Genetically encoded stress indicator for noninvasively imaging endogenous corticosterone in living mice

Physical and emotional stress is one of the major controllers of physiological reactions and homeostasis in living animals. A stress hormone, corticosterone, is secreted from the adrenal cortex into the blood vessels when animals sense the stress. The quantitative evaluation of corticosterone in living animals has been limited because of the unavailability of suitable methods in vivo. For a noninvasive molecular imaging of the stress, we developed a method for detecting physiological increases in the endogenous corticosterone caused by exo- and endogenous stress in living animals. We constructed a pair of genetically encoded indicators composed of cDNAs of glucocorticoid receptor (GR), split Renilla luciferase (RLuc), and a Synechocystis sp. DnaE intein. The GR fused with C-terminal halves of RLuc and DnaE is localized in the cytosol, whereas a fusion protein of N-terminal halves of RLuc and DnaE is localized in the nucleus. If corticosterone induces GR translocation into the nucleus, the C-terminal RLuc meets the N-terminal one in the nucleus, and full-length RLuc is reconstituted by protein splicing with DnaE. Cell-based methods provided a quantitative bioluminescence assay of the extent of GR translocation into the nucleus. We further demonstrated that the indicator enabled noninvasive imaging against two different types of imposed stress: a forced swimming and metabolic perturbation caused by 2-deoxy-D-glucose. This stress indicator should be valuable for screening pharmacological compounds and for tools to study the mechanism of physiological stress.     

Genetically encoded fluorescent indicators to visualize protein phosphorylation by extracellular signal-regulated kinase in single living cells

Extracellular signal-regulated kinase (ERK) is a serine/threonine protein kinase that regulates a wide variety of cell functions, such as cell growth and differentiation. To study the spatiotemporal dynamics of protein phosphor-ylation by activated ERK in living cells, we have developed genetically encoded fluorescent indicators for ERK. The present indicators are based on fluorescence resonance energy transfer (FRET) between two green fluorescent protein mutants. Phosphorylation of the indicators by activated ERK changes the FRET efficiency due to their conformational alterations. We visualized the cytosolic and nuclear activity of ERK using the present indicators. We thus found that the activation duration of ERK is considerably different between the cytosol and nucleus in living cells. The subcellular difference in the ERK activity may be fundamental to the regulation of cell functions by ERK. The present fluorescent indicators provide a powerful tool to reveal the spatiotemporal dynamics of protein phosphorylation by ERK in single living cells.     

Split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells based on protein splicing

We describe a new method for detecting protein-protein interactions in intact mammalian cells; the approach is based on protein splicing-induced complementation of rationally designed fragments of firefly luciferase. The protein splicing is a posttranslational protein modification through which inteins (internal proteins) are excised out from a precursor fusion protein, ligating the flanking exteins (external proteins) into a contiguous polypeptide. As the intein, naturally split DnaE from Synechocystis sp. PCC6803 was used: The N- and C-terminal DnaE, each fused respectively to N- and C-terminal fragments of split luciferase, were connected to proteins of interest. In this approach, protein-protein interactions trigger the folding of DnaE intein, wherein the protein splicing occurs and thereby the extein of ligated luciferase recovers its enzymatic activity. To test the applicability of this split luciferase complementation, we used insulin-induced interaction between known binding partners, phosphorylated insulin receptor substrate 1 (IRS-1) and its target N-terminal SH2 domain of PI 3-kinase. Enzymatic luciferase activity triggered by insulin served to monitor the interaction between IRS-1 and the SH2 domain in an insulin dose-dependent manner, of which amount was assessed by the luminescent intensity. This provides a convenient method to study phosphorylation of any protein or interactions of integral membrane proteins, a class of molecules that has been difficult to study by existing biochemical or genetic methods. High-throughput drug screening and quantitative analysis for a specific pathway in tyrosine phosphorylation of IRS-1 in insulin signaling are also made possible in this system.     

