Some Secrets of Fluorescent Proteins: Distinct Bleaching in Various Mounting Fluids and Photoactivation of cyan fluorescent proteins at YFP-Excitation

Background
The use of spectrally distinct variants of green fluorescent protein (GFP) such as cyan or yellow mutants (CFP and YFP, respectively) is very common in all different fields of life sciences, e.g. for marking specific proteins or cells or to determine protein interactions. In the latter case, the quantum physical phenomenon of fluorescence resonance energy transfer (FRET) is exploited by specific microscopy techniques to visualize proximity of proteins.

Methodology/Principal Findings
When we applied a commonly used FRET microscopy technique - the increase in donor (CFP)-fluorescence after bleaching of acceptor fluorophores (YFP), we obtained good signals in live cells, but very weak signals for the same samples after fixation and mounting in commercial microscopy mounting fluids. This observation could be traced back to much faster bleaching of CFP in these mounting media. Strikingly, the opposite effect of the mounting fluid was observed for YFP and also for other proteins such as Cerulean, TFP or Venus. The changes in photostability of CFP and YFP were not caused by the fixation but directly dependent on the mounting fluid. Furthermore we made the interesting observation that the CFP-fluorescence intensity increases by about 10 - 15% after illumination at the YFP-excitation wavelength – a phenomenon, which was also observed for Cerulean. This photoactivation of cyan fluorescent proteins at the YFP-excitation can cause false-positive signals in the FRET-microscopy technique that is based on bleaching of a yellow FRET acceptor.

Conclusions/Significance
Our results show that photostability of fluorescent proteins differs significantly for various media and that CFP bleaches significantly faster in commercial mounting fluids, while the opposite is observed for YFP and some other proteins. Moreover, we show that the FRET microscopy technique that is based on bleaching of the YFP is prone to artifacts due to photoactivation of cyan fluorescent proteins under these conditions

On two superintegrable nonlinear oscillators in N dimensions

We consider the classical superintegrable Hamiltonian system given by $H=T+U={p^2}/{2(1+\lambda q^2)}+{{\omega}^2 q^2}/{2(1+\lambda q^2)}$, where U is known to be the "intrinsic" oscillator potential on the Darboux spaces of nonconstant curvature determined by the kinetic energy term T and parametrized by {\lambda}. We show that H is Stackel equivalent to the free Euclidean motion, a fact that directly provides a curved Fradkin tensor of constants of motion for H. Furthermore, we analyze in terms of {\lambda} the three different underlying manifolds whose geodesic motion is provided by T. As a consequence, we find that H comprises three different nonlinear physical models that, by constructing their radial effective potentials, are shown to be two different nonlinear oscillators and an infinite barrier potential. The quantization of these two oscillators and its connection with spherical confinement models is briefly discussed.Comment: 11 pages; based on the contribution to the Manolo Gadella Fest-60 years-in-pucelandia, "Recent advances in time-asymmetric quantum mechanics, quantization and related topics" hold in Valladolid (Spain), 14-16th july 201

Phase-space formulation of quantum mechanics and quantum state reconstruction for physical systems with Lie-group symmetries

We present a detailed discussion of a general theory of phase-space distributions, introduced recently by the authors [J. Phys. A {\bf 31}, L9 (1998)]. This theory provides a unified phase-space formulation of quantum mechanics for physical systems possessing Lie-group symmetries. The concept of generalized coherent states and the method of harmonic analysis are used to construct explicitly a family of phase-space functions which are postulated to satisfy the Stratonovich-Weyl correspondence with a generalized traciality condition. The symbol calculus for the phase-space functions is given by means of the generalized twisted product. The phase-space formalism is used to study the problem of the reconstruction of quantum states. In particular, we consider the reconstruction method based on measurements of displaced projectors, which comprises a number of recently proposed quantum-optical schemes and is also related to the standard methods of signal processing. A general group-theoretic description of this method is developed using the technique of harmonic expansions on the phase space.Comment: REVTeX, 18 pages, no figure

Justification of the symmetric damping model of the dynamical Casimir effect in a cavity with a semiconductor mirror

