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 color-opponent 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.

Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes.

The quality of genetically encoded calcium indicators (GECIs) has improved dramatically in recent years, but high-performing ratiometric indicators are still rare. Here we describe a series of fluorescence resonance energy transfer (FRET)-based calcium biosensors with a reduced number of calcium binding sites per sensor. These 'Twitch' sensors are based on the C-terminal domain of Opsanus troponin C. Their FRET responses were optimized by a large-scale functional screen in bacterial colonies, refined by a secondary screen in rat hippocampal neuron cultures. We tested the in vivo performance of the most sensitive variants in the brain and lymph nodes of mice. The sensitivity of the Twitch sensors matched that of synthetic calcium dyes and allowed visualization of tonic action potential firing in neurons and high resolution functional tracking of T lymphocytes. Given their ratiometric readout, their brightness, large dynamic range and linear response properties, Twitch sensors represent versatile tools for neuroscience and immunology.

Correlating calcium binding, Förster resonance energy transfer, and conformational change in the biosensor TN-XXL.

Genetically encoded calcium indicators have become instrumental in imaging signaling in complex tissues and neuronal circuits in vivo. Despite their importance, structure-function relationships of these sensors often remain largely uncharacterized due to their artificial and multimodular composition. Here, we describe a combination of protein engineering and kinetic, spectroscopic, and biophysical analysis of the Förster resonance energy transfer (FRET)-based calcium biosensor TN-XXL. Using fluorescence spectroscopy of engineered tyrosines, we show that two of the four calcium binding EF-hands dominate the FRET output of TN-XXL and that local conformational changes of these hands match the kinetics of FRET change. Using small-angle x-ray scattering and NMR spectroscopy, we show that TN-XXL changes from a flexible elongated to a rigid globular shape upon binding calcium, thus resulting in FRET signal output. Furthermore, we compare calcium titrations using fluorescence lifetime spectroscopy with the ratiometric approach and investigate potential non-FRET effects that may affect the fluorophores. Thus, our data characterize the biophysics of TN-XXL in detail and may form a basis for further rational engineering of FRET-based biosensors.

Dynamic tuning of FRET in a green fluorescent protein biosensor.

Förster 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.

Large Scale Bacterial Colony Screening of Diversified FRET Biosensors.

Biosensors based on Förster 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.

In vivo odourant response properties of migrating adult-born neurons in the mouse olfactory bulb.

Juxtaglomerular neurons (JGNs) of the mammalian olfactory bulb are generated throughout life. Their integration into the preexisting neural network, their differentiation and survival therein depend on sensory activity, but when and how these adult-born cells acquire responsiveness to sensory stimuli remains unknown. In vivo two-photon imaging of retrovirally labelled adult-born JGNs reveals that ~90% of the cells arrive at the glomerular layer after day post injection (DPI) 7. After arrival, adult-born JGNs are still migrating, but at DPI 9, 52% of them have odour-evoked Ca(2+) signals. Their odourant sensitivity closely resembles that of the parent glomerulus and surrounding JGNs, and their spontaneous and odour-evoked spiking is similar to that of their resident neighbours. Our data reveal a remarkably rapid functional integration of adult-born cells into the preexisting neural network. The mature pattern of odour-evoked responses of these cells strongly contrasts with their molecular phenotype, which is typical of immature, migrating neuroblasts.

Real-time in vivo analysis of T cell activation in the central nervous system using a genetically encoded calcium indicator.

To study T cell activation in vivo in real time, we introduced a newly developed fluorescence resonance energy transfer-based, genetically encoded calcium indicator into autoantigen-specific and non-autoantigen-specific CD4(+) T cells. Using two-photon microscopy, we explored the responses of retrovirally transduced calcium indicator-expressing T cells to antigen in the lymph nodes and the central nervous system. In lymph nodes, the administration of exogenous antigen caused an almost immediate arrest of T cells around antigen-presenting cells and an instant rise of cytosolic calcium. In contrast, encephalitogenic T cells entering the leptomeningeal space, one main portal into the central nervous system parenchyma during experimental autoimmune encephalomyelitis, showed elevated intracellular calcium concentrations while still meandering through the space. This approach enabled us to follow the migration and activation patterns of T cells in vivo during the course of the disease.