Analysis of early apoptotic events in individual cells by fluorescence intensity and polarization measurements

Apoptosis is a dynamic process of variable duration. The ability to continuously detect the death process occurring in single or subgroups of cells is therefore very important in identifying apoptotic cells within a complex population. The Individual Cell Scanner (ICS), a multiparametric, multilaser-based scanning static cytometer, was used in the present report for the continuous monitoring of the apoptosis process. Fluorescence intensity (FI), polarization (FP), kinetic measurements, and cluster analysis of subpopulations were carried out utilizing various fluorescent probes. Hydrogen peroxide-induced apoptosis was monitored online in intact live lymphocytes by continuous sequential measurements of intracellular hyperpolarization. Plasma membrane asymmetry, mitochondrial membrane potential, and lysosomal rupture were monitored in individual cells. Cytoplasmic condensations, due to cell shrinkage and early lysosomal rupture, were found to be very early events of apoptosis. The new analytical capabilities suggested here may provide simple and convenient methods for detecting apoptosis from its earlier stages.     

Two-dimensional fluorescence intensity distribution analysis: theory and applications

A method of sample analysis is presented which is based on fitting a joint distribution of photon count numbers. In experiments, fluorescence from a microscopic volume containing a fluctuating number of molecules is monitored by two detectors, using a confocal microscope. The two detectors may have different polarizational or spectral responses. Concentrations of fluorescent species together with two specific brightness values per species are determined. The two-dimensional fluorescence intensity distribution analysis (2D-FIDA), if used with a polarization cube, is a tool that is able to distinguish fluorescent species with different specific polarization ratios. As an example of polarization studies by 2D-FIDA, binding of 5'-(6-carboxytetramethylrhodamine) (TAMRA)-labeled theophylline to an anti-theophylline antibody has been studied. Alternatively, if two-color equipment is used, 2D-FIDA can determine concentrations and specific brightness values of fluorescent species corresponding to individual labels alone and their complex. As an example of two-color 2D-FIDA, binding of TAMRA-labeled somatostatin-14 to the human type-2 high-affinity somatostatin receptors present in stained vesicles has been studied. The presented method is unusually accurate among fluorescence fluctuation methods. It is well suited for monitoring a variety of molecular interactions, including receptors and ligands or antibodies and antigens.     

Analysis of enzyme kinetics in individual living cells utilizing fluorescence intensity and polarization measurements

BACKGROUND: The Cellscan mark-S (CS-S) scanning cytometer was used for tracing enzymatic reactions in the same individual cells under various physiological conditions over periods of minutes. On-line reagent addition and changes in the experimental conditions (buffers, ions, substrates and inhibitors) were performed. METHODS: Kinetic events were monitored by fluorescence intensity (FI) and fluorescence polarization (FP) measurements of fluorescein diacetate (FDA) and chloromethyl fluorescein diacetate (CMFDA) intracellular hydrolysis. FP measurements have been used to assess the intracellular marker's mobility restrictions. RESULTS: Kinetic measurement along 1000 s of FDA labeled individual Jurkat T cells, indicated variation of 65% for FI(t) and approximately 10% for FP(t). While FI increased linearly with time, FP(t) decreased nonlinearly and asymptotically, reaching a constant value. The FP(t) of CMFDA-labeled cells was different from that of FDA-labeled cells. Average cellular Km of 3.9 microM was calculated from individual cell FDA hydrolysis curves. CONCLUSIONS: (1) Analysis of the reaction kinetics of intracellular enzymes can be refined by using FP measurements of the products of fluorogenic substrates in addition to the FI measurements. (2) Subpopulations or individual cells could be classified according to their reaction rates. (3) A specific dependence of FP(t) on type of enzyme substrate is suggested.     