A "microscopic" justification of the "symmetric damping" model of a quantum oscillator with time-dependent frequency and time-dependent damping is given. This model is used to predict results of experiments on simulating the dynamical Casimir effect in a cavity with a photo-excited semiconductor mirror. It is shown that the most general bilinear time-dependent coupling of a selected oscillator (field mode) to a bath of harmonic oscillators results in two equal friction coefficients for the both quadratures, provided all the coupling coefficients are proportional to a single arbitrary function of time whose duration is much shorter than the periods of all oscillators. The choice of coupling in the rotating wave approximation form leads to the "mimimum noise" model of the quantum damped oscillator, introduced earlier in a pure phenomenological way.Comment: 9 pages, typos corrected, corresponds to the published version, except for the reference styl

Imaging fluorescence lifetime modulation of a ruthenium-based dye in living cells: the potential for oxygen sensing

Fluorescence lifetime measurements of long excited-state lifetime, oxygen-quenched ruthenium dyes are emerging as methods for intracellular oxygen sensing. Fluorescence lifetime imaging microscopy (FLIM) studies in cells have been reported previously. Many current FLIM systems use high repetition rate (∼107 Hz) lasers optimized for nanosecond lifetime measurements, making measurement of long, microsecond lifetime fluorophores difficult. Here, we present an experimental approach for obtaining a large temporal dynamic range in a FLIM system by using a low repetition rate (101 Hz), high output, nitrogen pumped dye laser and a wide-field, intensified CCD camera for image detection. We explore the feasibility of the approach by imaging the oxygen-sensitive dye tris(2,2′-bipyridyl)dichloro-ruthenium(II) hexahydrate (RTDP) in solution and in living cells. We demonstrate the ability of the system to resolve 60% variations in RTDP fluorescence lifetime upon oxygen cycling in solution. Furthermore, the FLIM system was able to resolve an increase in RTDP fluorescence lifetime in cultured human epithelial cells under diminished oxygen conditions. The technique may be useful in developing methods for quantifying intracellular oxygen concentrations.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/48916/2/d31406.pd

Fertilization induces a transient exposure of phosphatidylserine in mouse eggs

Phosphatidylserine (PS) is normally localized to the inner leaflet of the plasma membrane and the requirement of PS translocation to the outer leaflet in cellular processes other than apoptosis has been demonstrated recently. In this work we investigated the occurrence of PS mobilization in mouse eggs, which express flippase Atp8a1 and scramblases Plscr1 and 3, as determined by RT-PCR; these enzyme are responsible for PS distribution in cell membranes. We find a dramatic increase in binding of flouresceinated-Annexin-V, which specifically binds to PS, following fertilization or parthenogenetic activation induced by SrCl2 treatment. This increase was not observed when eggs were first treated with BAPTA-AM, indicating that an increase in intracellular Ca2+ concentration was required for PS exposure. Fluorescence was observed over the entire egg surface with the exception of the regions overlying the meiotic spindle and sperm entry site. PS exposure was also observed in activated eggs obtained from CaMKIIγ null females, which are unable to exit metaphase II arrest despite displaying Ca2+ spikes. In contrast, PS exposure was not observed in TPEN-activated eggs, which exit metaphase II arrest in the absence of Ca2+ release. PS exposure was also observed when eggs were activated with ethanol but not with a Ca2+ ionophore, suggesting that the Ca2+ source and concentration are relevant for PS exposure. Last, treatment with cytochalasin D, which disrupts microfilaments, or jasplakinolide, which stabilizes microfilaments, prior to egg activation showed that PS externalization is an actin-dependent process. Thus, the Ca2+ rise during egg activation results in a transient exposure of PS in fertilized eggs that is not associated with apoptosis.Fil: Curia, Claudio Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Ernesto, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Stein, Paula. University of Pennsylvania; Estados UnidosFil: Busso, Dolores. Pontificia Universidad Católica de Chile; ChileFil: Schultz, Richard. University of Pennsylvania; Estados UnidosFil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Cohen, Debora Juana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentin

Spermatogonial stem cell sensitivity to capsaicin: An in vitro study

<p>Abstract</p> <p>Background</p> <p>Conflicting reports have been published on the sensitivity of spermatogenesis to capsaicin (CAP), the pungent ingredient of hot chili peppers. Here, the effect of CAP on germ cell survival was investigated by using two testis germ cell lines as a model. As CAP is a potent agonist of the transient receptor potential vanilloid receptor 1 (TRPV1) and no information was available of its expression in germ cells, we also studied the presence of TRPV1 in the cultured cells and in germ cells in situ.</p> <p>Methods</p> <p>The rat spermatogonial stem cell lines Gc-5spg and Gc-6spg were used to study the effects of different concentrations of CAP during 24 and 48 h. The response to CAP was first monitored by phase-contrast microscopy. As germ cells appear to undergo apoptosis in the presence of CAP, the activation of caspase 3 was studied using an anti activated caspase 3 antibody or by quantifying the amount of cells with DNA fragmentation using flow cytometry. Immunolocalization was done with an anti-TRPV1 antibody either with the use of confocal microscopy to follow live cell labeling (germ cells) or on Bouin fixed paraffin embedded testicular tissues. The expression of TRPV1 by the cell lines and germ cells was confirmed by Western blots.</p> <p>Results</p> <p>Initial morphological observations indicated that CAP at concentrations ranging from 150 uM to 250 uM and after 24 and 48 h of exposure, had deleterious apoptotic-like effects on both cell lines: A large population of the CAP treated cell cultures showed signs of DNA fragmentation and caspase 3 activation. Quantification of the effect demonstrated a significant effect of CAP with doses of 150 uM in the Gc-5spg cell line and 200 uM in the Gc-6spg cell line, after 24 h of exposure. The effect was dose and time dependent in both cell lines. TRPV1, the receptor for CAP, was found to be expressed by the spermatogonial stem cells in vitro and also by premeiotic germ cells in situ.</p> <p>Conclusion</p> <p>CAP adversely affects spermatogonial survival in vitro by inducing apoptosis to those cells and TRPV-1, a CAP receptor, may be involved in this effect as this receptor is expressed by mitotic germ cells.</p

Real-time visualization of heterotrimeric G protein Gq activation in living cells

Contains fulltext : 97296.pdf (publisher's version ) (Open Access)BACKGROUND: Gq is a heterotrimeric G protein that plays an important role in numerous physiological processes. To delineate the molecular mechanisms and kinetics of signalling through this protein, its activation should be measurable in single living cells. Recently, fluorescence resonance energy transfer (FRET) sensors have been developed for this purpose. RESULTS: In this paper, we describe the development of an improved FRET-based Gq activity sensor that consists of a yellow fluorescent protein (YFP)-tagged Ggamma2 subunit and a Galphaq subunit with an inserted monomeric Turquoise (mTurquoise), the best cyan fluorescent protein variant currently available. This sensor enabled us to determine, for the first time, the kon (2/s) of Gq activation. In addition, we found that the guanine nucleotide exchange factor p63RhoGEF has a profound effect on the number of Gq proteins that become active upon stimulation of endogenous histamine H1 receptors. The sensor was also used to measure ligand-independent activation of the histamine H1 receptor (H1R) upon addition of a hypotonic stimulus. CONCLUSIONS: Our observations reveal that the application of a truncated mTurquoise as donor and a YFP-tagged Ggamma2 as acceptor in FRET-based Gq activity sensors substantially improves their dynamic range. This optimization enables the real-time single cell quantification of Gq signalling dynamics, the influence of accessory proteins and allows future drug screening applications by virtue of its sensitivity

Direct interactions of early and late assembling division proteins in Escherichia coli cells resolved by FRET

The bacterial cell division machinery is organized in the so-called divisome composed of highly dynamic but low abundant interacting (membrane-bound) proteins. In order to elucidate the molecular interactions between these proteins, we developed a robust background-insensitive quantitative spectral unmixing method for estimating FRET efficiencies at near endogenous protein levels using fluorescent protein fusions. The assembly of the division machinery of Escherichia coli occurs in two steps that are discrete in time: first the FtsZ-ring and the so-called early localizing proteins that together seem to prepare the division assembly at midcell. Subsequently, the late localizing protein complexes that contain the peptidoglycan-synthesizing proteins PBP1B and FtsI (PBP3) are recruited to the division site, which initiates septation. Physical interactions were observed between members within each group but also between the early and late localizing proteins strongly suggesting that these proteins despite their differential localization in time are linked at the molecular and functional level. Interestingly, we find FtsN, one of the latest proteins in the divisome assembly, interacting with late assembling proteins FtsI and FtsW, but also with early (proto-ring) protein ZapA. This is in line with the recently described role of FtsN in divisome stabilization including the proto-ring elements