Blue-Light Receptors for Optogenetics Losi, Aba; Gardner, Kevin H; Möglich, Andreas
Chemical reviews,
11/2018, Letnik:
118, Številka:
21
Journal Article
Recenzirano
Odprti dostop
Sensory photoreceptors underpin light-dependent adaptations of organismal physiology, development, and behavior in nature. Adapted for optogenetics, sensory photoreceptors become genetically encoded ...actuators and reporters to enable the noninvasive, spatiotemporally accurate and reversible control by light of cellular processes. Rooted in a mechanistic understanding of natural photoreceptors, artificial photoreceptors with customized light-gated function have been engineered that greatly expand the scope of optogenetics beyond the original application of light-controlled ion flow. As we survey presently, UV/blue-light-sensitive photoreceptors have particularly allowed optogenetics to transcend its initial neuroscience applications by unlocking numerous additional cellular processes and parameters for optogenetic intervention, including gene expression, DNA recombination, subcellular localization, cytoskeleton dynamics, intracellular protein stability, signal transduction cascades, apoptosis, and enzyme activity. The engineering of novel photoreceptors benefits from powerful and reusable design strategies, most importantly light-dependent protein association and (un)folding reactions. Additionally, modified versions of these same sensory photoreceptors serve as fluorescent proteins and generators of singlet oxygen, thereby further enriching the optogenetic toolkit. The available and upcoming UV/blue-light-sensitive actuators and reporters enable the detailed and quantitative interrogation of cellular signal networks and processes in increasingly more precise and illuminating manners.
Light–oxygen–voltage sensitive (LOV) flavoproteins are ubiquitous photoreceptors that mediate responses to environmental cues. Photosensory inputs are transduced into signaling outputs via structural ...rearrangements in sensor domains that consequently modulate the activity of an effector domain or multidomain clusters. Establishing the diversity in effector function and sensor–effector topology will inform what signaling mechanisms govern light-responsive behaviors across multiple kingdoms of life and how these signals are transduced. Here,we report the bioinformatics identification of over 6,700 candidate LOV domains (including over 4,000 previously unidentified sequences from plants and protists), and insights from their annotations for ontological function and structural arrangements. Motif analysis identified the sensors from ∼42 million ORFs, with strong statistical separation from other flavoproteins and non-LOV members of the structurally related Per-aryl hydrocarbon receptor nuclear translocator (ARNT)-Sim family. Conserved-domain analysis determined putative light-regulated function and multidomain topologies. We found that for certain effectors, sensor–effector linker length is discretized based on both phylogeny and the preservation of α-helical heptad repeats within an extended coiled-coil linker structure. This finding suggests that preserving sensor–effector orientation is a key determinant of linker length, in addition to ancestry, in LOV signaling structure–function. We found a surprisingly high prevalence of effectors with functions previously thought to be rare among LOV proteins, such as regulators of G protein signaling, and discovered several previously unidentified effectors, such as lipases. This work highlights the value of applying genomic and transcriptomic technologies to diverse organisms to capture the structural and functional variation in photosensory proteins that are vastly important in adaptation, photobiology, and optogenetics.
The outer segments of vertebrate rod photoreceptors are renewed every 10 d. Outer segment components are transported from the site of synthesis in the inner segment through the connecting cilium, ...followed by assembly of the highly ordered discs. Two models of assembly of discrete discs involving either successive fusion events between intracellular rhodopsin-bearing vesicles or the evagination of the plasma membrane followed by fusion of adjacent evaginations have been proposed. Here we use immuno-electron microscopy and electron tomography to show that rhodopsin is transported from the inner to the outer segment via the ciliary plasma membrane, subsequently forming successive evaginations that “zipper” up proximally, but at their leading edges are free to make junctions containing the protocadherin, PCDH21, with the inner segment plasma membrane. Given the physical dimensions of the evaginations, coupled with likely instability of the membrane cortex at the distal end of the connecting cilium, we propose that the evagination occurs via a process akin to blebbing and is not driven by actin polymerization. Disassembly of these junctions is accompanied by fusion of the leading edges of successive evaginations to form discrete discs. This fusion is topologically different to that mediated by the membrane fusion proteins, SNAREs, as initial fusion is between exoplasmic leaflets, and is accompanied by gain of the tetraspanin rim protein, peripherin.
Ambient temperature regulates many aspects of plant growth and development, but its sensors are unknown. Here, we demonstrate that the phytochrome B (phyB) photoreceptor participates in temperature ...perception through its temperature-dependent reversion from the active Pfr state to the inactive Pr state. Increased rates of thermal reversion upon exposing Arabidopsis seedlings to warm environments reduce both the abundance of the biologically active Pfr-Pfr dimer pool of phyB and the size of the associated nuclear bodies, even in daylight. Mathematical analysis of stem growth for seedlings expressing wild-type phyB or thermally stable variants under various combinations of light and temperature revealed that phyB is physiologically responsive to both signals. We therefore propose that in addition to its photoreceptor functions, phyB is a temperature sensor in plants.