Oligomerization of the EGF receptor investigated by live cell fluorescence intensity distribution analysis

Recent evidence suggests that the EGF receptor oligomerizes or clusters in cells even in the absence of agonist ligand. To assess the status of EGF receptors in live cells, an EGF receptor fused to eGFP was stably expressed in CHO cells and studied using fluorescence correlation spectroscopy and fluorescent brightness analysis. By modifying FIDA for use in a two-dimensional system with quantal brightnesses, a method was developed to quantify the degree of clustering of the receptors on the cell surface. The analysis demonstrates that under physiological conditions, the EGF receptor exists in a complex equilibrium involving single molecules and clusters of two or more receptors. Acute depletion of cellular cholesterol enhanced EGF receptor clustering whereas cholesterol loading decreased receptor clustering, indicating that receptor aggregation is sensitive to the lipid composition of the membrane.     

Detection and analysis of tumor fluorescence using a two-photon optical fiber probe

The utility of a two-photon optical fiber fluorescence probe (TPOFF) for sensing and quantifying tumor fluorescent signals was tested in vivo. Xenograft tumors were developed in athymic mice using MCA207 cells expressing green fluorescent protein (GFP). The TPOFF probe was able to detect ex vivo fluorescence from excised tumors containing as little as 0.3% GFP-expressing cells. TPOFF results were similar to both flow-cytometric analysis of tumor cells after isolation and suspension, and fluorescence determined by microscope images of cryosectioned tumors. TPOFF was then used to measure GFP fluorescence from tumors in live mice. The fiber probe detected fluorescently-labeled Herceptin antibody targeted to HER2-expressing tumors in severe combined immunodeficient mice. Dendrimer nanoparticles targeted by folic acid and having 6-TAMRA as a fluorescent probe were also used to label KB cell tumors in vivo. The fiber probe documented a fourfold increase in tumor fluorescence in animals that received the targeted dendrimer. These results suggest TPOFF can be used as a minimally invasive system for identifying tumor markers and monitoring drug therapy.     

The trace and subgrouping of lymphocyte activation by dynamic fluorescence intensity and polarization measurements.

Cell activation involves conformational changes of cytosolic enzymes, and/or their regulatory proteins, as well as intracellular matrix re-organization. In this work, these changes were monitored by dynamic measurements of fluorescence polarization in single cells incubated with or without phytohaemagglutinin (PHA), using the Cellscan mark S (CS-S) cytometer. This instrument and the procedure used proved to be a powerful tool for distinguishing subpopulations of cells. Grouping of cells by their staining rates (the time rate of change of the fluorescence intensity) yielded three major subgroups. For each subgroup, the fluorescence depolarization (FDP) induced by the incubation with PHA was measured. The kinetics of the subgroups indicate that the major FDP is contributed by the cells with the lowest staining rate. This FDP is approximately 1.5 times greater than that of a bulk population. It is believed that the analysis of kinetic probing might yield an important and more sensitive method for functional marking of subgroups of cells by their response characteristics.     

Development of a 1-microl scale assay for mitogen-activated kinase kinase 7 using 2-D fluorescence intensity distribution analysis anisotropy

This paper describes the development of a robust, miniaturizable, and quantitative fluorescence-based assay for mitogen-activated protein kinase kinase 7 (MKK7). As a first step, the basic steady-state kinetics of the MKK7-catalyzed phosphorylation of c-Jun N-terminal kinases (JNKs) 1, 2, and 3 were defined using standard radiometric methods. Subsequently, the authors found that in addition to the holo JNKs, a series of novel small peptides (based on the region around the JNK phosphorylation site) are also substrates, provided that these were prephosphorylated on the Y residue of the TPY motif. One of these peptide substrates was used in the development of a fluorescence polarization-based assay using an antibody as a sensor. The assay was successfully miniaturized for use with conventional fluorescence polarization (FP) reader technology in 8.5 microl and on the single microl scale using Evotec proprietary 2-dimensional fluorescence intensity distribution analysis (2D-FIDA) anisotropy and liquid handling technology. The steady-state kinetic parameters derived using the FP or 2D-FIDA anisotropy format assays correlated well with those generated using a radiometric assay. Moreover, the quantitative sensitivity to known inhibitors was maintained independent of the format and assay volume. In addition, the authors found that the 2D-FIDA anisotropy assay exhibited superior performance statistics (typical Z' = approximately 0.5) relative to conventional FP (typical Z' = 0.3) and yielded the additional benefit of order-of-magnitude savings in terms of reagent costs. The 2D-FIDA anisotropy assay was used to carry out a successful high-throughput screening in 1-microl final volume against company file compounds.     

