a-step-beyond-bret-fluorescence-unbound-excitation-from-luminescence

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence (FUEL)

Fluorescence by Unbound Excitation from Luminescence (FUEL) is a radiative excitation-emission process that produces increased signal and contrast ...

Institut Pasteur

A novel system that has enabled the measurement of single-cell oxygen consumption rates is presented. The experimental apparatus includes a temperature controlled environmental chamber, an array of microwells etched in glass, and a lid actuator used to seal cells in the microwells. Each microwell contains an oxygen sensitive platinum phosphor sensor used to monitor the cellular metabolic rates. Custom automation software controls the digital image data collection for oxygen sensor measurements, which are analyzed using an image-processing program to yield the oxygen concentration within each microwell versus time. Two proof-of-concept experiments produced oxygen consumption rate measurements for A549 human epithelial lung cancer cells of 5.39 and 5.27 fmol/min/cell, closely matching published oxygen consumption rates for bulk A549 populations.

A New Approach for Measuring Single-Cell Oxygen Consumption Rates.

Using an analogy with fed-batch heterotrophic growth, the algal photoautotrophic yield Φ(DW) (in grams of dry weight biomass synthesized per micromole of absorbed photons) was derived from the algae batch growth behavior in nutrient-replete medium. At known levels of incident light, the yield Φ(DW) enables the estimate of a maximum productivity, and is therefore critical to compare and select algal cultures and growth conditions for large-scale production. The algal culture maximum growth rate was shown to be an unreliable indicator of autotrophic biomass yield. The developed carbonate addition method (carbonate addition, neutralization, and sealing) alleviated carbon limitations otherwise seen in aerated batch cultures, leading to two to five fold higher yield estimates. The fully defined FLX growth medium with variable ionic strengths (FLX1-100) supported excellent growth in most cultures tested. The chosen experimental methods and versatile FLX medium proved well-suited for small sample volumes and a high number of samples.

Intrinsic autotrophic biomass yield and productivity in algae: experimental methods for strain selection.

Mutations in the myosin VIIa gene cause Usher syndrome type IB (USH1B), characterized by deaf-blindness. A delay of opsin trafficking has been observed in the retinal photoreceptor cells of myosin VIIa-deficient mice. We identified spectrin βV, the mammalian β-heavy spectrin, as a myosin VIIa- and rhodopsin-interacting partner in photoreceptor cells. Spectrin βV displays a polarized distribution from the Golgi apparatus to the base of the outer segment, which, unlike that of other β spectrins, matches the trafficking route of opsin and other phototransduction proteins. Formation of spectrin βV-rhodopsin complex could be detected in the differentiating photoreceptors as soon as their outer segment emerges. A failure of the spectrin βV-mediated coupling between myosin VIIa and opsin molecules thus probably accounts for the opsin transport delay in myosin VIIa-deficient mice. We showed that spectrin βV also associates with two USH1 proteins, sans (USH1G) and harmonin (USH1C). Spectrins are supposed to function as heteromers of α and β subunits, but fluorescence resonance energy transfer and in vitro binding experiments indicated that spectrin βV can also form homodimers, which likely supports its αII-independent βV functions. Finally, consistent with its distribution along the connecting cilia axonemes, spectrin βV binds to several subunits of the microtubule-based motor proteins, kinesin II and the dynein complex. We therefore suggest that spectrin βV homomers couple some USH1 proteins, opsin and other phototransduction proteins to both actin- and microtubule-based motors, thereby contributing to their transport towards the photoreceptor outer disks.

The giant spectrin βV couples the molecular motors to phototransduction and Usher syndrome type I proteins along their trafficking route.

Energy transfer mechanisms represent the basis for an array of valuable tools to infer interactions in vitro and in vivo, enhance detection or resolve interspecies distances such as with resonance. Based upon our own previously published studies and new results shown here we present a novel framework describing for the first time a model giving a view of the biophysical relationship between Fluorescence by Unbound Excitation from Luminescence (FUEL), a conventional radiative excitation-emission process, and bioluminescence resonance energy transfer. We show here that in homogeneous solutions and in fluorophore-targeted bacteria, FUEL is the dominant mechanism responsible for the production of red-shifted photons. The minor resonance contribution was ascertained by comparing the intensity of the experimental signal to its theoretical resonance counterpart. Distinctive features of the in vitro FUEL signal include a macroscopic depth dependency, a lack of enhancement upon targeting at a constant fluorophore concentration cf and a non-square dependency on cf. Significantly, FUEL is an important, so far overlooked, component of all resonance phenomena which should guide the design of appropriate controls when elucidating interactions. Last, our results highlight the potential for FUEL as a means to enhance in vivo and in vitro detection through complex media while alleviating the need for targeting.

Article	Year
A New Approach for Measuring Single-Cell Oxygen Consumption Rates. IEEE transactions on automation science and engineering : a publication of the IEEE Robotics and Automation Society\| PubMed ID: 21057593	2008
Intrinsic autotrophic biomass yield and productivity in algae: experimental methods for strain selection. Biotechnology journal\| PubMed ID: 21381200	2011
The giant spectrin βV couples the molecular motors to phototransduction and Usher syndrome type I proteins along their trafficking route. Human molecular genetics\| PubMed ID: 23704327	2013
In vitro characterization of Fluorescence by Unbound Excitation from Luminescence: broadening the scope of energy transfer. Methods (San Diego, Calif.)\| PubMed ID: 24045025	2014

Joseph Dragavon

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