Papers by Zygmunt Gryczynski
At the Center for Fluorescence Spectroscopy, we have taken advantage of the favorable properties ... more At the Center for Fluorescence Spectroscopy, we have taken advantage of the favorable properties of surface plasmon-coupled emission (SPCE) to improve fluores- cence-based immunoassays. SPCE occurs when excited fluorophores near conducting metallic structures effi- ciently couple to surface plasmons. These surface plas- mons, appearing as free electron oscillations in the metallic layer, produce electromagnetic radiation that preserves the spectral properties
Springer Series in Optical Sciences, 2007
Molecular Cancer, 2015
Migration and invasion enhancer 1 (MIEN1) is a novel gene found to be abundantly expressed in bre... more Migration and invasion enhancer 1 (MIEN1) is a novel gene found to be abundantly expressed in breast tumor tissues and functions as a critical regulator of tumor cell migration and invasion to promote systemic metastases. Previous studies have identified post-translational modifications by isoprenylation at the C-terminal tail of MIEN1 to favor its translocation to the inner leaflet of plasma membrane and its function as a membrane-bound adapter molecule. However, the exact molecular events at the membrane interface activating the MIEN1-driven tumor cell motility are vaguely understood. MIEN1 was first studied using in-silico analysis on available RNA sequencing data of human breast tissues and its expression was ascertained in breast cells. We performed several assays including co-immunoprecipitation, wound healing, western blotting and immunofluorescence to decipher the molecular events involved in MIEN1-mediated tumor cell migration. Clinically, MIEN1 is predominantly overexpressed in Her-2 and luminal B subtypes of breast tumors, and its increased expression correlates with poor disease free survival. Molecular studies identified a phosphorylation-dependent activation signal in the immunoreceptor tyrosine based activation motif (ITAM) of MIEN1 and the phosphorylation-deficient MIEN1-mutants (Y39F/50 F) to regulate filopodia generation, migration and invasion. We found that ITAM-phosphorylation of MIEN1 is significantly impaired in isoprenylation-deficient MIEN1 mutants indicating that prenylation of MIEN1 and membrane association is required for cross-phosphorylation of tyrosine residues. Furthermore, we identified MIEN1 as a novel interactor of Annexin A2 (AnxA2), a Ca(2+) -dependent phospholipid binding protein, which serves as an extracellular proteolytic center regulating plasmin generation. Fluorescence resonance energy transfer (FRET) confirmed that MIEN1 physically interacts with AnxA2 and functional studies revealed that they mutually cooperate to accentuate tumor cell motility. Interestingly, our study identified that ectopic overexpression of MIEN1 significantly enhances Tyr23-phosphorylation on AnxA2, thereby stimulating cell surface translocation of AnxA2 and catalyzing the activation of its proteolytic activity. Our data show that the presence and interaction of both MIEN1 and AnxA2 in breast tumors are crucial drivers of cell motility. Our study has now deciphered a novel regulatory network governing the vicious process of breast tumor cell invasion-metastasis, and findings suggest MIEN1-AnxA2 as prospective targets to counter the deadly disease.
