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In JoVE (1)
Other Publications (12)
- American Journal of Pharmacogenomics : Genomics-related Research in Drug Development and Clinical Practice
- The European Journal of Neuroscience
- Molecular Imaging
- PloS One
- PloS One
- Journal of Biomedical Optics
- Journal of Biomedical Optics
- Evidence-based Complementary and Alternative Medicine : ECAM
- Methods in Cell Biology
- Cell Host & Microbe
- Proceedings of the National Academy of Sciences of the United States of America
- The Journal of Experimental Medicine
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Articles by Kelly L. Rogers in JoVE
JoVE Immunology and Infection
Использование изображений Биолюминесцентный по расследованию синергизм между Пневмококк И вируса гриппа А в младенческой Мыши
1Department of Microbiology and Immunology, University of Melbourne, 2Laboratory of Pediatric Infectious Diseases, Radboud University Nijmegen Medical Centre, 3The Centre for Dynamic Imaging, The Walter and Eliza Hall Institute for Medical Research
Одновременное инфицирование вирусом гриппа типа А является одним из факторов, обуславливающих индукции инвазивных пневмококковых инфекций во время бессимптомной
Other articles by Kelly L. Rogers on PubMed
Molecular Mechanisms of Migraine: Prospects for Pharmacogenomics
Migraine is a common complex disorder that affects a large portion of the population and thus incurs a substantial economic burden on society. The disorder is characterized by recurrent headaches that are unilateral and usually accompanied by nausea, vomiting, photophobia, and phonophobia. The range of clinical characteristics is broad and there is evidence of comorbidity with other neurological diseases, complicating both the diagnosis and management of the disorder. Although the class of drugs known as the triptans (serotonin 5-HT(1B/1D) agonists) has been shown to be effective in treating a significant number of patients with migraine, treatment may in the future be further enhanced by identifying drugs that selectively target molecular mechanisms causing susceptibility to the disease.Genetically, migraine is a complex familial disorder in which the severity and susceptibility of individuals is most likely governed by several genes that may be different among families. Identification of the genomic variants involved in genetic predisposition to migraine should facilitate the development of more effective diagnostic and therapeutic applications. Genetic profiling, combined with our knowledge of therapeutic response to drugs, should enable the development of specific, individually-tailored treatment.
Visualization of Local Ca2+ Dynamics with Genetically Encoded Bioluminescent Reporters
Measurements of local Ca2+ signalling at different developmental stages and/or in specific cell types is important for understanding aspects of brain functioning. The use of light excitation in fluorescence imaging can cause phototoxicity, photobleaching and auto-fluorescence. In contrast, bioluminescence does not require the input of radiative energy and can therefore be measured over long periods, with very high temporal resolution. Aequorin is a genetically encoded Ca(2+)-sensitive bioluminescent protein, however, its low quantum yield prevents dynamic measurements of Ca2+ responses in single cells. To overcome this limitation, we recently reported the bi-functional Ca2+ reporter gene, GFP-aequorin (GA), which was developed specifically to improve the light output and stability of aequorin chimeras [V. Baubet, et al., (2000) PNAS, 97, 7260-7265]. In the current study, we have genetically targeted GA to different microdomains important in synaptic transmission, including to the mitochondrial matrix, endoplasmic reticulum, synaptic vesicles and to the postsynaptic density. We demonstrate that these reporters enable 'real-time' measurements of subcellular Ca2+ changes in single mammalian neurons using bioluminescence. The high signal-to-noise ratio of these reporters is also important in that it affords the visualization of Ca2+ dynamics in cell-cell communication in neuronal cultures and tissue slices. Further, we demonstrate the utility of this approach in ex-vivo preparations of mammalian retina, a paradigm in which external light input should be controlled. This represents a novel molecular imaging approach for non-invasive monitoring of local Ca2+ dynamics and cellular communication in tissue or whole animal studies.