Quantitative analysis of c-myc-tagged protein in crude cell extracts using fluorescence polarization

A fluorescence polarization (FP) assay was developed to determine the concentration of a c-myc-tagged recombinant protein in a crude cell extract. The basis of the assay was a competition between a c-myc-tagged protein and a fluorescein-labeled c-myc peptide for a c-myc antibody Fab. Fluorescein-labeled c-myc peptide produced a high-fluorescence polarization signal upon binding to the c-myc antibody, which can be inhibited in the presence of a c-myc-tagged protein. Quantitation of a c-myc-tagged protein was realized by measuring the decrease in fluorescence polarization. The observed IC(50) values in the competition FP assay were similar among all monomeric c-myc-tagged proteins tested, indicating that the interaction of the c-myc tag with the antibody was independent of the fusion protein sequence. The c-myc-tagged protein concentrations measured by FP were found to correlate well with values derived from a spike experiment and with values obtained by quantitative immunoblot. This assay was not perturbed by the presence of crude cell lysate, dithiothreitol or detergents, and worked with both native and denatured samples from several expression systems, including Escherichia coli, Pichia, insect cells, and mammalian cells. The assay under the current condition can detect as low as 0.05% expression level of c-myc-tagged protein with regards to total proteins, depending on the expression system. This assay is both quantitative and rapid (less than 15min) and is therefore suitable for the optimization of recombinant protein expression conditions as well as for the monitoring of protein purification procedures.     

Investigation of fluorescence intensity and distribution of wheat leaf on exposure to temperature

It was established that exposure to extreme temperatures changed the distribution and intensity of fluorescence of pigments in the wheat leaf, which is likely related to the changes of both the leaf morphology and pigment state.     

Fluorescence intensity and lifetime distribution analysis: toward higher accuracy in fluorescence fluctuation spectroscopy

Fluorescence fluctuation methods such as fluorescence correlation spectroscopy and fluorescence intensity distribution analysis (FIDA) have proven to be versatile tools for studying molecular interactions with single molecule sensitivity. Another well-known fluorescence technique is the measurement of the fluorescence lifetime. Here, we introduce a method that combines the benefits of both FIDA and fluorescence lifetime analysis. It is based on fitting the two-dimensional histogram of the number of photons detected in counting time intervals of given width and the sum of excitation to detection delay times of these photons. Referred to as fluorescence intensity and lifetime distribution analysis (FILDA), the technique distinguishes fluorescence species on the basis of both their specific molecular brightness and the lifetime of the excited state and is also able to determine absolute fluorophore concentrations. The combined information yielded by FILDA results in significantly increased accuracy compared to that of FIDA or fluorescence lifetime analysis alone. In this paper, the theory of FILDA is elaborated and applied to both simulated and experimental data. The outstanding power of this technique in resolving different species is shown by quantifying the binding of calmodulin to a peptide ligand, thus indicating the potential for application of FILDA to similar problems in the life sciences.     

