Coral reefs are a critical resource to developing and developed nations world wide. Providing shelter, food, monetary value, and a vast resource of ecological wealth, the corals of the reefs underpin an entire ecosystem. Climate change, driven by increased greenhouse gases, is raising the temperature of Earth’s waters and atmosphere, while making the planet’s oceans increasingly acidic. Brightly lit and increasingly warm tropical waters present a potentially challenging environment in which scleractinian corals grow. In attempting to cope with the competing stresses of intense photon flux density (PFD) and anomalously high sea surface temperatures, corals and dinoflagellates exhibit myriad biochemical and physiological adaptations. Pocilloporins, a diverse group of non-fluorescent green fluorescent protein (GFP) homologs found across Cnidaria and beyond, are one such adaptation within the tissues of heavily pigmented scleractinian corals. Chemically unique amongst pigments, GFP-like pigments exist as pure protein chromophores and exhibit little to no cytotoxicity when naturally occurring. This non-fluorescent class of GFP-like pigments has found popularity in biochemical and biotechnological applications, though an ecological and evolutionary explanation for the heavy conservation of pocilloporins across a broad range of scleractinian corals and related cnidaria is still a subject of scientific research and debate. This thesis supports the hypothesis that pocilloporins act as a naturally occurring photoprotective pigment in reef-building corals, specifically acting to filter and regulate the light environment within coral polyps. In examining the role of pocilloporins in Scleractinia, the need to examine environmental sources of pigment production induction and suppression, the localization of pigments within coral tissues and cells, and the ability of coral colonies to direct resource allocation with regards to pocilloporin production were identified as lines of inquiry. Briefly, for experiments examining either pocilloporin induction or suppression, the following aspects were studied: holobiont responses in the form of mRNA signal expression, host pigment isolation and analysis, dinoflagellate density and pigmentation sampling, and chlorophyll fluorescence of live corals. Blue morph Acropora aspera, common to the reef flat of Heron Island (Great Barrier Reef, Australia), were subjected to 99% shade and thermal bleaching threshold temperatures in separate attempts to suppress pocilloporin expression, while red morph Montipora monasteriata was transplanted at equivalent depth from their natural cave environments to exposed portions of the spur and groove formations of the northern face of Wistari Reef (Great Barrier Reef, Australia). Both ambient temperature and heat-stressed A. aspera were concurrently collected during the thermal stress experiment and placed in preservatives for immuno-histochemical localization of pocilloporins with their tissues. Finally, radio-labelled glycine, a very common amino acid in the primary sequence of pocilloporin, was injected into artificially injured tan morph Montipora monasteriata, also on the northern face of Wistrai Reef to study the uptake of dissolved organic materials (DOM) and incorporation of metabolic resources into newly generated pigments. Pocilloporins proved easier to induce in this work than suppress, and the location of these pigments in A. aspera tissues suggests a potential mechanism. The data demonstrated the presence of pocilloporins in the most directly exposed epidermal and gastrodermal tissues of the coral polyp, specifically the outermost layers of epidermis and gastrodermal layers bordering directly upon the gastrovascular cavity. Closer inspection through anti-pocilloporin-gold stained TEM images was highly suggestive of pocilloporin secretion in coral mucus, a theory separately supported by observations of coral mucus in collected live corals. Neither suppression experiment induced heavy mucus sloughing in A. aspera, so despite multi-fold reductions in pocilloporin mRNA as a result of applied stimuli, the continued presence of pocilloporin aaCP592 in blue morph A. aspera is not surprising. Conversely, pocilloporin msCP576 in plating Montipora monasteriata was induced in response to both general increases in PFD and specific increases of PFD at the sites of physical injury. Additionally, tan morph Montipora monasteriata demonstrated the capacity to collect and allocate DOM from the environment to assist in the production of new pigments and tissues, an energetically expensive process. The reduction of the orange-red spectrum in favour of the blue light ranges is generally beneficial to the photosynthetic systems of both higher plants and the resident dinoflagellates of corals. msCP576 and aa592, both positively identified as pocilloporins within this work, absorb within the orange-red region and apparently act as a photoprotective filter in all exposed surfaces of heavily pigmented corals, enhancing the blue spectrum of incident and reflected PFD and generally regulating the internal light environment.