Genetically encoded bright Ca2+ probe applicable for dynamic Ca2+ imaging of dendritic spines

G-CaMP is a Ca2+ probe based on a single green fluorescent protein (GFP). G-CaMP shows a large fluorescence increase upon Ca2+ binding, but its fluorescence is dim and pH sensitive, similar to other single GFP-based probes. Here we report an improved G-CaMP, named G-CaMP1.6, which enables easier detection of intracellular Ca2+ signals. G-CaMP1.6 was approximately 40 times more fluorescent than G-CaMP, mainly due to an increase in quantum yield. Furthermore, compared with G-CaMP, G-CaMP1.6 had not only a lower pH sensitivity but also a higher selectivity for divalent cations having an ionic radius similar to Ca2+. Ca2+ sensitivity of G-CaMP1.6 (Kd = 146 nM, Hill coefficient = 3.8, Fmax/Fmin = 4.9) was slightly shifted toward higher affinity compared with that of G-CaMP. When expressed in mammalian cells, G-CaMP1.6 showed large fluorescence changes with drug applications. Notably, local Ca2+ changes in such tiny structures as dendritic spines of neurons were successfully observed with G-CaMP1.6, this being the first observation using a GFP-based probe. Additional mutations in Ca2+-binding sites of G-CaMP1.6 shifted the affinity for Ca2+ and reduced the Ca2+-buffering effect. G-CaMP1.6-CaM(E140K), which has a mutation in the Ca2+ binding site, is an improved probe with its increased brightness and reduced Ca2+-buffering capacity.     

A genetically encoded fluorescent indicator capable of discriminating estrogen agonists from antagonists in living cells

A genetically encoded fluorescent indicator was developed for the detection and characterization of estrogen agonists and antagonists. Two different color mutants of green fluorescent protein were joined by a tandem fusion domain composed of LXXLL (L = leucine, X = any amino acid) motif from the nuclear receptor-box II of steroid receptor coactivator 1, a flexible linker sequence, and the estrogen receptor alpha ligand binding domain (ERalpha LBD). Monitoring real-time ligand-induced conformational change in the ERalpha LBD to recruit the LXXLL motif allowed screening of natural and synthetic estrogens in single living cells using fluorescence resonance energy-transfer technique. The indicator was named SCCoR (single cell-coactivator recruitment). The high sensitivity of the present indicator made it possible to distinguish between estrogen strong and weak agonists in a dose-dependent fashion, immediately after adding ligand to live cells. Discrimination of agonists from antagonists was efficiently achieved using the present study. The approach described here can be applied to develop biosensors for other hormone receptors as well.     

Subcellular tracking of drug release from carbon nanotube vehicles in living cells

The direct observation of drug release from carbon nanotube vehicles in living cells is realized through a unique two-dye labeling approach. Single-walled carbon nanotubes (SWNTs) are firstly marked with fluorescein isothiocyanate (FITC) to track their location and movement inside the cell. Then a fluorescent anticancer drug doxorubicin (DOX) is attached by means of π-stacking onto SWNTs. Delivered by SWNTs into cells, DOX will detach from the vehicle in an acidic environment due to the pH-dependent π-π stacking interaction between DOX and SWNTs. From observation of the two different kinds of fluorescence (green and red) that respectively represent the carrier SWNTs and drug DOX, the process of drug release inside the living cell can be monitored under a confocal microscope. Results show that the drug DOX detaches from SWNTs inside the lysosomes to yield free molecules and escape into the cytoplasm and finally into the nucleus, while the vehicle SWNTs are trapped inside the lysosomes, without entering the nucleus. The current observations confirm previously proposed mechanisms for drug/DOX release inside cells. The experimental establishment of drug-release mechanisms in living cells here might provide important insights for future design of new drug-delivery and release systems.     

Visualization of nonengineered single mRNAs in living cells using genetically encoded fluorescent probes

Single mRNA imaging in live cells is a useful technique to elucidate its precise localization and dynamics. We developed a method for visualizing endogenous mRNAs in living cells with single molecule sensitivity using genetically encoded probes. An RNA-binding protein of human PUMILIO1 (PUM-HD) was used for recognizing base sequences of a target mRNA, β-actin mRNA. Two PUM-HDs were modified by amino acid mutations to bind specifically to tandem 8-base sequences of the target mRNA. Because each PUM-HD was connected with amino- and carboxyl-terminal fragments of enhanced green fluorescent protein (EGFP), the probes emit fluorescence by reconstitution of EGFP fragments upon binding to β-actin mRNAs. The EGFP reconstituted on the mRNAs was monitored with a total internal reflection fluorescence microscope. Results show that each fluorescent spot in live cells represented a single β-actin mRNA and that distinct spatial and temporal movement of the individual β-actin mRNAs was visualized. We also estimated the average velocity of the movement of the single mRNAs along microtubules in live cells. This method is widely applicable to tracking various mRNAs of interest in the native state of living cells with single-mRNA sensitivity.     