The substantial time taken for regaining visual sensitivity (dark adaptation) following bleaching exposures has been investigated for over a century. Psychophysical studies yielded the classic ...biphasic curve representing recovery of cone‐driven and rod‐driven vision. The electroretinogram (ERG) permits direct assessment of recovery at the level of the retina (photoreceptors, bipolar cells), with the first report over 70 years ago. Over the last two decades, ERG studies of dark adaptation have generated insights into underlying physiological processes. After large bleaches, rod photoreceptor circulating current, estimated from the rod‐isolated bright‐flash ERG a‐wave, takes 30 min to recover, indicating that products of bleaching, thought to be free opsin (unbound to 11‐cis‐retinal), continue to activate phototransduction, shutting off rod circulating current. In contrast, cone current, assessed with cone‐driven bright‐flash ERG a‐waves, recovers within 100 ms following similar exposures, suggesting that free opsin is less able to shut off cone current. The cone‐driven dim‐flash a‐wave can be used to track recovery of cone photopigment, showing regeneration is 'rate‐limited' rather than first order. Recoveries of the dim‐flash ERG b‐wave are consistent also with rate‐limited rod photopigment regeneration (where free opsin, desensitising the visual system as an 'equivalent background', is removed by rate‐limited delivery of 11‐cis‐retinal). These findings agree with psychophysical and retinal densitometry studies, although there are unexplained points of divergence. Post‐bleach ERG recovery has been explored in age‐related macular degeneration and in trials of visual cycle inhibitors for retinal diseases. ERG tracking of dark adaptation may prove useful in future clinical contexts.
figure legend The prolonged time course of recovery of human visual sensitivity (dark adaptation) following bright light exposure has been the subject of study for over one and a half centuries. The upper panels represent (illustratively rather than accurately) recovery in visual sensitivity over time following a bright light exposure (‘bleach’). Various techniques have been employed to investigate this, ranging from psychophysical to electrophysiological to densitometric. Psychophysical assessments were earliest and more accessible, assessing higher levels of the visual system (conscious perception). Densitometric assessments came later and allowed direct measurement of photopigment levels. Electrophysiological measurements are somewhere in between. The years shown represent approximately the earliest year in which a deliberate quantitative investigation of recovery following bleaching was performed or published. This review mainly considers those ERG studies conducted in the last 20–25 years, and the range of insights gained from these studies (summarised in the right‐hand box). The lowest row of panels gives an example: recovery of the dim‐flash ERG b‐wave amplitude with time in the dark after a bright light exposure (traces are schematic responses expected in a healthy human subject). The panels in this row show the response to the same strength of flash (ERG amplitude plotted against time after flash in each panel) delivered at a progressively later time in the dark following extinction of a bright background (in this case a steady‐state exposure to approximately 3000 scotopic trolands). Abbreviations: ERG, electroretinogram; ERP, early receptor potential; VEP, visual‐evoked potential.
Structure of cone photoreceptors Mustafi, Debarshi; Engel, Andreas H.; Palczewski, Krzysztof
Progress in retinal and eye research,
07/2009, Letnik:
28, Številka:
4
Journal Article
Recenzirano
Odprti dostop
Although outnumbered more than 20:1 by rod photoreceptors, cone cells in the human retina mediate daylight vision and are critical for visual acuity and color discrimination. A variety of human ...diseases are characterized by a progressive loss of cone photoreceptors but the low abundance of cones and the absence of a macula in non-primate mammalian retinas have made it difficult to investigate cones directly. Conventional rodents (laboratory mice and rats) are nocturnal rod-dominated species with few cones in the retina, and studying other animals with cone-rich retinas presents various logistic and technical difficulties. Originating in the early 1900s, past research has begun to provide insights into cone ultrastructure but has yet to afford an overall perspective of cone cell organization. This review summarizes our past progress and focuses on the recent introduction of special mammalian models (transgenic mice and diurnal rats rich in cones) that together with new investigative techniques such as atomic force microscopy and cryo-electron tomography promise to reveal a more unified concept of cone photoreceptor organization and its role in retinal diseases.
Inosine monophosphate dehydrogenase (IMPDH) is a key regulatory enzyme in the de novo synthesis of the purine base guanine. Dominant mutations in human IMPDH1 cause photoreceptor degeneration for ...reasons that are unknown. Here, we sought to provide some foundational information on Impdh1a in the zebrafish retina. We found that in zebrafish, gene subfunctionalization due to ancestral duplication resulted in a predominant retinal variant expressed exclusively in rod and cone photoreceptors. This variant is structurally and functionally similar to the human IMPDH1 retinal variant and shares a reduced sensitivity to GTP-mediated inhibition. We also demonstrated that Impdh1a forms prominent protein filaments in vitro and in vivo in both rod and cone photoreceptor cell bodies, synapses, and to a lesser degree, in outer segments. These filaments changed length and cellular distribution throughout the day consistent with diurnal changes in both mRNA and protein levels. The loss of Impdh1a resulted in a substantial reduction of guanine levels, although cellular morphology and cGMP levels remained normal. Our findings demonstrate a significant role for IMPDH1 in photoreceptor guanine production and provide fundamental new information on the details of this protein in the zebrafish retina.
PDF