Thesis (Ph. D.)--Cornell University, May, 2007. / Vita. Includes bibliographical references (leaves 101-107).

In the current work, the photophysics and photochemistry of the phototoxic red fluorescent protein (RFP) KillerRed was investigated. KillerRed's phototoxicity makes it useful for studying oxidative stress on cell physiology and for cell killing in photodynamic therapy. Spectroscopic probes were used to show that the phototoxicity of KillerRed stems primarily from a type I photosensitization mechanism producing radicals. The production of radicals was supported by electron paramagnetic resonance (EPR) studies, where a long-lived radical was observed in KillerRed and two other RFPs (mRFP and DsRed) following excitation. Transient absorption spectroscopy, various other spectroscopic techniques, and the published crystal structure of KillerRed indicate that the long-filled water channel is likely responsible for the increased phototoxicity of KillerRed. In the blue fluorescent protein (BFP) mKalama1, some of the same techniques were applied to understand the photophysics and photochemistry on the timescale ranging from femtoseconds to seconds. Transient absorption spectroscopy and previously published results demonstrate that two-photon excitation of mKalama1 likely results in the formation of a radical cation and solvated electrons. This may explain the blinking behavior which has been observed on the single molecule level for many fluorescent proteins, the identity of which has remained elusive. It was also shown that the chromophore, while neutral in the ground state, does not exhibit excited-state proton transfer (ESPT) during its nanosecond excited-state lifetime; however, the chromophore undergoes a deprotonation in the ground state after electronic relaxation. This work plays a key role in our understanding of fluorescent proteins and will help pave the way to developing new ones. The research on the BFPs was extended to improve them for cellular imaging. This was accomplished by identification of dark states in the BFPs which are longer in wavelength than the collected fluorescence. Using dual lasers, it was shown that these dark states could be optically depleted, thereby increasing the overall fluorescence without enhancing the background fluorescence. Rational site-directed mutagenesis was carried out on the BFPs and the mutants were screened for fluorescence enhancement. These proteins were then analyzed using transient absorption spectroscopy to elucidate the identity of the dark state(s) used for fluorescence enhancement.

Fluorescence resonance energy transfer (FRET)-based biosensors have been designed to fluorometrically detect everything from proteolytic activity to receptor-ligand interactions and structural changes in proteins. While a wide variety of fluorophores have demonstrated effectiveness in FRET probes, several potential sensor components are particularly notable. Semiconductor quantum dots (QDs) are attractive FRET donors because they are rather bright, exhibit high quantum yields, and their nanoparticulate structure enables the attachment of multiple acceptor molecules. Fluorescent proteins (FPs) are also of particular interest for fluorescent biosensors because design elements necessary for signal transduction, probe assembly, and device delivery and localization for intracellular applications can all be genetically incorporated into the FP polypeptide. The studies described in this thesis elucidate the important parameters for concerted QD-FP FRET probe design. Experimental results clarify issues of FRET pair selection, probe assembly, and donor-acceptor distance for the multivalent systems. Various analysis approaches are compared and guidelines asserted based on the results. To demonstrate the effectiveness of the QD-FP FRET probe platform, a ratiometric pH sensor is presented. The sensor, which uses the intrinsic pH-sensitivity of the FP mOrange to modulate the FP/QD emission ratio, exhibits a 20-fold change in its ratiometric measurement over a physiologically interesting pH range, making it a prime candidate for intracellular imaging applications.

