Monthly Archives: April 2013

Photoswitchable protein degradation using a LOV2 domain-based single construct

Plants have developed elaborate light-sensing pathways to be able to adequately react on the quality and quantity of light. These signal transduction pathways regulate among others growth directionality (phototropism), flowering, stomatal opening, and chloroplast movement. Light-signalling pathways in plants converge on the ubiquitin-protein ligase COP1 that regulates stability of many transcription factors involved in light-dependent responses. This puts regulated proteolysis by the ubiquitin proteasome system at the heart of light-depending signaling. Recently, Renicke et al., reported in Chemistry & Biology on the engineering of a synthetic tool that concentrates light-regulated proteolysis into a single construct. The module developed by the authors comprises the light-sensing LOV2 domain of Arabidopsis thaliana phot1 and the ornithine decarboxylase (ODC) like degradation sequence (degron) cODC1. Activity of this so-called photo-sensitive degron (psd) module depends on the presence of blue light: the conformational changes within the LOV2 domain expose the degron, which induces proteasomal degradation. In contrast to the majority of proteasomal substrates, the cODC1 degron is directly recognized by the proteasome, without polyubiquitylation by an ubiquitin-protein ligase. Thus, the psd module is a minimized plant light-signalling pathway that comprises a light-responsive domain fused with a degradation-inducing sequence. The module can be fused to target proteins … Continue reading

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Holographic optogenetic control of retinal neural activity

Sensory information is typically represented in distributed patterns of activity across large populations of neurons. Therefore, many emerging neuro-photonic applications, such as optogenetic retinal prostheses require systems capable of delivering intense, parallel and dynamic light patterns. Such a system will ideally allow photo-control with single-cell selectivity of large neural populations expressing optogenetic probes, rather than nonspecific flashed illumination of the whole population (as provided by many current optogenetic light delivery systems). In a recent Nature Communications report, Reutsky-Gefen, Shoham and colleagues demonstrate holographic optogenetic control of retinal neural activity which is shown to provide rapid cellular-resolution, massively parallel excitation across macroscopic (millimeter-scale) coverage areas. The study illustrates that diffractive wavefront shaping (holographic) tools offer a powerful modality for dynamic patterned photo-stimulation as they naturally combine the high intensity, efficiency and resolution that are characteristic of sequential laser deflectors (like acousto-optical deflectors) with the simultaneous scan-less parallel illumination of multiple locations of microdisplay array projectors, but without their respective limitations. Holographic tools were previously shown to allow structured excitation of dendritic arbors and neurons using neurotransmitter photolysis, as well as two-photon optogenetic stimulation of proximal neurons in brain slices.

The study’s main goal was to develop a prosthetic system that would … Continue reading

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A multimodal micro-optrode

Microelectrodes are powerful tools for in vivo functional studies. However they are limited in the number of information they provide. In January 2011, LeChasseur and colleagues (Nature Methods 8(4), 319-325, 2011) developed a glass microelectrode which was integrating an optical micro-channel for light delivery and fluorescence collection. The probe serves for specific cellular fluorescence optical detection and activation/inhibition. In a recent issue of PLOS ONE, Dufour et al. extended the multimodal aspect of this micro-optrode. They introduce a, aluminum-coated, fibre optic-based glass microprobe (diameter ≤ 10 μm) with multiple electrical and optical detection capabilities. The probe enables optical separation from individual cells in transgenic mice expressing multiple fluorescent proteins in distinct populations of neurons within the same deep brain nucleus. It also enables color conversion of photoswitchable fluorescent proteins, which can be used for post-hoc identification of the recorded cells and finally it enables dual electrical recordings. Figure 1 shows a representation of the microp-optrode and the modalities described in this paper.

These modalities are in addition to the calcium monitoring and optogenetic cellular activation previously reported (Nature Methods 8(4), 319-325, 2011). In this study, two different excitation sources and detection pathways were used simultaneously to differentiate two different populations … Continue reading

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Luminopsins: bioluminescent opsins allowing combined opto- and chemogenetic control of neuronal activity

In optogenetic experiments aiming at controlling neuronal activity light-sensitive molecules such as channelrhodopsins or proton pumps are activated through application of light from a physical light source, such as arc lamps, lasers or LEDs. An interesting extension of such experiments could be achieved by controlling light-sensing molecules with a biological light source. Luciferases are such light producing proteins; they are enzymes, used by a variety of bioluminescent organisms, which produce light by oxidizing a substrate molecule. Replacing the physical light source with a biological one, i.e. a light-emitting protein, should allow non-invasive activation of light-sensitive molecules. In addition, the advantage of genetically encoding both light production and light sensing introduces unique combinatorial possibilities.

Using bioluminescence in combination with optogenetic actuators hinges on the ability of luciferase to activate these actuators. In a recent paper published in PLoS ONE, Berglund and colleagues demonstrate that this is indeed possible. In this study the authors engineered fusion proteins of a luciferase from the marine copepod Gaussia princeps (GLuc) and channelrhodopsins, creating luminescent opsins, or “luminopsins”. Their study brings the proof-of-concept that light produced upon supplying the luciferase with its substrate, coelenterazine (CTZ), can activate the fused channelrhodopsin, thereby modulating neuronal activity.

Because luminopsins … Continue reading

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