Use of laurdan fluorescence intensity and polarization to distinguish between changes in membrane fluidity and phospholipid order

Laurdan is a fluorescent probe that detects changes in membrane phase properties through its sensitivity to the polarity of its environment in the bilayer. Variations in membrane water content cause shifts in the laurdan emission spectrum, which are quantified by calculating the generalized polarization (GP). We tested whether laurdan fluorescence could be used to distinguish differences in phospholipid order from changes in membrane fluidity by examining the temperature dependence of laurdan GP and fluorescence anisotropy in dipalmitoylphosphatidylcholine (DPPC) vesicles. The phase transition from the solid ordered phase to the liquid disordered phase was observed as a decrease in laurdan GP values from 0.7 to −0.14 and a reduction in anisotropy from 0.25 to 0.12. Inclusion of various amounts of cholesterol in the membranes to generate a liquid ordered phase caused an increase in the apparent melting temperature detected by laurdan GP. In contrast, cholesterol decreased the apparent melting temperature estimated from anisotropy measurements. Based on these results, it appeared that laurdan anisotropy detected changes in membrane fluidity while laurdan GP sensed changes in phospholipid order. Thus, the same fluorescent probe can be used to distinguish effects of perturbations on membrane order and fluidity by comparing the results of fluorescence emission and anisotropy measurements.     

Use of laurdan fluorescence intensity and polarization to distinguish between changes in membrane fluidity and phospholipid order

Sialyltransferase activity has been determined in membrane preparations containing the Golgi apparatus that were isolated from atherosclerotic and normal human aortic intima as well as in plasma of patients with documented atherosclerosis and healthy donors by measuring the transfer of N-acetylneuraminic acid (NeuAc) from CMP-NeuAc to asialofetuin. The asialofetuin sialyltransferase activity was found to be 2 times higher in the atherosclerotic intima as compared to the normal intima and 2-fold higher in patients' plasma than in that from healthy donors. The mean values of the apparent Michaelis constant (K(m)) for the sialylating enzyme for both tissues did not differ and were close for the intima and plasma. In contrast, the maximal velocity (V(max)) was 2 times higher for the atherosclerotic intima than for the normal intima and 3 times higher for patients' plasma than for that of the donors. These results suggest that the activity of asialofetuin sialyltransferases of aortal intima is enhanced in atherosclerosis as is the secretion of their soluble forms into patients' plasma.     

Analysis of protein-peptide interaction by a miniaturized fluorescence polarization assay using cyclin-dependent kinase 2/cyclin E as a model system.

As a result of the increasing size of chemical libraries, more rapid and highly sensitive strategies are needed to accelerate the process of drug discovery without increasing the cost. One means of accomplishing this is to miniaturize the assays that enter high-throughput screening (HTS). Miniaturization requires an assay design that has few steps, has a large degree of separation between the signal and background, and has a low well to well signal variation. Fluorescence polarization (FP) is an assay type that, in many cases, meets all of the above requirements. FP is a homogenous method that allows interactions between molecules to be measured directly in solution. This article demonstrates the application of FP in a miniaturized HTS format, using 1536-well plates, to measure direct binding between cyclin-dependent kinase 2/cyclin E complex (CDK2/E) and an 8-mer-peptide kinase inhibitor. The data indicate that low variability and high specificity allow rapid and precise identification of antagonist compounds affecting CDK2/E-peptide interactions.     

Development of a specific siRNA delivery system into HeLa cells using an IgG-binding fusion protein

Stable carriers are required for delivering siRNA to cells. The use of polyethyleneimine (PEI) as gene carrier has been researched extensively; however, it does not provide sufficient protection from RNase degradation and is not suitable for targeted siRNA delivery to specific cells. In this study, two repeats of Fc binding domain of protein G (C2) were used to introduce a specific antibody to PEI-based carrier of siRNA. In addition, we used the double-stranded RNA binding domain (DRBD) that can bind to siRNA. The complex, consisting of PEI, siRNA and constructed fusion protein, TrxC2DRBD including C2 and DRBD domains, could protect siRNA from RNase degradation. Furthermore, cell specific siRNA delivery into HeLa cells could be performed by the complex fusion with specific antibodies via C2 domain.     

