Browsing by Subject "Fluorescence"

Now showing 1 - 7 of 7
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    Benzoxazole and Benzothiazole Derivatives as Potential Fluorescence Imaging Agents
    (2019-08) Geppert, Sarah
    Abstract: Substituted Benzoxazole (1-hydroxy-2-naphthoic benzoxazole and 2-hydroxy- 3-naphthoic benzoxazole) and benzothiazole (1-hydroxy-2-naphthoic benzoxazole and 2- hydroxy-3-naphthoic benzothiazole) derivatives previously complexed with Boroncontaining fragments in our lab, were reviewed for potential use in Fluorescence Lifetime Imaging Microscopy (FLIM) with Differential Interference Contrast (DIC) microscopy in adherent cells. The Excited State Intramolecular Proton Transfer (ESIPT), excited state tautomerization, and potential chelating properties inherent to these compounds prompted our research. The flexible nature of the molecules in two key bonds, when compared to that of other fluorophores, contributed to an inherent quantum inefficiency resulting in reduced brightness when compared to commercially available fluorescent dyes like fluorescein. The quantum chemical study of the photochemistry and conformational preference of these compounds was not able to accurately predict absorbance characteristics, but provided insight into the ESIPT process and established a strong conformational preference for one conformer. The unique properties of these ligands and similar compounds may still be of biophotochemical interest and warrant further investigation.
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    Examining the Role of Phospholamban Phosphorylation on Interaction with SERCA Using Fluorescence Microscopy
    (2018-07) Haydon, Suzanne
    Regulation of the Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) by Phospholamban (PLB) plays a crucial role in normal cardiomyocyte function through controlling the speed and extent of myocyte relaxation. The interaction between PLB and SERCA is altered in many forms of heart failure (HF), making these proteins potential targets for the treatment of HF. Both proteins have been extensively studied in vitro, where their basic structure and function were determined, and in animal models, where their role in disease was examined. However, key information connecting the in vitro experiments and animal models is needed to better understand the PLB-SERCA interaction and to design effective HF treatment strategies. In particular, we wanted to examine two conflicting in vitro models of how the PLB-SERCA interaction changes after PLB phosphorylation: the dissociation model and the structural model. In the dissociation model, phosphorylation causes PLB to dissociate from SERCA, while in the structural model, phosphorylation causes a shift in the PLB binding position along SERCA. In order to determine the correct model of PLB-SERCA interaction in live cells, we expressed cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to the N-termini of SERCA and PLB respectively, in HEK293 cells for fluorescence resonance energy transfer (FRET) microscopy experiments. We were able to use the native beta-adrenergic signaling system in the cells to control the state of PLB phosphorylation in a time-dependent manner. For the dissociation model to be true, we expected to see a significant reduction in FRET between CFP-SERCA and YFP-PLB after PLB phosphorylation. While significant increases in PLB phosphorylation were produced in the cells, FRET did not decrease. Instead, FRET increased with PLB phosphorylation at serine 16, indicating either a shorter distance between PLB and SERCA, or higher binding of PLB to SERCA. As the beta-adrenergic signal progressed through the cells, causing phosphorylation of PLB at threonine 17, FRET returned towards basal levels, but did not show the decrease below basal FRET levels that would indicate PLB dissociation from SERCA. Thus, we determined that there is a subtle change in the PLB-SERCA interaction due to PLB phosphorylation rather than a large scale dissociation. In order to differentiate changes in distance from changes in binding time-resolved (TR)-FRET experiments were required. Fluorescence lifetime imaging microscopy (FLIM) is a variant of TR-FRET that measures fluorescence decay curves with a fast-pulsed laser and photon counting board attached to a confocal microscope. These fluorescence decay curves provide more information than intensity measurements since they can be fit to multiple exponentials to test different interaction models. FLIM is a relatively new technique, thus we worked on developing appropriate experimental conditions for acquiring fluorescence decays that contained enough photons for multi-exponential fitting while still measuring individual cells. We were able to use FLIM to measure FRET values similar to those acquired on standard fluorescence microscopes and confirmed that phosphorylation of PLB did not cause dissociation from SERCA. However, further improvements to FLIM acquisition and analysis are needed for the multi-exponential fitting to provide a better model of PLB-SERCA interaction in live cells.