Biochemistry, 2009
Clinical studies have revealed that the D166V mutation in the ventricular myosin regulatory light... more Clinical studies have revealed that the D166V mutation in the ventricular myosin regulatory light chain (RLC) can cause a malignant phenotype of familial hypertrophic cardiomyopathy (FHC). It has been proposed that RLC induced FHC in the heart originates at the level of the myosin crossbridge due to alterations in the rates of cross-bridge cycling. In this report we examine whether the environment of an active cross-bridge in cardiac myofibrils from transgenic (Tg) mice is altered by the D166V mutation in RLC. The cross-bridge environment was monitored by tracking the fluorescence lifetime (τ) of Alexa488-phalloidin labeled actin. The fluorescence lifetime is the averaged rate of decay of a fluorescent species from the excited state, which strongly depends on various environmental factors. We observed that the lifetime was high when cross-bridges were bound to actin and low when they were dissociated from it. The lifetime was measured every 50 msec from the center half of the I-band during 60 sec of rigor, relaxation and contraction of muscle. We found no differences between lifetimes of Tg-WT and Tg-D166V muscle during rigor, relaxation and contraction. The duty ratio expressed as a fraction of time that cross-bridges spend attached to the thin filaments during isometric contraction was similar in Tg-WT and Tg-D166V muscles. Since independent measurements showed a large decrease in the cross-bridge turnover rate in Tg-D166V muscle compared to Tg-WT, the fact that the duty cycle remains constant suggests that the D166V mutation of RLC causes a decrease in the rate of cross-bridge attachment to actin. Despite significant clinical advances in the treatment of various cardiovascular diseases, mortality rates remain high. Familial hypertrophic cardiomyopathy (FHC) is one pathological manifestation of the heart resulting from its inability to adequately pump blood, thus leading to premature fatigue, dyspnea, hypertrophy and/or cardiac failure (1,2). FHC is an autosomal dominant disease originating from mutations in genes that encode for the major contractile proteins of the heart, including the ventricular myosin regulatory light chain (RLC) (for review see (3,4)). It is characterized by ventricular and septal hypertrophy, myofibrillar disarray, abnormal ECG findings and sudden cardiac death (SCD) at a young age (5,6). Clinical studies have revealed that the D166V mutation in myosin RLC is associated with a malignant FHC disease phenotype (7). Our objective was to examine the molecular determinants of the D166V
Methods in Enzymology, 1997
G protein-coupled receptors represent the largest class of drug discovery targets. Drugs that act... more G protein-coupled receptors represent the largest class of drug discovery targets. Drugs that activate G protein-coupled receptors are classified as either agonists or partial agonists. To study the mechanism whereby these different classes of activating ligands modulate receptor function, we directly monitored ligand-induced conformational changes in the G protein-coupling domain of the  2 adrenergic receptor. Fluorescence lifetime analysis of a reporter fluorophore covalently attached to this domain revealed that, in the absence of ligands, this domain oscillates around a single detectable conformation. Binding to an antagonist does not change this conformation but does reduce the flexibility of the domain. However, when the  2 adrenergic receptor is bound to a full agonist, the G protein coupling domain exists in two distinct conformations. Moreover, the conformations induced by a full agonist can be distinguished from those induced by partial agonists. These results provide new insight into the structural consequence of antagonist binding and the basis of agonism and partial agonism.
Methods in Enzymology, 2003
Novel approaches to sensor design, based on the use of an internal standard with appropriate spec... more Novel approaches to sensor design, based on the use of an internal standard with appropriate spectral properties, provide new possibilities for designing simple devices for fluorescence sensing. Detection of combined emission from the reference and an analyte-sensitive fluorophore has been achieved in numerous measurements in cuvettes,7–10,42,43 tissues,46,47 and high-throughput formats.48 These methods have been used with a long-lifetime reference to
Nanoscale Imaging, Sensing, and Actuation for Biomedical Applications VII, 2010
Scattering from noble metallic nanoparticles with specific structures are strongly depolarized in... more Scattering from noble metallic nanoparticles with specific structures are strongly depolarized in contrast to dielectric particles. The effect depends on the shape and symmetry of the nanoparticles and can be explained by induced plasmonic multi-resonances along different axes of ...