Measurement of fluorescence intensity in microscope preparations using a photodiode detector

The development of a photodiode detector to measure fluorescence intensity in biological samples is described and the circuit is given for the amplication of the signal.     

Detection of asymmetric distribution of phospholipids by fluorescence resonance energy transfer

It is well established that the plasma membrane exhibits an asymmetric distribution of lipids between the inner and outer leaflets of the lipid bilayer. Recent studies suggest that the asymmetric distribution changes locally and temporarily, accompanied by cellular events. However, available methods to detect lipid asymmetry lack spatio-temporal resolution. As a technique of potential use for real-time imaging of lipid asymmetry, we a novel method that utilizes fluorescence resonance energy transfer (FRET) between NBD-labeled phospholipids (donor) and extracellular rhodamine (acceptor). When cell apoptosis was induced by staurosporine, the fluorescence intensity of NBD-labeled phosphatidylserine decreased owing to FRET from NBD to rhodamine. This method provides a simple way to detect lipid asymmetry and may be useful for observing dynamic changes in asymmetric distribution of lipids.     

A maximum entropy analysis of protein orientations using fluorescence polarization data from multiple probes

Techniques have recently become available to label protein subunits with fluorescent probes at predetermined orientation relative to the protein coordinates. The known local orientation enables quantitative interpretation of fluorescence polarization experiments in terms of orientation and motions of the protein within a larger macromolecular assembly. Combining data obtained from probes placed at several distinct orientations relative to the protein structure reveals functionally relevant information about the axial and azimuthal orientation of the labeled protein segment relative to its surroundings. Here we present an analytical method to determine the protein orientational distribution from such data. The method produces the broadest distribution compatible with the data by maximizing its informational entropy. The key advantages of this approach are that no a priori assumptions are required about the shape of the distribution and that a unique, exact fit to the data is obtained. The relative orientations of the probes used for the experiments have great influence on information content of the maximum entropy distribution. Therefore, the choice of probe orientations is crucial. In particular, the probes must access independent aspects of the protein orientation, and two-fold rotational symmetries must be avoided. For a set of probes, a "figure of merit" is proposed, based on the independence among the probe orientations. With simulated fluorescence polarization data, we tested the capacity of maximum entropy analysis to recover specific protein orientational distributions and found that it is capable of recovering orientational distributions with one and two peaks. The similarity between the maximum entropy distribution and the test distribution improves gradually as the number of independent probe orientations increases. As a practical example, ME distributions were determined with experimental data from muscle fibers labeled with bifunctional rhodamine at known orientations with respect to the myosin regulatory light chain (RLC). These distributions show a complex relationship between the axial orientation of the RLC relative to the fiber axis and the azimuthal orientation of the RLC about its own axis. Maximum entropy analysis reveals limitations in available experimental data and supports the design of further probe angles to resolve details of the orientational distribution.     

Detection of phosphopeptides by fluorescence polarization in the presence of cationic polyamino acids: application to kinase assays

We have studied the interaction of several phosphopeptides with cationic polyamino acids such as polyarginine and polylysine by fluorescence polarization. The phosphopeptides used were labeled with fluorescein, and their net charges at the experimental pH of 7. 5 were 0, -1, -2, and -3. These phosphopeptides represent the products of enzymatic phosphorylation reactions of the corresponding nonphosphorylated precursors by the protein kinase A, Akt1 (protein kinase Balpha), and protein kinase C. We found that these phosphopeptides bind more strongly to the cationic polyamino acids studied than their nonphosphorylated analogs. This preferential binding of the phosphorylated peptides could be conveniently detected by an increase in the fluorescence polarization signal of the attached fluorescein residue. We have exploited this observation to develop a new approach for the detection of kinase activity that does not require radioactivity or separation of substrate from product. We have successfully used this method to perform K(m) determinations of the kinase enzymes for their substrates and K(i) determinations of one of their inhibitors. This method for measuring kinase activity might be particularly useful for high-throughput screening applications.