Fu-Jen Kao

Differentiation of apoptosis from necrosis by dynamic changes of reduced nicotinamide adenine dinucleotide fluorescence lifetime in live cells

Direct monitoring of cell death (i.e., apoptosis and necrosis) during or shortly after treatment is desirable in all cancer therapies to determine the outcome. Further differentiation of apoptosis from necrosis is crucial to optimize apoptosis-favored treatment protocols. We investigated the potential modality of using tissue intrinsic fluorescence chromophore, reduced nicotinamide adenine dinucleotide (NADH), for cell death detection. We imaged the fluorescence lifetime changes of NADH before and after staurosporine (STS)-induced mitochondria-mediated apoptosis and hydrogen peroxide (H2O2)-induced necrosis, respectively, using two-photon fluorescence lifetime imaging in live HeLa cells and 143B osteosarcoma. Time-lapsed lifetime images were acquired at the same site of cells. In untreated cells, the average lifetime of NADH fluorescence was approximately 1.3 ns. The NADH average fluorescence lifetime increased to approximately 3.5 ns within 15 min after 1 microM STS treatment and gradually decreased thereafter. The NADH fluorescence intensity increased within 15 min. In contrast, no significant dynamic lifetime change was found in cells treated with 1 mM H2O2. Our findings suggest that monitoring the NADH fluorescence lifetime may be a valuable noninvasive tool to detect apoptosis and distinguish apoptosis from necrosis for the optimization of apoptosis-favored treatment protocols and other clinical applications.

Reduced nicotinamide adenine dinucleotide fluorescence lifetime separates human mesenchymal stem cells from differentiated progenies

The metabolic changes of human mesenchymal stem cells (hMSCs) during osteogenic differentiation were accessed by reduced nicotinamide adenine dinucleotide (NADH) fluorescence lifetime. An increase in mean fluorescence lifetime and decrease in the ratio between free NADH and protein-bound NADH correlated with our previously reported increase in the adenosine triphosphate (ATP) level of hMSCs during differentiation. These findings suggest that NADH fluorescence lifetime may serve as a new optical biomarker for noninvasive selection of stem cells from differentiated progenies.

Forward-collected simultaneous fluorescence lifetime imaging and coherent anti-Stokes Raman scattering microscopy

We demonstrate the simultaneous collection and separation of femtosecond-laser-based forward-collected coherent anti-Stokes Raman scattering (F-CARS) and two-photon-excitation-induced fluorescence lifetime images (FLIM) using time-correlated single photon counting (TCSPC). We achieve this in a nondescanned geometry using a single multimode fiber without significant loss of light, field of view, and most importantly, TCSPC timing fidelity. In addition to showing the ability to separate CARS images from FLIM images using time gating, we also demonstrate composite multimodal epicollected FLIM imaging with fiber-collected F-CARS imaging in live cells.

Dominant Role of Orai1 with STIM1 on the Cytosolic Entry and Cytotoxicity of Lead Ions

Pb(2+) ions cause severe damages to living cells. In particular, our previous study showed that the Orai-STIM1 (stromal interacting protein 1)-formed store-operated Ca(2+) channels (SOCs) allow Pb(2+) entry. In relation to this, the present study investigates the molecular gating mechanism of Pb(2+) entry by Orai1 with STIM1, as well as the resulting cytotoxicity on human embryonic kidney HEK293 cells. The store-operated Ca(2+) entry (SOCE, activity of SOCs) and Pb(2+) entry were measured using the fura-2 imaging method and indo-1 quenching strategy, as well as through an atomic absorption spectrophotometer. The results of RT-PCR, Western blot, fast confocal, and fluorescent lifetime imaging microscopy indicated the endogenous expression of Orai1 and STIM1 in HEK cells and the functional interaction between these two proteins during SOCE. Both SOCE and Pb(2+) entry largely increased when Orai1 and STIM1 were overexpressed (3- and 1.64-folds, respectively) compared with nonfluorescent cells, and they were significantly attenuated when the E106Q mutation of Orail with STIM1 was cotransfected (6- and 2.25-folds decrease, respectively) compared with Orai1-STIM1 coexpressed cells. The ion gating for Pb(2+) could be governed by the E106 region of Orai1. After sorting and subsequent cultures, the Orai1-STIM1 positive expressed cells behaved more sensitively to Pb(2+) than the Orai1-STIM1 negative cells. In summary, the data suggest that Orai1, together with STIM1, plays a critical role in Pb(2+) entry and the toxicity of Pb(2+).