PloS one, 2015
Astrocytes outnumber neurons and serve many metabolic and trophic functions in the mammalian brai... more Astrocytes outnumber neurons and serve many metabolic and trophic functions in the mammalian brain. Preserving astrocytes is critical for normal brain function as well as for protecting the brain against various insults. Our previous studies have indicated that methylene blue (MB) functions as an alternative electron carrier and enhances brain metabolism. In addition, MB has been shown to be protective against neurodegeneration and brain injury. In the current study, we investigated the protective role of MB in astrocytes. Cell viability assays showed that MB treatment significantly protected primary astrocytes from oxygen-glucose deprivation (OGD) & reoxygenation induced cell death. We also studied the effect of MB on cellular oxygen and glucose metabolism in primary astrocytes following OGD-reoxygenation injury. MB treatment significantly increased cellular oxygen consumption, glucose uptake and ATP production in primary astrocytes. In conclusion our study demonstrated that MB pro...
Methods in enzymology, 2003
Novel approaches to sensor design, based on the use of an internal standard with appropriate spec... more Novel approaches to sensor design, based on the use of an internal standard with appropriate spectral properties, provide new possibilities for designing simple devices for fluorescence sensing. Detection of combined emission from the reference and an analyte-sensitive fluorophore has been achieved in numerous measurements in cuvettes, tissues, and high-throughput formats. These methods have been used with a long-lifetime reference to measure pH, O2, pCO2, glucose, and calcium by means of modulation-sensing methods as well as by the use of oriented films as the reference for polarization sensing of glucose, pH, oxygen, and lactate. Polarization sensing has also been developed with visual detection to measure the concentration of rhodamine B and pH. Modulation and polarization sensing was found to be effective in highly scattering media such as Intralipid or tissue. The applicability of these technologies to transdermal diagnostics depends on the availability of red fluorophores that...
Journal of nanophotonics, Jan 22, 2010
Fluorescence intensity changes were investigated theoretically and experimentally using self-asse... more Fluorescence intensity changes were investigated theoretically and experimentally using self-assembled colloidal structures on silver semitransparent mirrors. Using a simplified quasi-static model and finite element method, we demonstrate that near-field interactions of metallic nanostructures with a continuous metallic surface create conditions that produce enormously enhanced surface plasmon resonances. The results were used to explain the observed enhancements and determine the optimal conditions for the experiment. The theoretical parts of the studies are supported with reports on detailed emission intensity changes which provided multiple fluorescence hot spots with 2-3 orders of enhancements. We study two kinds of the fluorophores: dye molecules and fluorescent nanospheres characterized with similar spectral emission regions. Using a lifetime-resolved fluorescence/reflection confocal microscopy technique, we find that the largest rate for enhancement (~1000-fold) comes from lo...
Molecular cell, Jan 21, 2005
During eukaryotic translation initiation, ribosomal 43S complexes scan mRNAs for the correct AUG ... more During eukaryotic translation initiation, ribosomal 43S complexes scan mRNAs for the correct AUG codon at which to begin translation. Start codon recognition triggers GTP hydrolysis, committing the complex to engagement at that point on the mRNA. While fidelity at this step is essential, the nature of the codon recognition event and the mechanism by which it activates GTP hydrolysis are poorly understood. Here we report the changes that occur within the 43S.mRNA complex in response to AUG codon recognition. eIF1 and eIF1A are key players in assembly of 43S.mRNA complexes capable of locating initiation codons. We observed FRET between these two factors when bound to the 40S subunit. Using steady-state FRET, anisotropy, and kinetic analyses, we demonstrate that start codon recognition results in a conformational change and release of eIF1 from the ribosome. These rearrangements probably play a role in triggering GTP hydrolysis and committing the complex to downstream events.
Proceedings - Society of Photo-Optical Instrumentation Engineers, 2004
We describe the development of a novel generic approach to fluorescence sensing based on metal-en... more We describe the development of a novel generic approach to fluorescence sensing based on metal-enhanced fluorescence (MEF). This work follows our initial reports of radiative decay engineering (RDE), where we experimentally demonstrated dramatic signal enhancements of fluorophores positioned close to surface-bound silver nanostructures. The attractive changes in spectral properties of fluorophores includes increased rates of excitation, increased quantum yields, decreased fluorescence lifetimes with an increased photostability, and drastically increased rates of multi-photon excitation. In this report we present a new class of fluorescent biomarkers which are strongly enhanced by metallic particles. This has afforded the development of a novel generic approach for ultra-sensitive fluorescence assay technology. The assay platform utilizes metal particles deposited on glass/quartz surfaces, covered with sub-nanometer layers of a fluorescent biomarker. As such the fluorescence signal o...
