Photo Control of Protein Function Using Photoactive Yellow Protein
Photoswitchable proteins are becoming increasingly common tools for manipulating cellular processes with high spatial and temporal precision. Photoactive yellow protein (PYP) is a small, water-soluble protein that undergoes a blue light induced change in conformation. It can serve as a scaffold for designing new tools to manipulate biological processes, but with respect to other protein scaffolds it presents some technical challenges. Here, we present practical information on how to overcome these, including how to synthesize the PYP chromophore, how to express and purify PYP, and how to screen for desired activity. (Sourc...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Controlling Protein Activity and Degradation Using Blue Light
Regulation of protein stability is a fundamental process in eukaryotic cells and pivotal to, e.g., cell cycle progression, faithful chromosome segregation, or protein quality control. Synthetic regulation of protein stability requires conditional degradation sequences (degrons) that induce a stability switch upon a specific signal. Fusion to a selected target protein permits to influence virtually every process in a cell. Light as signal is advantageous due to its precise applicability in time, space, quality, and quantity. Light control of protein stability was achieved by fusing the LOV2 photoreceptor domain of Arabidops...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Reversible Photoregulation of Gene Expression and Translation
Several methods for controlling gene expression by light illumination have been reported. Most of these methods control transcription by regulating the interaction between DNA and transcription factors. The use of a photolabile protecting compound (cage compound) is another promising approach for controlling gene expression, although typically in an irreversible manner. We here describe a new approach for reversibly controlling translation using a photoresponsive 8-styryl cap (8ST-cap) that can be reversibly isomerized by illumination with light of a specific wavelength. (Source: Springer protocols feed by Neuroscience)
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Algal Photobiology: A Rich Source of Unusual Light Sensitive Proteins for Synthetic Biology and Optogenetics
The light absorption system in eukaryotic (micro)algae includes highly sensitive photoreceptors, which change their conformation in response to different light qualities on a subsecond time scale and induce physiological and behavioral responses. Some of the light sensitive modules are already in use to engineer and design photoswitchable tools for control of cellular and physiological activities in living organisms with various degrees of complexity. Thus, identification of new light sensitive modules will not only extend the source material for the generation of optogenetic tools but also foster the development of new li...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Natural Resources for Optogenetic Tools
Photoreceptors are found in all kingdoms of life and mediate crucial responses to environmental challenges. Nature has evolved various types of photoresponsive protein structures with different chromophores and signaling concepts for their given purpose. The abundance of these signaling proteins as found nowadays by (meta-)genomic screens enriched the palette of optogenetic tools significantly. In addition, molecular insights into signal transduction mechanisms and design principles from biophysical studies and from structural and mechanistic comparison of homologous proteins opened seemingly unlimited possibilities for cu...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Erratum to: Optogenetics in Plants: Red/Far-Red Light Control of Gene Expression
(Source: Springer protocols feed by Neuroscience)
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Guidelines for Photoreceptor Engineering
Sensory photoreceptors underpin optogenetics by mediating the noninvasive and reversible perturbation of living cells by light with unprecedented temporal and spatial resolution. Spurred by seminal optogenetic applications of natural photoreceptors, the engineering of photoreceptors has recently garnered wide interest and has led to the construction of a broad palette of novel light-regulated actuators. Photoreceptors are modularly built of photosensors that receive light signals, and of effectors that carry out specific cellular functions. These modules have to be precisely connected to allow efficient communication, such...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Optogenetic Manipulation of Selective Neural Activity in Free-Moving Drosophila Adults
Activating selected neurons elicits specific behaviors in Drosophila adults. By combining optogenetics and laser-tracking techniques, we have recently developed an automated laser-tracking and optogenetic manipulation system (ALTOMS) for studying how brain circuits orchestrate complex behaviors. The established ALTOMS can independently target three lasers (473-nm blue laser, 593.5-nm yellow laser, and 1064-nm infrared laser) on any specified body part of two freely moving flies. Triggering light-sensitive proteins in real time, the blue laser and yellow laser can respectively activate and inhibit target neurons in artifici...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Protein Inactivation by Optogenetic Trapping in Living Cells