Optical modulation has shown the selective and sensitive signal improvement in high background systems in cell imaging; however, cell applications are still limited due to biocompatibility and delivery issues. Fluorescent proteins have a variety of optically accessible states that make them ideal candidates for investigation of modulatability. Combining the optical modulation technique with the biocompatibility of fluorescent proteins is a major advance. This work focuses on evaluation fluorescent proteins and their optical states for modulation, as well demonstrations of cellular imaging. Herein, we evaluate a green fluorescent protein with interesting photophysical properties favorable for optical modulation. Positive for optical modulation, further investigation of the state dictating modulation reveals the presence of a slow component on the order of milliseconds. To better understand the mechanism responsible modulation, blue fluorescent proteins are created to modify the chromophore environment. Extraction of photophysics confirm the alteration timescales of the modulated state. Motivated by the ability to improve imaging and decode hidden dynamics, demodulation of these proteins demonstrates the selective recovery of signal in the presence of high cellular background. The continued investigation of several other fluorescent proteins identifies modulatable proteins across the visible wavelength region. Additionally, solvent environmental factors show varying timescales which, when combined with mutagenesis, suggest a cis/trans isomerization coupled with a proton transfer. This information of the properties dictating optical modulation allows for the engineering of improved modulatable proteins to study cellular dynamics.

Genetically identical cells show a heterogeneous response to a multitude of signals such as growth factors and DNA damage. While this heterogeneity has been shown to be a major determinant of treatment success in several diseases including cancer, little is known about how differences in biochemical signalling networks underlie such heterogeneity. State-of-the-art methodologies to study biochemical networks are often invasive and enable to quantify biochemical events only on cell populations or at a single point in time for a single cell, and therefore, cannot adequately quantify the fast, asynchronous and heterogeneous responses. In order to address these limitations, we have developed a unique sensing platform based on fluorescence lifetime imaging microscopy (FLIM) capable to multiplex at least three biosensors by utilizing Förster Resonance Energy Transfer (FRET) efficiently. After an overall introduction in Chapter 1, I describe the rational design and characterization of novel FRET pairs aiming to utilize the visible spectrum efficiently in combination with FLIM in Chapter 2. We combined blue, green and red donor fluorescent proteins that are excited at the same wavelength (840 nm for two-photon excitation) with genetically encoded quenchers, i.e. non-fluorescent chromoproteins as acceptors. This sensing platform enables the simultaneous detection of three biochemical reactions within single living cells providing new opportunities to characterize and understand non-genetic heterogeneity. In Chapter 3, I will demonstrate the first application of this novel platform by studying the activity of three key enzymes in DNA damage-induced cell death, caspase-2, -3, and -9. We confirm the heterogeneous nature of Cisplatin-induced cell death in genetically identical cells but reveal the existence of at least three subpopulations of cells characterized by distinct caspase dynamics. By combining biochemical and morphological information we infer the existence of different biochemical network topologies that are associated with alternative death phenotypes each cell adopts, such as apoptosis and programmed necrosis. Finally, deconvolution of cellular populations and direct measurement of a three-node caspase network - formerly impossible - permitted us to design perturbations of cell fate choices utilizing clinically relevant inhibitors. These perturbations resulted in changes in cell fate in response to Cisplatin, a clinically desirable outcome that suggests new avenues for combinatorial drugging and a new strategy to reveal cancer vulnerabilities that may be otherwise confounded by typical genetic and non-genetic heterogeneity.

Oxidative stress is a hallmark of several aging and trauma related neurological disorders, but the precise details of how altered neuronal activity elicits subcellular redox changes have remained difficult to resolve. Current redox sensitive dyes and fluorescent proteins can quantify spatially distinct changes in reactive oxygen species levels, but multicolor probes are needed to accurately analyze compartment-specific redox dynamics in single cells that can be masked by population averaging. Our lab previously engineered a genetically-encoded red-shifted redox-sensitive fluorescent protein sensors using a Förster resonance energy transfer relay strategy. Here, we developed a second-generation excitation ratiometric sensor called rogRFP2 with improved red emission for quantitative live-cell imaging. Using this sensor to measure activity-dependent redox changes in individual cultured neurons, we observed an anticorrelation in which mitochondrial oxidation was accompanied by a concurrent reduction in the cytosol. This behavior was dependent on the activity of Complex I of the mitochondrial electron transport chain and could be modulated by the presence of co-cultured astrocytes. We also demonstrated that the red fluorescent rogRFP2 facilitates ratiometric redox imaging in Drosophila retinas. The proof-of-concept studies reported here demonstrate that this new rogRFP2 redox sensor can be a powerful tool for understanding redox biology both in vitro and in vivo across model organisms. In addition, we have used these tools that monitor cellular redox, to study oxidative stress and ROS changes in Parkinson’s disease models. Here, we have established cellular models for studying Parkinson’s disease causing LRRK2 mutations to create a platform for future work to explore the relationship between PD associated LRRK2 variants and oxidative stress.