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    Exploring anaerobic fluorescence in Methanococcus maripaludis with the fluorescence-activating and absorption-shifting tag (FAST)
    (2022-06) Hernandez, Eric
    Live-cell fluorescence imaging in methanogenic archaea has been limited due to the strictly anoxic conditions required for growth and issues with autofluorescence associated with electron carriers in central metabolism. Here, we show that the fluorescence-activating and absorption-shifting tag (FAST) when complexed with the fluorogenic ligand 4-hydroxy-3-methylbenzylidene-rhodanine (HMBR) overcomes these issues and displays robust fluorescence in Methanococcus maripaludis. We also describe a mechanism to visualize cells under anoxic conditions using a fluorescence microscope. Derivatives of FAST were successfully applied for protein abundance analysis, subcellular localization, and determination of protein-protein interactions. FAST fusions to both formate dehydrogenase (Fdh) and F420-reducing hydrogenase (Fru) displayed increased fluorescence in cells grown on formate containing medium, consistent with previous studies suggesting increased abundance of these proteins in the absence of H2. Additionally, FAST fusions to both Fru and the ATPase associated with the archaellum (FlaI) showed membrane localization in single cells observed using anoxic fluorescence microscopy. Additionally, split reporter translationally fused to the alpha and beta subunits of Fdh reconstituted a functionally fluorescent molecule in vivo via bimolecular fluorescence complementation. Lastly, we demonstrate an example in which FAST is able to validate the localization of an uncharacterized protein and provide a framework for future multi-color imaging that would empower further studies going forward. Together, these observations demonstrate the utility of FAST as a tool for studying members of the methanogenic archaea. Portions of this thesis have been submitted for publication in The Journal of Bacteriology.
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    A Fluorescence Assessment of the Intracellular Trafficking of the Amyloid Precursor Protein within the Secretory Pathway
    (2020-08) Rodriguez, Kassidy
    Amyloid-β (Aβ) plaque formation in the brain is a major hallmark of Alzheimer’s disease (AD) and has been linked to known symptoms. According to the amyloid cascade hypothesis, Aβ is generated from the cleavage of amyloid-β precursor protein (APP) by BACE1 followed by the regulated intramembrane proteolysis (RIP) by γ-secretase. Aβ plaques are then formed through the extracellular deposition and aggregation of Aβ proteins. Current research suggests that BACE1 dimerizes in cells in a substrate-mediated manner, and that these dimers exhibit a much higher catalytic activity than their monomeric forms within acidic environments, such as endosomal compartments. It was also reported that the interaction between APP and BACE1 increased Aβ production. To date, there has been no research completed using HEK293 cells that compares the degree of co-localization of APP within specific endosomal compartments in the secretory pathway. The co-residence of APP and BACE1 in the same subcellular location would likely favor their interaction and facilitate the proteolysis of APP. Therefore, the study of their intracellular trafficking and localization should indicate where BACE1 is likely to process APP. BACE1 is an attractive therapeutic target for slowing the production of Aβ in the early stages of AD, further highlighting the importance of its study. In this contribution, we describe our efforts to examine where APP-GFP resides along the secretory pathway using fluorescently labeled Rab proteins that target distinct regions of the secretory pathway as points of reference. In addition, we observed an increase in the overall fluorescence intensity in cells co-transfected with APP-GFP and BACE1-YFP. HEK293 cells were transiently transfected with APP-GFP and either Rab5-DsRed, Rab7-DsRed, Rab11-DsRed, or BACE1-YFP fluorescent fusion proteins then imaged using widefield fluorescence and differential interference contrast (DIC) microscopy. The subcellular localization and distribution of each fluorescent fusion protein construct was determined by the colocalization of GFP and DsRed by evaluating Pearson’s correlation coefficients (PCC), Manders’ overlap coefficients (MOC), and Li’s intensity correlation quotients (ICQ). Fluorescence images were analyzed using the JACoP plug-in within FIJI/ImageJ. The results of this study provide evidence that APP-GFP has the highest degree of colocalization with Rab11-DsRed, suggesting that the APP concentration is highest within the recycling endosomes. HEK293 cells co-expressing APP-GFP and BACE1-YFP exhibited an increased fluorescence intensity compared to cells expressing only APP-GFP. These results establish baseline measurements for future cell-based studies of BACE1 with APP-GFP as its substrate.