RSC Adv., 2015
A symmetrical BODIPY-BODIPY dyad with a diyne linker was prepared in two steps; the lifetime deca... more A symmetrical BODIPY-BODIPY dyad with a diyne linker was prepared in two steps; the lifetime decay of this rotor appeared to correlate with the viscosity of the media, thus making this dyad a suitable small molecule viscometer for molecular solvents. The potential of using the rotor to probe the viscosity of ionic liquids was also investigated.
Metallic surfaces can have unusual effects on fluoro- phores such as increasing or decreasing the... more Metallic surfaces can have unusual effects on fluoro- phores such as increasing or decreasing the rates of radiative decay and the rates of resonance energy trans- fer (RET). In the present article we describe the effects of metallic silver island films on the emission spectra, lifetimes, and energy transfer for several fluorophores. The fluorophores are not covalently coupled to the
Methods in Cell Biology, 2004
... Fluorescence methods have been extended to high-throughput studies of gene expression using t... more ... Fluorescence methods have been extended to high-throughput studies of gene expression using the so-called gene ... For thick dielectric, both p and s polarizations present positive imaginary admittance, so multiple plasmon resonance may ... Surface-PlasmonCoupled Emission. ...
Thin Solid Films, 2006
Surface plasmon-coupled emission (SPCE) phenomenon is the coupling of excited fluorophores near a... more Surface plasmon-coupled emission (SPCE) phenomenon is the coupling of excited fluorophores near a silver film with surface plasmons, resulting in directional emission into the underlying glass substrates. We report a complex coupling of Nile Blue fluorophore with 50 nm silver mirror, resulting in emission at several angles in the glass substrate, with either s or p polarization. This complex pattern of directional and polarized emission appears to be due to optical waveguide effects occurring when the sample thickness becomes comparable to the emission wavelength. We expect waveguide-modulated SPCE to have applications to biophysics and sensing. D
Biopolymers, 2003
The fluorescence properties of a monolayer of labeled avidin molecules were studied near silver i... more The fluorescence properties of a monolayer of labeled avidin molecules were studied near silver island films. We first adsorbed a monolayer of biotinylated-BSA as a base that was used to capture labeled avidin molecules. For labeled avidin on silver island films, we observed an increase of the fluorescence intensity of between 18 and 80 with one-photon excitation and up to
Plasmonics in Biology and Medicine, 2004
Directional fluorescence emission of a sulforhodamine 101 in polyvinyl alcohol film has been obse... more Directional fluorescence emission of a sulforhodamine 101 in polyvinyl alcohol film has been observed from samples deposited on semi-transparent silver mirror. The fully p-polarized fluorescence emerges through the glass prism in form of hollow cone. The angle of this cone of emission depends on the thickness of the sample, and does not depend on the mode of excitation. The angular dependence of surface plasmon-coupled emission (SPCE) on the sample thickness has been discussed as well as its relevance to the surface plasmon resonance (SPR) analysis.
Plasmonics in Biology and Medicine II, 2005
We have recently shown that metallic particles or colloids when deposited on the transparent surf... more We have recently shown that metallic particles or colloids when deposited on the transparent surface can enhance fluorescence properties of nearby fluorophores. We obtained the fluorophore-metal colloid complexes that display significant fluorescence signal enhancement in solution. Silver nanoparticles (about 20-50 nm size) were synthesized as a stable yellow colloidal solution, and coated with proteins labeled with fluorophores. A several-fold amplification
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Papers by Zygmunt Gryczynski