Red-shifted Aequorin-based Bioluminescent Reporters for in Vivo Imaging of Ca2 Signaling
Real-time visualization of calcium (Ca(2+)) dynamics in the whole animal will enable important advances in understanding the complexities of cellular function. The genetically encoded bioluminescent Ca(2+) reporter green fluorescent protein-aequorin (GA) allows noninvasive detection of intracellular Ca(2+) signaling in freely moving mice. However, the emission spectrum of GA is not optimal for detection of activity from deep tissues in the whole animal. To overcome this limitation, two new reporter genes were constructed by fusing the yellow fluorescent protein (Venus) and the monomeric red fluorescent protein (mRFP1) to aequorin. Transfer of aequorin chemiluminescence energy to Venus (VA) is highly efficient and produces a 58 nm red shift in the peak emission spectrum of aequorin. This substantially improves photon transmission through tissue, such as the skin and thoracic cage. Although the Ca(2+)-induced bioluminescence spectrum of mRFP1-aequorin (RA) is similar to that of aequorin, there is also a small peak above 600 nm corresponding to the peak emission of mRFP1. Small amounts of energy transfer between aequorin and mRFP1 yield an emission spectrum with the highest percentage of total light above 600 nm compared with GA and VA. Accordingly, RA is also detected with higher sensitivity from brain areas. VA and RA will therefore improve optical access to Ca(2+) signaling events in deeper tissues, such as the heart and brain, and offer insight for engineering new hybrid molecules.
In Vivo Bioluminescence Imaging of Ca Signalling in the Brain of Drosophila
Many different cells' signalling pathways are universally regulated by Ca(2+) concentration [Ca(2+)] rises that have highly variable amplitudes and kinetic properties. Optical imaging can provide the means to characterise both the temporal and spatial aspects of Ca(2+) signals involved in neurophysiological functions. New methods for in vivo imaging of Ca(2+) signalling in the brain of Drosophila are required for probing the different dynamic aspects of this system. In studies here, whole brain Ca(2+) imaging was performed on transgenic flies with targeted expression of the bioluminescent Ca(2+) reporter GFP-aequorin (GA) in different neural structures. A photon counting based technique was used to undertake continuous recordings of cytosolic [Ca(2+)] over hours. Time integrals for reconstructing images and analysis of the data were selected offline according to the signal intensity. This approach allowed a unique Ca(2+) response associated with cholinergic transmission to be identified by whole brain imaging of specific neural structures. Notably, [Ca(2+)] transients in the Mushroom Bodies (MBs) following nicotine stimulation were accompanied by a delayed secondary [Ca(2+)] rise (up to 15 min. later) in the MB lobes. The delayed response was sensitive to thapsigargin, suggesting a role for intra-cellular Ca(2+) stores. Moreover, it was reduced in dunce mutant flies, which are impaired in learning and memory. Bioluminescence imaging is therefore useful for studying Ca(2+) signalling pathways and for functional mapping of neurophysiological processes in the fly brain.
Non-invasive in Vivo Imaging of Calcium Signaling in Mice
Rapid and transient elevations of Ca(2+) within cellular microdomains play a critical role in the regulation of many signal transduction pathways. Described here is a genetic approach for non-invasive detection of localized Ca(2+) concentration ([Ca(2+)]) rises in live animals using bioluminescence imaging (BLI). Transgenic mice conditionally expressing the Ca(2+)-sensitive bioluminescent reporter GFP-aequorin targeted to the mitochondrial matrix were studied in several experimental paradigms. Rapid [Ca(2+)] rises inside the mitochondrial matrix could be readily detected during single-twitch muscle contractions. Whole body patterns of [Ca(2+)] were monitored in freely moving mice and during epileptic seizures. Furthermore, variations in mitochondrial [Ca(2+)] correlated to behavioral components of the sleep/wake cycle were observed during prolonged whole body recordings of newborn mice. This non-invasive imaging technique opens new avenues for the analysis of Ca(2+) signaling whenever whole body information in freely moving animals is desired, in particular during behavioral and developmental studies.
