Caspase-2 is important for the mitochondrial apoptotic pathway, however, the mechanism by which caspase-2 executes apoptosis remains obscure. We carry out the first measurements of the dynamics of caspase-2 activation in a single living cell by a FRET (fluorescence resonance energy transfer) probe. Two FRET probes are constructed that each encoded a CRS (caspase-2 or caspase-3 recognition site) fused with a cyan fluorescent protein (CFP) and a red fluorescent protein (DsRed) (CFP-CRS-DsRed). Using these probes, we found that during cisplatin-induced apoptosis, caspase-2 activation occurred more slowly than did activation of caspase-3; additionally, caspase-2 activation was initiated much earlier than that of caspase-3.
Caspases-3 is a kind of cysteine proteases and plays an important role in cell apoptosis. It has been reported that caspase-3 activation can be real-time detected in living cells by fluorescence resonance energy transfer (FRET) between an enhanced cyan fluorescent protein and enhanced yellow fluorescent protein. However, the large spectral overlap between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) emission and the highly sensitivity to pH of YFP restricted their detecting sensitivity and reliability. CFP and red fluorescent protein (DsRed) possess superb wavelength separation of donor and acceptor emission spectra and DsRed was insensitive to pH, so the FRET probe composed of CFP and DsRed would be more suitable for imaging caspase-3 activation than the FRET probe composed of CFP and YFP. We constructed a vector that encoded CRS (caspase-3 recognition site) fused with CFP and DsRed (CFP-CRS-DsRed). In CFP-CRS-DsRed expressing tumor cells, FRET from CFP to DsRed could be detected. In the Clinical applications of cancer chemotherapy, cisplatin is one of the most broadly used drugs. It was already confirmed that caspase-3 was activated in HeLa cell treated by cisplatin. When the cells were stimulated with cisplatin, we found that the FRET efficient was remarkably decreased and then disappeared. It indicated that actived caspase-3 cleaved the CFP-CRS-DsRed fusion protein at CRS site. Thus, the FRET probe of CFP-CRS-DsRed could sensitively and reliably monitor caspase-3 activation in living cell. This probe will be highly useful for rapid-screening potential drugs that may target the apoptotic process and for imaging tumors in vivo.
Degradation of the extracellular matrix by Matrix metalloproteinases (MMPs) not only enhances tumor invasion, but also affects tumor cell behaviour and leads to cancer progression. To monitor gelatinases (contain MMP2 and MMP9) activity in living cells, we constructed a vector that encoded a gelatinases recognition site (GRS) between citrine (mutation of EYFP Q69M) in N terminal and ECFP in C terminal. Because Gelatinases are secretory proteins and act outside of cell, an expressing vector displayed the fusion protein on cellular surface was used for this FRET gene probe. On expression of YFP-GRS-ECFP in MCF-7 cells that expressed no gelatinases, we were able to observe the efficient transfer of energy from excited ECFP to YFP within the YFP-GRS-ECFP molecule. However, the fusion protein YFP-GRS-ECFP was expressed in MDA-MB 453s cell line with high secretory gelatinases, so YFP-GRS-ECFP was cleaved by gelatinases, no such transfer of energy was detected and fluorescence signal disappeared in YFP channel since YFP protein was cut down. Moreover, Doxycycline, a MMP inhibitor, could make FRET signal increase and fluorescence signal appeared in YFP channel. Thus, the FRET probe YFP-GRS-ECFP can sensitively and reliably monitor gelatinases activation in living cells and can be used for screening MMP inhibitors.
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.
A large number of biological information has been available from genome sequencing and bioinformatics. To further understand the qualities of the biological networks (such as metabolic network) in the complex biological system, representations of integrated function in silico have been widely investigated, and various modeling approaches have been designed, most of which are based on detailed kinetic information except flux balance analysis (FBA). FBA, just based on stoichimetrical information of reactions, is a suitable method for the study of metabolic pathways, and it analyzes the behaviors of the network from the viewpoint of the whole system. Herein, this modeling approach has been utilized to reconstruct the mitochondrial metabolic network to integrate and analyze its capability of producing energy. Besides, extreme pathways analysis (EPA) and shadow prices analysis have also been integrated to study the interior characters of the network. Our modeling results have indicated for the first time that the covalent regulative property of pyruvate dehydrogenase is restrained by the feedback of acetyl-CoA. Combined with the biological experiments, these simulations in silico could be pretty useful for the further understanding of functions and characters of the biological network as a complex system.