Metabolism of HeLa cells revealed through autofluorescence lifetime upon infection with enterohemorrhagic Escherichia coli.

Abstract. Fluorescence lifetime imaging microscopy (FLIM) is a sensitive technique in monitoring functional and conformational states of nicotinamide adenine dinucleotide reduced (NADH) and flavin adenine dinucleotide (FAD),main compounds participating in oxidative phosphorylation in cells. In this study, we have applied FLIM to characterize the metabolic changes in HeLa cells upon bacterial infection and made comparison with the results from the cells treated with staurosporine (STS), a well-known apoptosis inducer. The evolving of NADH’s average autofluorescence lifetime during the 3 h after infection with enterohemorragic Escherichia coli (EHEC) or STS treatment has been observed. The ratio of the short and the long lifetime components’ relative contributions of NADH increases with time, a fact indicating cellular metabolic activity, such as a decrease of oxidative phosphorylation over the course of infection, while opposite dynamics is observed in FAD. Being associated with mitochondria, FAD lifetimes and redox ratio could indicate heterogeneous mitochondrial function, microenvironment with bacterial infection, and further pathway to cell death. The redox ratios for both EHEC-infected and STS-treated HeLa cells have been observed and these observations also indicate possible apoptosis induced by bacterial infection.

Orai1–STIM1 formed store-operated Ca2+ channels (SOCs) as the molecular components needed for Pb2+ entry in living cells

Heavy metal lead (Pb2+) is a pollutant and causes severe toxicity when present in human tissues especially the nervous system. Recent reviews have suggested that Pb2+ can target Ca2+-related proteins within neurons and that Ca2+ channels might be a candidate for Pb2+ entry. This studys main aim was to identify the functional entry pathway of Pb2+ into living cells. We firstly characterized the endogenous expression of Orai1 and STIM1 mRNA together with the level of thapsigargin (TG) stimulated capacitative Ca2+ entry in PC12 and HeLa cells; this was done by RT-PCR and time-lapse Ca2+ imaging microscopy, respectively. Our data supported Orai1 and STIM1 as contributing to store-operated Ca2+ channel (SOC) basal activity. Secondly, using the indo-1 quenching method with the SOC blocker 2-APB, we observed that Pb2+ was able to enter cells directly through unactivated SOCs without TG pretreatment. Thirdly, we further demonstrated that co-expression of Orai1 and STIM1 differentially enhanced SOC functional activity (4-fold with PC12 and 5-fold with HeLa cells) and Pb2+ entry (5- to 7-fold with PC12 and 2-fold with HeLa cells). Furthermore, after a 1 h of Pb2+ exposure, the depolarization- and histamine-induced Ca2+ responses were significantly decreased in both PC12 and HeLa cells in a dose-dependent manner. This result indicated that the decreased Ca2+ responses were, in part, due to Pb2+ entry. In summary, our results suggest that SOCs are responsible for Pb2+ permeation and that the Orai1-STIM1 protein complex formed by functional SOCs is one of the molecular components involved in Pb2+ entry.

In-Depth Fluorescence Lifetime Imaging Analysis Revealing SNAP25A-Rabphilin 3A Interactions

The high sensitivity and spatial resolution enabled by two-photon excitation fluorescence lifetime imaging microscopy/fluorescence resonance energy transfer (2PE-FLIM/FRET) provide an effective approach that reveals protein-protein interactions in a single cell during stimulated exocytosis. Enhanced green fluorescence protein (EGFP)-labeled synaptosomal associated protein of 25 kDa (SNAP25A) and red fluorescence protein (mRFP)-labeled Rabphillin 3A (RPH3A) were co-expressed in PC12 cells as the FRET donor and acceptor, respectively. The FLIM images of EGFP-SNAP25A suggested that SNAP25A/RPH3A interaction was increased during exocytosis. In addition, the multidimensional (three-dimensional with time) nature of the 2PE-FLIM image datasets can also resolve the protein interactions in the z direction, and we have compared several image analysis methods to extract more accurate and detailed information from the FLIM images. Fluorescence lifetime was fitted by using one and two component analysis. The lifetime FRET efficiency was calculated by the peak lifetime (taupeak) and the left side of the half-peak width (tau1/2), respectively. The results show that FRET efficiency increased at cell surface, which suggests that SNAP25A/RPH3A interactions take place at cell surface during stimulated exocytosis. In summary, we have demonstrated that the 2PE-FLIM/FRET technique is a powerful tool to reveal dynamic SNAP25A/RPH3A interactions in single neuroendocrine cells.

