation light source. The bacteria, expressing the DsRed2, are sufficiently bright as to be clearly visible from outside the infected animal and recorded with simple equipment. Introduced bacteria were observed in the abdomen. Instantaneous real-time images of the infectious process were acquired by using a digital camera by simply illuminating nude mice with mercury lamp. The development of infection over 48 hours and its regression after kanamycin treatment were visualized by whole-body imaging. The DsRed2 was excited directly by mercury lamp with EF500/50 nm band-pass filter and fluorescence was recorded by digital camera with CB580 nm long-pass filter. By this easy operation tool, the authors imaged, in real time, fluorescent tumors growing in live mice. The imaging system is external and noninvasive. For one year our experiments suggested the imaging scheme was feasible, which affords a powerful approach to visualizing the infection process.

Five-color fluorescent imaging in living tumor cells

The fluorescent probes based on fluorescent proteins (FP) have been widely used to investigate the molecules of interest in living cells. It is well-known that the molecular events in the living cells are very complicate and all of the cell activities are involved by multi-molecular interaction. With the development of novel fluorescent protein mutants and imaging technology, the molecular signal in living cells could be detected accurately. In this study, with the appropriate targeting signals, the fluorescent proteins were localized to plasma membrane (Rac1-mCerulean), Golgi membrane (EYFP-go), ER membrane (RFP2-er), mitochondrial membrane (RFP1-mt). Cultured Hela cells were cotransfected with these four plasmids, and 36 h later, labeled with Hoechst33258 which located in the nucleus of a living cell. Using a confocal microscopy, with 405 nm, 458 nm and 514 nm laser lines employed respectively, a five-color fluorescent image was obtained in which five subcellular structures were clearly shown in living cells. The technique of multi-color imaging in a single cell provides a powerful tool to simultaneously study the multi-molecular events in living cells.

Visualization of EGFR and Grb2 interaction using bimolecular fluorescence complementation

The dimerization of epidermal growth factor receptor (EGFR) and its endocytic transport are important in regulating signal transduction. In the present study, we applied the strategy of Bimolecular Fluorescence Complementation (BiFC), EGFR homodimer and hetrodimer of EGFR and its partner Grb2 in cells were visualized. This strategy takes advantage of the ability of two nonfluorescent fragments of Venus fluorescent protein to form a fluorescent signal when fused to the amino termini of EGFR and Grb2. Since EGFR is a large protein contains more than 1800 amido acids, proper fold of the fusion protein is essential for the BiFC assay of EGFR and its partners. Our results indicate that BiFC is a suitable application for research of EGFR interaction with other proteins.

Compact whole-body fluorescent imaging of nude mice bearing EGFP expressing tumor

Issue of tumor has been a hotspot of current medicine. It is important for tumor research to detect tumors bearing in animal models easily, fast, repetitively and noninvasivly. Many researchers have paid their increasing interests on the detecting. Some contrast agents, such as green fluorescent protein (GFP) and Discosoma red fluorescent protein (Dsred) were applied to enhance image quality. Three main kinds of imaging scheme were adopted to visualize fluorescent protein expressing tumors in vivo. These schemes based on fluorescence stereo microscope, cooled charge-coupled-device (CCD) or camera as imaging set, and laser or mercury lamp as excitation light source. Fluorescence stereo microscope, laser and cooled CCD are expensive to many institutes. The authors set up an inexpensive compact whole-body fluorescent imaging tool, which consisted of a Kodak digital camera (model DC290), fluorescence filters(B and G2;HB Optical, Shenyang, Liaoning, P.R. China) and a mercury 50-W lamp power supply (U-LH50HG;Olympus Optical, Japan) as excitation light source. The EGFP was excited directly by mercury lamp with D455/70 nm band-pass filter and fluorescence was recorded by digital camera with 520nm long-pass filter. By this easy operation tool, the authors imaged, in real time, fluorescent tumors growing in live mice. The imaging system is external and noninvasive. For half a year our experiments suggested the imaging scheme was feasible. Whole-body fluorescence optical imaging for fluorescent expressing tumors in nude mouse is an ideal tool for antitumor, antimetastatic, and antiangiogenesis drug screening.