Putting a finishing touch on GEC's

More than a decade ago genetically encoded calcium indicators (GECIs) entered the stage as new promising tools to image calcium dynamics and neuronal activity in living tissues and designated cell types in vivo. From a variety of initial designs two have emerged as promising prototypes for further optimization: FRET (Forster Resonance Energy Transfer)based sensors and single fluorophore sensors of the GCaMP family. Recent efforts in structural analysis, engineering and screening have broken important performance thresholds in the latest generation for both classes. While these improvements have made GECIs a powerful means to perform physiology in living animals, a number of other aspects of sensor function deserve attention. These aspects include indicator linearity, toxicity and slow response kinetics. Furthermore creating high performance sensors with optically more favorable emission in red or infrared wavelengths as well as new stably or conditionally GECI-expressing animal lines are on the wish list. When the remaining issues are solved, imaging of GECIs will finally have crossed the last milestone, evolving from an initial promise into a fully matured technology

Large Scale Bacterial Colony Screening of Diversified FRET Biosensors

Biosensors based on Forster Resonance Energy Transfer (FRET) between fluorescent protein mutants have started to revolutionize physiology and biochemistry. However, many types of FRET biosensors show relatively small FRET changes, making measurements with these probes challenging when used under sub-optimal experimental conditions. Thus, a major effort in the field currently lies in designing new optimization strategies for these types of sensors. Here we describe procedures for optimizing FRET changes by large scale screening of mutant biosensor libraries in bacterial colonies. We describe optimization of biosensor expression, permeabilization of bacteria, software tools for analysis, and screening conditions. The procedures reported here may help in improving FRET changes in multiple suitable classes of biosensors

Color processing in the early visual system of Drosophila

Color vision extracts spectral information by comparing signals from photoreceptors with different visual pigments. Such comparisons are encoded by color-opponent neurons that are excited at one wavelength and inhibited at another. Here, we examine the circuit implementation of color-opponent processing in the Drosophila visual system by combining two-photon calcium imaging with genetic dissection of visual circuits. We report that coloropponent processing of UVshort/blue and UVlong/green is already implemented in R7/R8 inner photoreceptor terminals of "pale'' and "yellow'' ommatidia, respectively. R7 and R8 photoreceptors of the same type of ommatidia mutually inhibit each other directly via HisCl1 histamine receptors and receive additional feedback inhibition that requires the second histamine receptor Ort. Color-opponent processing at the first visual synapse represents an unexpected commonality between Drosophila and vertebrates; however, the differences in the molecular and cellular implementation suggest that the same principles evolved independently

High-Throughput Platform for Optoacoustic Probing of Genetically Encoded Calcium Ion Indicators

Functional optoacoustic (OA) imaging assisted with genetically encoded calcium ion indicators (GECIs) holds promise for imaging large-scale neuronal activity at depths and spatiotemporal resolutions not attainable with existing optical microscopic techniques. However, currently available GECIs optimized for fluorescence (FL) imaging lack sufficient contrast for OA imaging and respond at wavelengths having limited penetration into the mammalian brain. Here we present an imaging platform capable of rapid assessment and cross-validation between OA and FL responses of sensor proteins expressed in Escherichia coli colonies. The screening system features optimized pulsed light excitation combined with ultrasensitive ultrasound detection to mitigate photobleaching while further allowing the dynamic characterization of calcium ion responses with millisecond precision. Targeted probing of up to six individual colonies per second in both calcium-loaded and calcium-unloaded states was possible with the system. The new platform greatly facilitates optimization of absorption-based labels, thus setting the stage for directed evolution of OA GECIs

Follicle-stimulating hormone and luteinizing hormone increase Ca2+ in the granulosa cells of mouse ovarian follicles

In mammalian ovarian follicles, follicle stimulating hormone (FSH) and luteinizing hormone (LH) signal primarily through the G-protein G(s) to elevate cAMP, but both of these hormones can also elevate Ca2+ under some conditions. Here, we investigate FSH- and LH-induced Ca2+ signaling in intact follicles of mice expressing genetically encoded Ca2+ sensors, Twitch-2B and GCaMP6s. At a physiological concentration (1 nM), FSH elevates Ca2+ within the granulosa cells of preantral and antral follicles. The Ca2+ rise begins several minutes after FSH application, peaks at similar to 10 min, remains above baseline for another similar to 10 min, and depends on extracellular Ca2+. However, suppression of the FSH-induced Ca2+ increase by reducing extracellular Ca2+ does not inhibit FSH-induced phosphorylation ofMAP kinase, estradiol production, or the acquisition of LH responsiveness. Like FSH, LH also increases Ca2+, when applied to preovulatory follicles. At a physiological concentration (10 nM), LH elicits Ca2+ oscillations in a subset of cells in the outer mural granulosa layer. These oscillations continue for at least 6 h and depend on the activity of G(q) family G-proteins. Suppression of the oscillations by G(q) inhibition does not inhibit meiotic resumption, but does delay the time to 50% ovulation by about 3 h. In summary, both FSH and LH increase Ca2+ in the granulosa cells of intact follicles, but the functions of these Ca2+ rises are only starting to be identified. Summary Sentence Both FSH and LH increase Ca2+ in the granulosa cells of intact ovarian follicles from mice expressing genetically encoded sensors

Dynamic tuning of FRET in a green fluorescent protein biosensor.