Electron-multiplying Charge-coupled Detector-based Bioluminescence Recording of Single-cell Ca2+
The construction and application of genetically encoded intracellular calcium concentration ([Ca2+]i) indicators has a checkered history. Excitement raised over the creation of new probes is often followed by disappointment when it is found that the initial demonstrations of [Ca2+]i sensing capability cannot be leveraged into real scientific advances. Recombinant apo-aequorin cloned from Aequorea victoria was the first Ca2+ sensitive protein genetically targeted to subcellular compartments. In the jellyfish, bioluminescence resonance energy transfer (BRET) between Ca2+ bound aequorin and green fluorescent protein (GFP) emits green light. Similarly, Ca2+ sensitive bioluminescent reporters undergoing BRET have been constructed between aequorin and GFP, and more recently with other fluorescent protein variants. These hybrid proteins display red-shifted spectrums and have higher light intensities and stability compared to aequorin alone. We report BRET measurement of single-cell [Ca2+]i based on the use of electron-multiplying charge-coupled-detector (EMCCD) imaging camera technology, mounted on either a bioluminescence or conventional microscope. Our results show for the first time how these new technologies make facile long-term monitoring of [Ca2+]i at the single-cell level, obviating the need for expensive, fragile, and sophisticated equipment based on image-photon-detectors (IPD) that were until now the only technical recourse to dynamic BRET experiments of this type.
New Device for Real-time Bioluminescence Imaging in Moving Rodents
Bioluminescence imaging (BLI) allows detection of biological functions in genetically modified cells, bacteria, or animals expressing a luciferase (i.e., firefly, Renilla, or aequorin). Given the high sensitivity and minimal toxicity of BLI, in vivo studies on molecular events can be performed noninvasively in living rodents. To date, detection of bioluminescence in living animals has required long exposure times that are incompatible with studies on dynamic signaling pathways or nonanaesthetised freely moving animals. Here we develop an imaging system that allows: 1. bioluminescence to be recorded at a rate of 25 images/s using a third generation intensified charge-coupled device (CCD) camera running in a photon counting mode, and 2. coregistration of a video image from a second CCD camera under infrared lighting. The sensitivity of this instrument permits studies with subsecond temporal resolution in nonanaesthetized and unrestrained mice expressing firefly luciferase and imaging of calcium signaling in transgenic mice expressing green fluorescent protein (GFP) aequorin. This imaging system enables studies on signal transduction, tumor growth, gene expression, or infectious processes in nonanaesthetized and freely moving animals.
Isolation of Bioactive Compounds That Relate to the Anti-platelet Activity of Cymbopogon Ambiguus
Infusions and decoctions of Cymbopogon ambiguus have been used traditionally in Australia for the treatment of headache, chest infections and muscle cramps. The aim of the present study was to screen and identify bioactive compounds from C. ambiguus that could explain this plant's anti-headache activity. A dichloromethane extract of C. ambiguus was identified as having activity in adenosine-diphosphate-induced human platelet aggregation and serotonin-release inhibition bioassays. Subsequent fractionation of this extract led to the isolation of four phenylpropenoids, eugenol, elemicin, eugenol methylether and trans-isoelemicin. While both eugenol and elemicin exhibited dose-dependent inhibition of ADP-induced human platelet serotonin release, only eugenol displayed potent inhibitory activity with an IC(50) value of 46.6 microM, in comparison to aspirin, with an IC(50) value of 46.1 microM. These findings provide evidence to support the therapeutic efficacy of C. ambiguus in the non-conventional treatment of headache and inflammatory conditions.
The Use of Aequorins to Record and Visualize Ca(2+) Dynamics: from Subcellular Microdomains to Whole Organisms
In this chapter, we describe the practical aspects of measuring [Ca(2+)] transients that are generated in a particular cytoplasmic domain, or within a specific organelle or its periorganellar environment, using bioluminescent, genetically encoded and targeted Ca(2+) reporters, especially those based on apoaequorin. We also list examples of the organisms, tissues, and cells that have been transfected with apoaequorin or an apoaequorin-BRET complex, as well as of the organelles and subcellular domains that have been specifically targeted with these bioluminescent Ca(2+) reporters. In addition, we summarize the various techniques used to load the apoaequorin cofactor, coelenterazine, and its analogs into cells, tissues, and intact organisms, and we describe recent advances in the detection and imaging technologies that are currently being used to measure and visualize the luminescence generated by the aequorin-Ca(2+) reaction within these various cytoplasmic domains and subcellular compartments.
