Showing 1 - 8 of 8 results
Optical control of transcription - genetically encoded photoswitchable variants of T7 RNA polymerase.
Light-sensing protein domains that link an exogenous light signal to the activity of an enzyme have attracted much notice for engineering new regulatory mechanisms into proteins and for studying the dynamic behavior of intracellular reactions as well as reaction cascades. Light-oxygen-voltage (LOV) photoreceptors are blue light-sensing modules that have been intensely characterized for this purpose and linked to several proteins of interest. For successful application of these tools it is crucial to identify appropriate fusion strategies for combining sensor and enzyme domains that sustain activity and light-induced responsivity. Terminal fusion of LOV domains is the natural strategy; however, this is not transferrable to T7 RNA polymerase since both of its termini are involved in catalysis. We show here that it is possible to covalently insert LOV domains into the polymerase protein while preserving its activity and generating new light-responsive allosteric coupling.
Optogenetics: A Primer for Chemists.
The field of optogenetics uses genetically encoded, light-responsive proteins to control physiological processes. This technology has been hailed as the one of the ten big ideas in brain science in the past decade, the breakthrough of the decade, and the method of the year in 2010 and again in 2014. The excitement evidenced by these proclamations is confirmed by a couple of impressive numbers. The term "optogenetics" was coined in 2006. As of December 2017, "optogenetics" is found in the title or abstract of almost 1600 currently funded National Institutes of Health grants. In addition, nearly 600 reviews on optogenetics have appeared since 2006, which averages out to approximately one review per week! However, in spite of these impressive numbers, the potential applications and implications of optogenetics are not even close to being fully realized. This is due, in large part, to the challenges associated with the design of optogenetic analogs of endogenous proteins. This review is written from a chemist's perspective, with a focus on the molecular strategies that have been developed for the construction of optogenetic proteins.
Induction of signal transduction using non-channelrhodopsin-type optogenetic tools.
Signal transductions are the basis for all cellular functions. Previous studies investigating signal transductions mainly relied on pharmacological inhibition, RNA interference, and constitutive active/dominant negative protein expression systems. However, such studies do not allow the modulation of protein activity in cells, tissues, and organs in animals with high spatial and temporal precision. Recently, non-channelrhodopsin-type optogenetic tools for regulating signal transduction have emerged. These photoswitches address several disadvantages of previous techniques, and allow us to control a variety of signal transductions such as cell membrane dynamics, calcium signaling, lipid signaling, and apoptosis. In this review, we summarize recent advances in the development of such photoswitches and how these optotools are applied to signaling processes.
Near-infrared light-controlled gene expression and protein targeting in neurons and non-neuronal cells.
Near-infrared (NIR) light-inducible binding of bacterial phytochrome BphP1 to its engineered partner QPAS1 is used for optical protein regulation in mammalian cells. However, there are no data on the application of the BphP1-QPAS1 pair in cells derived from various mammalian tissues. Here, we tested functionality of two BphP1-QPAS1-based optogenetic tools, such as an NIR and blue light-sensing system for control of protein localization (iRIS) and an NIR light-sensing system for transcription activation (TA), in several cell types including cortical neurons. We found that the performance of these optogenetic tools often rely on physiological properties of a specific cell type, such as nuclear transport, which may limit applicability of blue light-sensitive component of iRIS. In contrast, the NIR-light-sensing part of iRIS performed well in all tested cell types. The TA system showed the best performance in HeLa, U-2 OS and HEK-293 cells. Small size of the QPAS1 component allows designing AAV viral particles, which were applied to deliver the TA system to neurons.
Optogenetic Control by Pulsed Illumination.
Sensory photoreceptors evoke numerous adaptive responses in Nature and serve as light-gated actuators in optogenetics to enable the spatiotemporally precise, reversible and noninvasive control of cellular events. The output of optogenetic circuits can often be dialed in by varying illumination quality, quantity and duration. Here, we devise a programmable matrix of light-emitting diodes to efficiently probe the response of optogenetic systems to intermittently applied light of varying intensity and pulse frequency. Circuits for light-regulated gene expression markedly differed in their responses to pulsed illumination of a single color which sufficed for sequentially triggering them. In addition to quantity and quality, the pulse frequency of intermittent light hence provides a further input variable for output control in optogenetics and photobiology. Pulsed illumination schemes allow the reduction of overall light dose and facilitate the multiplexing of several light-dependent actuators and reporters.
Light-dependent cytoplasmic recruitment enhances the dynamic range of a nuclear import photoswitch.
Cellular signal transduction is often regulated at multiple steps in order to achieve more complex logic or precise control of a pathway. For instance, some signaling mechanisms couple allosteric activation with localization to achieve high signal to noise. Here, we create a system for light activated nuclear import that incorporates two levels of control. It consists of a nuclear import photoswitch, Light Activated Nuclear Shuttle (LANS), and a protein engineered to preferentially interact with LANS in the dark, Zdk2. First, Zdk2 is tethered to a location in the cytoplasm, which sequesters LANS in the dark. Second, LANS incorporates a nuclear localization signal (NLS) that is sterically blocked from binding to the nuclear import machinery in the dark. When activated with light, LANS both dissociates from its tethered location and exposes its NLS, which leads to nuclear accumulation. We demonstrate that this coupled system improves the dynamic range of LANS in mammalian cells, yeast, and C. elegans and provides tighter control of transcription factors that have been fused to LANS.
A miniaturized E. coli green light sensor with high dynamic range.
Genetically-engineered photoreceptors enable unrivaled control over gene expression. Previously, we ported the Synechocystis PCC 6803 CcaSR two-component system, which is activated by green light and de-activated by red, into E. coli, resulting in a sensor with 6-fold dynamic range. Later, we optimized pathway protein expression levels and the output promoter sequence to decrease transcriptional leakiness and increase the dynamic range to approximately 120-fold. These CcaSR v1.0 and 2.0 systems have been used for precise quantitative, temporal, and spatial control of gene expression for a variety of applications. Recently, others have deleted two PAS domains of unknown function from the CcaS sensor histidine kinase in a CcaSR v1.0-like system. Here, we apply these deletions to CcaSR v2.0, resulting in a v3.0 light sensor with 4-fold lower leaky output and nearly 600-fold dynamic range. We demonstrate that the PAS domain deletions have no deleterious effect on CcaSR green light sensitivity or response dynamics. CcaSR v3.0 is the best performing engineered bacterial green light sensor available, and should have broad applications in fundamental and synthetic biology studies.
Photocontrolled Exposure of Pro-apoptotic Peptide Sequences in LOV Proteins Modulates Bcl-2 Family Interactions.
LOV domains act as biomolecular sensors for light, oxygen or the environment's redox potential. Conformational changes upon the formation of a covalent cysteinyl flavin adduct are propagated through hydrogen-bonding networks in the core of designed hybrid phototropin LOV2 domains that incorporate the Bcl homology region 3 (BH3) of the key pro-apoptotic protein BH3-interacting-domain death agonist (BID). The resulting change in conformation of a flanking amphiphilic α-helix creates a light-dependent optogenetic tool for the modulation of interactions with the anti-apoptotic B-cell leukaemia-2 (Bcl-2) family member Bcl-xL .