Optogenetic modules that use genetically encoded elements to control protein function in response to light allow for precise spatiotemporal modulation of signaling pathways. As one of optical approaches, LARIAT (Light-Activated Reversible Inhibition by Assembled Trap) is a unique light-inducible inhibition system that reversibly sequesters target proteins into clusters, generated by multimeric proteins and a blue light-induced heterodimerization module. Here we present a method based on LARIAT for optical inhibition of targets in living mammalian cells. In the protocol, we focus on the inhibition of proteins that modulate ...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Enhancing Channelrhodopsins: An Overview
After the discovery of Channelrhodopsin, a light-gated ion channel, only a few people saw the diverse range of applications for such a protein. Now, more than 10 years later Channelrhodopsins have become widely accepted as the ultimate tool to control the membrane potential of excitable cells via illumination. The demand for more application-specific Channelrhodopsin variants started a race between protein engineers to design improved variants. Even though many engineered variants have undisputable advantages compared to wild-type variants, many users are alienated by the tremendous amount of new variants and their perplex...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Combined Optogenetic and Chemogenetic Control of Neurons
We describe applications of this approach in cultured neurons in vitro, in brain slices ex vivo, and in awake and anesthetized animals in vivo. (Source: Springer protocols feed by Neuroscience)
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Optogenetic Modulation of Locomotor Activity on Free-Behaving Rats
The technology of optogenetics provides a new method to modulate neural activity with spatial specificity and millisecond-temporal scale. This nonelectrical modulation method also gives chance for simultaneous electrophysiological recording during stimulations. Here, we describe our locomotor activity modulation on free-behaving rats using optogenetic techniques. The target sites of the rat brain were dorsal periaqueductal gray (dPAG) and ventral tegmental area (VTA) for the modulation of defensive and reward behaviors, respectively. (Source: Springer protocols feed by Neuroscience)
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Optogenetic Control of Mammalian Ion Channels with Chemical Photoswitches
In neurons, ligand-gated ion channels decode the chemical signal of neurotransmitters into an electric response, resulting in a transient excitation or inhibition. Neurotransmitters act on multiple receptor types and subtypes, with spatially and temporally precise patterns. Hence, understanding the neural function of a given receptor requires methods for its targeted, rapid activation/inactivation in defined brain regions. To address this, we have developed a versatile optochemical genetic strategy, which allows the reversible control of defined receptor subtypes in designated cell types, with millisecond and micrometer pr...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

Optogenetics in Drosophila Neuroscience
Optogenetic techniques enable one to target specific neurons with light-sensitive proteins, e.g., ion channels, ion pumps, or enzymes, and to manipulate their physiological state through illumination. Such artificial interference with selected elements of complex neuronal circuits can help to determine causal relationships between neuronal activity and the effect on the functioning of neuronal circuits controlling animal behavior. The advantages of optogenetics can best be exploited in genetically tractable animals whose nervous systems are, on the one hand, small enough in terms of cell numbers and to a certain degree ste...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news

An Optimized Calcium-Phosphate Transfection Method for Characterizing Genetically Encoded Tools in Primary Neurons
In order to characterize genetically encoded tools under the most relevant conditions, the constructs need to be expressed in the cell type in which they will be used. This is a major hurdle in developing optogenetic tools for neuronal cells, due to the difficulty of gene transfer to these cells. Several protocols have been developed for transfecting neurons, focusing on improved transfection efficiency. However, obtaining healthy cells is as important. We monitored transfected cell health by measuring electrophysiological parameters, and used them as a guideline to optimize transfection. Here we describe an optimized tran...
Source: Springer protocols feed by Neuroscience - March 10, 2016 Category: Neuroscience Source Type: news