Molecular handles for the mechanical manipulation of single-membrane proteins in living cells

We have developed a procedure to selectively biotinylate a specific membrane protein, enabling its attachment to external force probes and thus allowing its mechanical manipulation within its native environment. Using potassium channels as model membrane proteins in oocytes, we have found that Maleimide-PEG3400-biotin is the crosslinker with highest conjugation selectivity and accessibility to external probes. Neutravidin-coated beads provide for directed attachment while avoiding nonspecific interactions with the cell. The technology was successfully tested by mechanical manipulation of biotinylated extracellular residues of channels in oocytes using an atomic force microscope under conditions which preserve function of the channels. Binding forces of /spl sim/80 pN at 100 nN/s were measured.     

Cell-based indicator to visualize picomolar dynamics of nitric oxide release from living cells

We report a novel cell-based indicator that is able to visualize picomolar dynamics of nitric oxide release from living cells. Cells from a pig kidney-derived cell line (PK15) endogenously express soluble guanylate cyclase (sGC), which is a receptor protein for the selective recognition of NO. Binding of NO by sGC causes the amplified generation of guanosine 3',5'-cyclic monophosphate (cGMP). To make the PK15 cells into NO indicators, the cells are transfected with a plasmid vector encoding a fluorescent indicator for cGMP and fluorescence resonance energy transfer is recorded at 480 +/- 15 and 535 +/- 12.5 nm upon excitation of the cells at 440 +/- 10 nm. The cell-based indicator exhibits exceptional sensitivity (detection limit of 20 pM), selectivity, reversibility, and reproducibility. The outstanding sensitivity of the present indicator has led us to uncover an oscillatory release of picomolar concentrations of NO from hippocampal neurons. We present evidence that Ca2+ oscillations in hippocampal neurons underlie the oscillatory NO release from the neurons during neurotransmission. We also have succeeded in visualizing the extent of diffusing NO from single vascular endothelial cells. The present cell-based indicator provides a powerful tool to uncover picomolar dynamics of NO that regulates a wide range of cell functions in biological systems.     

Layer-by-layer coated digitally encoded microcarriers for quantification of proteins in serum and plasma

The "layer-by-layer" (LbL) technology has been widely investigated for the coating of flat substrates and capsules with polyelectrolytes. In this study, LbL polyelectrolyte coatings applied at the surface of digitally encoded microcarriers were evaluated for the quantitative, sensitive, and simultaneous detection of proteins in complex biological samples like serum, plasma, and blood. LbL coated microcarriers were therefore coupled to capture antibodies, which were used as capture agents for the detection of tumor necrosis factor (TNF-alpha), P24, and follicle stimulating hormone (FSH). It was found that the LbL coatings did not disassemble upon incubating the microcarriers in serum and plasma. Also, nonspecific binding of target analytes to the LbL coating was not observed. We showed that the LbL coated microcarriers can reproducibly detect TNF-alpha, P24, and FSH down to the picogram per milliliter level, not only in buffer but also in serum and plasma samples. Microcarrier-to-microcarrier intratube variations were less then 30%, and interassay variations less than 8% were observed. This paper also shows evidence that the LbL coated digitally encoded microcarriers are ideally suited for assaying proteins in "whole" blood in microfluidic chips, which are of high interest for "point-of-care" diagnostics.     

An optical technique for detecting defects in thin plates and diaphragms

A non-destructive test is proposed for thin plates and diaphragm elements that consists of recording the images of a coarse grating reflected by the plates or elements when they are laterally loaded. A defective region is indicated by an obvious distortion of the lines constituting the grating image.     

Imaging of conformational changes of proteins with a new environment-sensitive fluorescent probe designed for site-specific labeling of recombinant proteins in live cells

We demonstrate herein a new method for imaging conformational changes of proteins in live cells using a new synthetic environment-sensitive fluorescent probe, 9-amino-6,8-bis(1,3,2-dithioarsolan-2-yl)-5H-benzo[a]phenoxazin-5-one. This fluorescent probe can be attached to recombinant proteins containing four cysteine residues at the i, i + 1, i + 4, and i + 5 positions of an alpha-helix. The specific binding of the fluorescent probe to this 4Cys motif enables fluorescent labeling inside cells by its extracellular administration. The high sensitivity of the fluorophore to its environment enables monitoring of the conformational changes of the proteins in live cells as changes in its fluorescence intensity. The present method was applied to calmodulin (CaM), a Ca2+-binding protein that was well-known to expose hydrophobic domains, depending on the Ca2+ concentration. A recombinant CaM fused at its C-terminal with a helical peptide containing a 4Cys motif was labeled with the fluorescent probe inside live cells. The fluorescence intensity changed reversibly depending on the intracellular Ca2+ concentration, which reflected the conformational change of the recombinant CaM in the live cells.     