No description available.

Live-cell imaging with fluorescent protein-based sensors allows us to monitor many dynamic changes in situ. The first genetic manipulation of green fluorescent protein to increase brightness initiated a boom, with a myriad of fluorescent protein sensors now available that span the UV, visible and near-IR range; capable of detecting a great number of metabolites, ions, and other biological signaling components with increased spatial and temporal precision. Used for both steady-state and time-resolved approaches, fluorescent proteins can be used in a wide variety of quantitative approaches. Steady-state sensors are typically characterized as intensiometric or ratiometric; and intensiometric sensors are characterized by an increase or decrease in emission intensity in response to analyte. However, moving in vivo, concentration and intensity dependence of the fluorophore, sample thickness, and photobleaching are limiting factors. Ratiometric probes respond by an inverse change in excitation or emission profiles in response to analyte, normalizing for bleaching or protein expression effects. As an intrinsic property of fluorophores, fluorescence lifetime does not rely on protein concentration, method of measurement or fluorescence intensity. By monitoring changes in lifetime using fluorescence lifetime spectroscopy, no special ratiometric fluorophores are needed, opening up a wider selection of potential fluorescent sensors. Lifetime and other time-resolved approaches are becoming more and more popular due to ease of quantitation and increased signal to background. Here we present the in vitro and live-cell characterization of genetically encoded, ratiometric and lifetime optimized red fluorescent protein pH sensors, a methodology for quantifying receptor trafficking in real time, as well as a lanthanide time resolved imaging approach.

Foram utilizadas proteínas da famÌlia das GFPs e nanocristais fluorescentes de CdSe/ZnS para caracterização da interação e verificação de transferência de energia por ressonância (FRET) entre estes compostos. Formou-se dois pares doador-receptor onde ora uma proteína figurava como doadora, ora um nanocristal ocupava este papel. Verificou-se que, em ambos os casos, o doador sofre supressão da fluorescência após a formação de complexo com o receptor, complexo este motivado por interação eletrostática e dependente de pH. Foi possível comprovar, através da observação de emissão sensitizada e redução da anisotropia, que entre o par formado por nanocristal com emissão no verde e proteína HcRed1 como receptora, de fato ocorre FRET. As distâncias aparentes entre doador e receptor foram determinadas a partir da eficiência da supressão da fluorescência do doador e da distância de Förster. As distâncias assim obtidas são compatíveis com as dimensões das proteínas e dos nanocristais / Proteins belonging to the GFP family were used to characterize their interaction with fluorescent CdSe/ZnS nanocrystals and to verify the occurrence of resonance energy transfer (FRET) among these elements. Two donor-acceptor pairs were established, one having a protein as donor and the other having a nanocrystal as donor. In both cases the donor suffers quenching of its fluorescence after the formation of a complex with the acceptor. The complex formation is dependent on pH and is due to electrostatic interaction. It was possible to prove the occurrence of FRET between CdSe/ZnS nanocrystals emitting green fluorescence as donors and the protein HcRed1 as acceptor, through the detection of sensitized emission and anisotropy reduction. Apparent donor-acceptor distances were determined from efficiency measurements and Förster distances. The obtained distances agreed with the protein and nanocrystal dimensions