Super-resolution Dissection of Coordinated Events During Malaria Parasite Invasion of the Human Erythrocyte
Erythrocyte invasion by the merozoite is an obligatory stage in Plasmodium parasite infection and essential to malaria disease progression. Attempts to study this process have been hindered by the poor invasion synchrony of merozoites from the only in vitro culture-adapted human malaria parasite, Plasmodium falciparum. Using fluorescence, three-dimensional structured illumination, and immunoelectron microscopy of filtered merozoites, we analyze cellular and molecular events underlying each discrete step of invasion. Monitoring the dynamics of these events revealed that commitment to the process is mediated through merozoite attachment to the erythrocyte, triggering all subsequent invasion events, which then proceed without obvious checkpoints. Instead, coordination of the invasion process involves formation of the merozoite-erythrocyte tight junction, which acts as a nexus for rhoptry secretion, surface-protein shedding, and actomyosin motor activation. The ability to break down each molecular step allows us to propose a comprehensive model for the molecular basis of parasite invasion.
Fas-mediated Neutrophil Apoptosis is Accelerated by Bid, Bak, and Bax and Inhibited by Bcl-2 and Mcl-1
During immune responses, neutrophils must integrate survival and death signals from multiple sources to regulate their lifespan. Signals that activate either the Bcl-2- or death receptor-regulated apoptosis pathways can provide powerful stimuli for neutrophils to undergo cell death, but whether they act cooperatively in parallel or directly cross-talk in neutrophils is not known. Previous studies suggested that Bcl-2 family proteins are not required for Fas-induced cell death in neutrophils, but did not examine whether they could modulate its rapid onset. By monitoring the rate of change in neutrophil viability associated with activation of the Fas-triggered death receptor pathway using real-time cell imaging, we show that the Bcl-2-related proteins Bid, Bax, and Bak accelerate neutrophil apoptosis but are not essential for cell death. Increased Bcl-2 or Mcl-1 expression prevents efficient induction of apoptosis by Fas stimulation indicating that the Bcl-2-regulated apoptosis pathway can directly interfere with Fas-triggered apoptosis. Fas has been shown to initiate NFκB activation and gene transcription in cell lines, however gene transcription is not altered in Fas-activated Bid(-/-) neutrophils, indicating that apoptosis occurs independently of gene transcription in neutrophils. The specification of kinetics of neutrophil apoptosis by Bid impacts on the magnitude of neutrophil IL-1β production, implicating a functional role for the Bcl-2-regulated pathway in controlling neutrophil responses to FasL. These data demonstrate that the intrinsic apoptosis pathway directly controls the kinetics of Fas-triggered apoptosis in neutrophils.
Megakaryocytes Possess a Functional Intrinsic Apoptosis Pathway That Must Be Restrained to Survive and Produce Platelets
It is believed that megakaryocytes undergo a specialized form of apoptosis to shed platelets. Conversely, a range of pathophysiological insults, including chemotherapy, are thought to cause thrombocytopenia by inducing the apoptotic death of megakaryocytes and their progenitors. To resolve this paradox, we generated mice with hematopoietic- or megakaryocyte-specific deletions of the essential mediators of apoptosis, Bak and Bax. We found that platelet production was unperturbed. In stark contrast, deletion of the prosurvival protein Bcl-x(L) resulted in megakaryocyte apoptosis and a failure of platelet shedding. This could be rescued by deletion of Bak and Bax. We examined the effect on megakaryocytes of three agents that activate the intrinsic apoptosis pathway in other cell types: etoposide, staurosporine, and the BH3 mimetic ABT-737. All three triggered mitochondrial damage, caspase activation, and cell death. Deletion of Bak and Bax rendered megakaryocytes resistant to etoposide and ABT-737. In vivo, mice with a Bak(-/-) Bax(-/-) hematopoietic system were protected against thrombocytopenia induced by the chemotherapeutic agent carboplatin. Thus, megakaryocytes do not activate the intrinsic pathway to generate platelets; rather, the opposite is true: they must restrain it to survive and progress safely through proplatelet formation and platelet shedding.