Forster resonance energy transfer (FRET) between mutants of green fluorescent protein is widely used to monitor protein-protein interactions and as a readout mode in fluorescent biosensors. Despite the fundamental importance of distance and molecular angles of fluorophores to each other, structural details on fluorescent protein FRET have been missing. Here, we report the high-resolution x-ray structure of the fluorescent proteins mCerulean3 and cpVenus within the biosensor Twitch-2B, as they undergo FRET and characterize the dynamics of this biosensor with B-0(2)-dependent paramagnetic nuclear magnetic resonance at 900 MHz and 1.1 GHz. These structural data provide the unprecedented opportunity to calculate FRET from the x-ray structure and to compare it to experimental data in solution. We find that interdomain dynamics limits the FRET effect and show that a rigidification of the sensor further enhances FRET

Practical three color live cell imaging by widefield microscopy

Live cell fluorescence microscopy using fluorescent protein tags derived from jellyfish and coral species has been a successful tool to image proteins and dynamics in many species. Multi-colored aequorea fluorescent protein (AFP) derivatives allow investigators to observe multiple proteins simultaneously, but overlapping spectral properties sometimes require the use of sophisticated and expensive microscopes. Here, we show that the aequorea coerulescens fluorescent protein derivative, PS-CFP2 has excellent practical properties as a blue fluorophore that are distinct from green or red fluorescent proteins and can be imaged with standard filter sets on a widefield microscope. We also find that by widefield illumination in live cells, that PS-CFP2 is very photostable. When fused to proteins that form concentrated puncta in either the cytoplasm or nucleus, PSCFP2 fusions do not artifactually interact with other AFP fusion proteins, even at very high levels of over-expression. PSCFP2 is therefore a good blue fluorophore for distinct three color imaging along with eGFP and mRFP using a relatively simple and inexpensive microscope

Engineering and Characterization of an Enhanced Fluorescent Protein Voltage Sensor

BACKGROUND: Fluorescent proteins have been used to generate a variety of biosensors to optically monitor biological phenomena in living cells. Among this class of genetically encoded biosensors, reporters for membrane potential have been a particular challenge. The use of presently known voltage sensor proteins is limited by incorrect subcellular localization and small or absent voltage responses in mammalian cells. RESULTS: Here we report on a fluorescent protein voltage sensor with superior targeting to the mammalian plasma membrane and high responsiveness to membrane potential signaling in excitable cells. CONCLUSIONS AND SIGNIFICANCE: This biosensor, which we termed VSFP2.1, is likely to lead to new methods of monitoring electrically active cells with cell type specificity, non-invasively and in large numbers, simultaneously

Red Fluorescent Protein-Aequorin Fusions as Improved Bioluminescent Ca2+ Reporters in Single Cells and Mice

Bioluminescence recording of Ca2+ signals with the photoprotein aequorin does not require radiative energy input and can be measured with a low background and good temporal resolution. Shifting aequorin emission to longer wavelengths occurs naturally in the jellyfish Aequorea victoria by bioluminescence resonance energy transfer (BRET) to the green fluorescent protein (GFP). This process has been reproduced in the molecular fusions GFP-aequorin and monomeric red fluorescent protein (mRFP)-aequorin, but the latter showed limited transfer efficiency. Fusions with strong red emission would facilitate the simultaneous imaging of Ca2+ in various cell compartments. In addition, they would also serve to monitor Ca2+ in living organisms since red light is able to cross animal tissues with less scattering. In this study, aequorin was fused to orange and various red fluorescent proteins to identify the best acceptor in red emission bands. Tandem-dimer Tomato-aequorin (tdTA) showed the highest BRET efficiency (largest energy transfer critical distance R0) and percentage of counts in the red band of all the fusions studied. In addition, red fluorophore maturation of tdTA within cells was faster than that of other fusions. Light output was sufficient to image ATP-induced Ca2+ oscillations in single HeLa cells expressing tdTA. Ca2+ rises caused by depolarization of mouse neuronal cells in primary culture were also recorded, and changes in fine neuronal projections were spatially resolved. Finally, it was also possible to visualize the Ca2+ activity of HeLa cells injected subcutaneously into mice, and Ca2+ signals after depositing recombinant tdTA in muscle or the peritoneal cavity. Here we report that tdTA is the brightest red bioluminescent Ca2+ sensor reported to date and is, therefore, a promising probe to study Ca2+ dynamics in whole organisms or tissues expressing the transgene

Glycine insertion makes yellow fluorescent protein sensitive to hydrostatic pressure

Fluorescent protein-based indicators for intracellular environment conditions such as pH and ion concentrations are commonly used to study the status and dynamics of living cells. Despite being an important factor in many biological processes, the development of an indicator for the physicochemical state of water, such as pressure, viscosity and temperature, however, has been neglected. We here found a novel mutation that dramatically enhances the pressure dependency of the yellow fluorescent protein (YFP) by inserting several glycines into it. The crystal structure of the mutant showed that the tyrosine near the chromophore flipped toward the outside of the β-can structure, resulting in the entry of a few water molecules near the chromophore. In response to changes in hydrostatic pressure, a spectrum shift and an intensity change of the fluorescence were observed. By measuring the fluorescence of the YFP mutant, we succeeded in measuring the intracellular pressure change in living cell. This study shows a new strategy of design to engineer fluorescent protein indicators to sense hydrostatic pressure