Linear optical setups for active and passive quantum error correction in polarization encoded qubits

In this work, we present active and passive linear optical setups for error correction in quantum communication systems that employ polarization of single-photon and mesoscopic coherent states. The proposed systems are analytically analysed and their applications in quantum communication systems are described. In particular, we show a security analysis of a QKD system employing the active error correction system when an eavesdropper uses the Fuchs–Peres–Brandt attack.     

Compact method for optical induction of proximal probe heating and elongation

A tapered, metal-coated, optical fiber probe will elongate when heated by light input through a fiber. The induced motion can be used for data storage or nanostructuring of a surface. The elongation produced by this alignment-free system is measured with force feedback in a near-field scanning optical microscope (NSOM). The input light intensity controls the elongation magnitude, which ranges from a few nanometers to more than 100 nm. A 0.5-mW input energy yields approximately 20 nm of probe elongation. The elongation quantified here can create artifacts in any experiment using pulsed laser light with a NSOM or an atomic force microscope.     

Design and synthesis of single-nanoparticle optical biosensors for imaging and characterization of single receptor molecules on single living cells

At the cellular level, a small number of protein molecules (receptors) can induce significant cellular responses, emphasizing the importance of molecular detection of trace amounts of protein on single living cells. In this study, we designed and synthesized silver nanoparticle biosensors (AgMMUA-IgG) by functionalizing 11.6 +/- 3.5-nm Ag nanoparticles with a mixed monolayer of 11-mercaptoundecanoic acid (MUA) and 6-mercapto-1-hexanol (1:3 mole ratio) and covalently conjugating IgG with MUA on the nanoparticle surface. We found that the nanoparticle biosensors preserve their biological activity and photostability and can be utilized to quantitatively detect individual receptor molecules (T-ZZ), map the distribution of receptors (0.21-0.37 molecule/microm(2)), and measure their binding affinity and kinetics at concentrations below their dissociation constant on single living cells in real time over hours. The dynamic range of detection is 0-50 molecules per cell. We also found that the binding rate (2-27 molecules/min) is highly dependent upon the coverage of receptors on living cells and their ligand concentration. The binding association and dissociation rate constants and affinity constant are k1 = (9.0 +/- 2.6) x 10(3) M(-1) s(-1), k(-1) = (3.0 +/- 0.4) x 10(-4) s(-1), and KB = (4.3 +/- 1.1) x 10(7) M(-1), respectively.     

Optical correlation of databases in conventionally encoded optical disks: techniques for improved accuracy

Optical correlation of multiple tracks of conventional optical-disk data, digitally encoded by transitions in reflectance rather than absolute reflectance, can be improved with a pulse-counting version of the digital-multiplication-by-analog-convolution algorithm, which also avoids analog weighting.     

A local optical probe for measuring motion and stress in a nanoelectromechanical system

Nanoelectromechanical systems can be operated as ultrasensitive mass sensors and ultrahigh-frequency resonators, and can also be used to explore fundamental physical phenomena such as nonlinear damping and quantum effects in macroscopic objects. Various dissipation mechanisms are known to limit the mechanical quality factors of nanoelectromechanical systems and to induce aging due to material degradation, so there is a need for methods that can probe the motion of these systems, and the stresses within them, at the nanoscale. Here, we report a non-invasive local optical probe for the quantitative measurement of motion and stress within a nanoelectromechanical system, based on Fizeau interferometry and Raman spectroscopy. The system consists of a multilayer graphene resonator that is clamped to a gold film on an oxidized silicon surface. The resonator and the surface both act as mirrors and therefore define an optical cavity. Fizeau interferometry provides a calibrated measurement of the motion of the resonator, while Raman spectroscopy can probe the strain within the system and allows a purely spectral detection of mechanical resonance at the nanoscale.     

Simultaneous characterization of the shape and refractive index of transparent living cells by an optical aperture

We report a method of using optical aperture diffraction to simultaneously detect the image and the refractive index of a living cell. By simulation, it is found that, when a suitable gap is introduced between the cell and the aperture, the image of the cell on the detection plane can be amplified hundreds to thousands of times, and a limit of detection of 3e-4 to 9e-5 can be reached for the refractive index of the cell. Experiments show that this method is feasible to realize, but the achievement of such a detection system is yet to be proved.