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    Fluorescence tools to identify Novel SERCA activators
    (2013-08) Gruber, Simon Joseph
    One of the universal hallmarks of heart failure is defective calcium cycling. The calcium concentration in a muscle cell must be high to cause contraction and low to allow relaxation, and most of the calcium removal is accomplished by the intracellular membrane pump known as the sarco-endoplasmic reticulum calcium ATPase (SERCA). When SERCA activity is too low in cardiac muscle, the heart does not fully relax and fill with blood, so the next contraction cannot pump enough blood through the body. The ubiquity of calcium cycling dysfunction in heart failure and other muscle diseases has made SERCA a major target for novel heart failure therapeutics since the late 1990s. All of the work presented in this thesis focuses on methods to activate SERCA as a treatment for heart failure. SERCA is regulated by phospholamban (PLB) in heart muscle, preventing the enzyme from being fully active all the time but allowing maximal activity when the body demands. Some methods of activating SERCA seek to remove the inhibitory effects of PLB, either partially or fully. In this thesis, PLB mutants are investigated as potential gene therapy vectors. PLB mutants that are less inhibitory but still bind to SERCA could allow the enzyme to be more active if they displace endogenous PLB. A FRET assay using genetically engineered fluorescent fusions of SERCA and PLB expressed stably in a human cell line was used to measure the ability of different mutants to compete for SERCA binding. Fluorescently labeled SERCA and PLB were also reconstituted in an in vitro lipid bilayer system to screen for small-molecule compounds that activate SERCA. Several compounds were found to decrease SERCA-PLB FRET and many of these turned out to be SERCA activators that improved myocyte contractility. However, none of the compounds were specific to the SERCA-PLB interaction. Finally, an intramolecular FRET assay was developed to detect changes in the relative distance between cytoplasmic domains within SERCA in living cells. This assay was used to screen a small-scale compound library to show that FRET between SERCA domains is sensitive to both activators and inhibitors of SERCA function. All of these FRET assays are being followed up in the Thomas lab to identify potential SERCA activators for heart failure and other diseases.
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    Supporting data for The fluorescence-activating and absorption-shifting tag (FAST) enables live-cell fluorescence imaging of Methanococcus maripaludis
    (2022-05-06) Hernandez, Eric; Costa, Kyle; kcosta@umn.edu; Costa, Kyle; University of Minnesota Costa Lab
    Microscopy images of Methanococcus maripaludis cells expressing FAST1 tagged to different genes involved in methanogenesis. Images were used for quantification of protein expression in live cells.
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    Thermo-activated drug release.
    (2010-09) Zeng, Pengyun
    Inhalation is an effective means of drug administration for treatment of respiratory diseases. Development of a respirable, stimuli-responsive aerosol formulation would further enhance the drug delivery efficiency. In this thesis, it is postulated that a magnetite/lipid formulation stimulated by alternating magnetic fields can be adapted for use as a thermal-activated delivery system to achieve the desired dose and temporal control of drug release. To test this hypothesis, the following specific aims were carried out: (1) Determine the thermal response of superparamagnetic nanoparticles (SPNs) to alternating magnetic fields, (2) Evaluate the release of solute from temperature sensitive aerosol particles, (3) Assess magnetic-activated release of drug from a lipid matrix, and (4) Study the feasibility of magnetic-activated release of solutes with varying polarity from lipid particles. SPNs heat production was found to be quantitatively consistent with theory, and incorporation of SPNs into solid lipid matrices allowed magnetic heating. For the second aim, thermal activation was shown to be necessary and sufficient for the release of encapsulated solute using naturally occurring lipids. For the third aim, stimuli sensitive release of a test solute was demonstrated, which coincided with melting of the matrix. As such, "on-off" drug release was shown to be controlled by a magnetic field. The release was diffusion controlled, such that existing transport theory can be used to guide the development of delivery systems with appropriate release characteristics. Finally, solid lipid particles containing test compounds were characterized and assessed in vitro for thermal and magnetic stimuli release. Surface release and particle erosion mechanism were suggested for nanoparticles containing a hydrophobic compound. For the release from microparticles, magnetic activation was observed in microscopic images. Magnetic activated release was detected for core-lipid shell particles containing a hydrophilic solute, which may be a consequence of physical rotation of the SPNs. A quantitative framework was established to judge the feasibility of developing a magnetic-sensitive drug delivery system that is also respirable. In light of this analysis, significant practical challenges were revealed that make this approach impractical with currently available technology.