Long-distance fluorescence lifetime imaging using stimulated emission.

Long-distance stimulated emission imaging has recently been demonstrated as a novel approach for the characterization and imaging of samples containing fluorescent moieties. We present an extension of this methodology through a pump-probe setup for fluorescence lifetime determination and imaging. We measure fluorescence lifetimes of Rhodamine 6G at different solutions and indocyanine green using long-distance fluorescence lifetime imaging.

Visualization of the Orai1 Homodimer and the Functional Coupling of Orai1-STIM1 by Live-Cell Fluorescence Lifetime Imaging

The Orai1-STIM1 constructed store-operated Ca2+ channels (SOCs) have been found to exert several essential Ca2+ entry/signaling cascades, e.g., the generation of immune response in T lymphocytes. Although biochemical and novel imaging evidence appear to indicate that Orai1 and STIM1 interact with each other to achieve store-operated Ca2+ entry (SOCE), the detailed mechanism of functional SOCE in situ has yet to be fully understood. In this study, green fluorescence protein (EGFP as donor) targeted to either the N- or C-terminal of Orai1 (wild type or delta1-90+delta267-301 double deletion type) and mOrange (as acceptor) tagged STIM1 were used to comprise a fluorescence resonance energy transfer (FRET) pair within living PC12 cells. The fluorescence lifetime map and histogram/distribution of each single cell, determined by one-photon excitation fluorescence lifetime imaging microscopy (FLIM), was used to visualize FRET and show the Orai1 homodimer and Orai1-STIM1 binding. Both the color-coded lifetime map and the distribution of EGFP-tagged Orai1 significantly changed after the administration of thapsigargin, the SOCE stimulating agent. The FRET efficiency from each experimental set was also calculated and compared using double exponential analysis. In summary, we show the detailed interactions Orai1-Orai1 and Orai1-STIM1 within intact living cells by using the FLIM-FRET technique.

Monitoring cellular metabolism of 3T3 upon wild type E. coli infection by mapping NADH with FLIM

Fluorescence lifetime imaging microscopy (FLIM) has gained popularity as a sensitive technique to monitor the functional/conformational states of reduced nicotinamide adenine dinucleotide (NADH), one of the main compounds of oxidative phosphorylation. In this letter, we apply the technique to characterize the metabolic changes in mouse embryonic fibroblast 3T3 cells upon bacterial infection. A gradual shortening of the decaying time constants in both the short and the long lifetime components of NADH’s autofluorescence is detected. The ratio of the short and the long lifetime components’ relative contributions, however, shows a rapid increase, indicating the rise of cellular metabolic activity over the course of infection. OCIS codes: 000.1430, 000.4920, 110.1080, 180.2520, 180.4315, 300.2530. doi: 10.3788/COL20100810.0931. Reduced nicotinamide adenine dinucleotide (NADH) serves as a co-enzyme and a principal electron donor within the cell for both oxidative phosphorylation (aerobic respiration) and glycolysis (anaerobic respiration). This molecule exists in two functional forms: free and bound, whereas the latter is associated mostly with the dehydrogenases of so called Complex I-one of 4 mitochondrial membrane protein complexes, which mediate electron transfer from NADH to O2 and use this flow to pump the hydrogen protons to the mitochondrial intermembrane space from the matrix [1] . This gradient of protons and electrical potential, termed proton-motive force, is utilized to synthesize new adenosine triphosphate (ATP) molecules at ATP-synthase via adenosine diphosphate (ADP) phosphorylation [2] . Thus NADH bound forms can be associated with the energy generation in the form of ATP, and the relative quantities of free and bound species of this coenzyme can give an insight on the metabolic state of a cell. Bacterial infections and their influence on cellular physiology are of critical interest to researchers in broad areas, such as microbiology, medicine, drug design, etc. It has been shown that O157:H7 serotype of Escherichia coli (E. coli ), an important human pathogen, closely adheres to the gut epithelium and activates an effacement of the brush border microvilli, a phenomenon known as attaching and effacing [3] . Adhesion of enteropathogenic E. coli to epithelial cells also triggers actin-rich pedestal formation beneath the bacteria [4] . By some uncharted mechanisms, E. coli can induce apoptosis in a number of epithelial cell lines [5] . All these studies suggest the importance of characterizing the cellular metabolism as a result of bacterial infection.