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Exposure of plants to ultraviolet-B (UV-B) radiation initiates transcriptional responses that modify metabolism, physiology and development to enhance viability in sunlight. Many of these regulatory responses to UV-B radiation are mediated by the photoreceptor UV RESISTANCE LOCUS 8 (UVR8). Following photoreception, UVR8 interacts directly with multiple proteins to regulate gene expression, but the mechanisms that control differential protein binding to initiate distinct responses are unknown. Here we show that UVR8 is phosphorylated at several sites and that UV-B stimulates phosphorylation at Serine 402. Site-directed mutagenesis to mimic Serine 402 phosphorylation promotes binding of UVR8 to REPRESSOR OF UV-B PHOTOMORPHOGENESIS (RUP) proteins, which negatively regulate UVR8 action. Complementation of the uvr8 mutant with phosphonull or phosphomimetic variants suggests that phosphorylation of Serine 402 modifies UVR8 activity and promotes flavonoid biosynthesis, a key UV-B-stimulated response that enhances plant protection and crop nutritional quality. This research provides a basis to understand how UVR8 interacts differentially with effector proteins to regulate plant responses to UV-B radiation., (© 2024. The Author(s).)

Social recognition memory (SRM) is a key determinant of social interactions. While the cerebellum emerges as an important region for social behavior, how cerebellar activity affects social functions remains unclear. We selectively increased the excitability of molecular layer interneurons (MLIs) to suppress Purkinje cell firing in the mouse cerebellar vermis. Chemogenetic perturbation of MLIs impaired SRM without affecting sociability, anxiety levels, motor coordination or object recognition. Optogenetic interference of MLIs during distinct phases of a social recognition test revealed the cerebellar engagement in the retrieval, but not encoding, of social information. c-Fos mapping after the social recognition test showed that cerebellar manipulation decreased brain-wide interregional correlations and altered network structure from medial prefrontal cortex and hippocampus-centered to amygdala-centered modules. Anatomical tracing demonstrated hierarchical projections from the central cerebellum to the social brain network integrating amygdalar connections. Our findings suggest that the cerebellum organizes the neural matrix necessary for SRM., (© 2023. Springer Nature Limited.)

Directional movements impact the ability of plants to respond and adjust their growth accordingly to the prevailing light environment. The plasma-membrane associated protein, ROOT PHOTOTROPISM 2 (RPT2) is a key signalling component involved in chloroplast accumulation movement, leaf positioning, and phototropism, all of which are regulated redundantly by the ultraviolet/blue light-activated AGC kinases phototropin 1 and 2 (phot1 and phot2). We recently demonstrated that members of the NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3)/RPT2-like (NRL) family in Arabidopsis thaliana, including RPT2, are directly phosphorylated by phot1. However, whether RPT2 is a substrate for phot2, and the biological significance of phot phosphorylation of RPT2 remains to be determined. Here, we show that RPT2 is phosphorylated by both phot1 and phot2 at a conserved serine residue (S591) within the C-terminal region of the protein. Blue light triggered the association of 14-3-3 proteins with RPT2 consistent with S591 acting as a 14-3-3 binding site. Mutation of S591 had no effect on the plasma membrane localization of RPT2 but reduced its functionality for leaf positioning and phototropism. Moreover, our findings indicate that S591 phosphorylation within the C-terminus of RPT2 is required for chloroplast accumulation movement to low level blue light. Taken together, these findings further highlight the importance of the C-terminal region of NRL proteins and how its phosphorylation contributes to phot receptor signalling in plants., (© 2023 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Procedural memories formed in the cerebellum in response to motor errors depend on changes to Purkinje cell (PC) spiking patterns that correct movement when the erroneous context is repeated. Because molecular layer interneurons (MLIs) inhibit PCs, learning-induced changes to MLI output may participate in reshaping PC spiking patterns. However, it remains unclear whether error-driven learning alters MLI activity and whether such changes are necessary for the memory engram. We addressed this knowledge gap by measuring and manipulating MLI activity in the flocculus of both sexes of mice before and after vestibulo-ocular reflex (VOR) adaptation. We found that MLIs are activated during vestibular stimuli and that their population response exhibits a phase shift after the instantiation of gain-increase VOR adaptation, a type of error-driven learning thought to require climbing-fiber-mediated instructive signaling. Although acute optogenetic suppression of MLI activity did not affect baseline VOR performance, it negated the expression of gain-increase learning, demonstrating a specific role of MLI activity changes in motor memory expression. This effect was transitory; after a multiday consolidation period, the expression of VOR gain-increase learning was no longer sensitive to MLI activity suppression. Together, our results indicate that error-driven alteration of MLI activity is necessary for labile, climbing-fiber-induced motor memory expression. SIGNIFICANCE STATEMENT In the cerebellum, motor learning induces an associative memory of the sensorimotor context of an erroneous movement that, when recalled, results in a new pattern of output that improves subsequent trials of performance. Our study shows that error-driven motor learning induces changes to the activity pattern of cerebellar molecular layer interneurons (MLIs) and that this new pattern of activity is required to express the corrective motor memory., (Copyright © 2023 the authors.)

The methylotrophic yeast Pichia pastoris is commonly used for the production of recombinant proteins at scale. The identification of an optimally overexpressing strain following transformation can be time and reagent consuming. Fluorescent reporters like GFP have been used to assist identification of superior producers, but their relatively big size, maturation requirements and narrow temperature range restrict their applications. Here, we introduce the use of iLOV, a flavin-based fluorescent protein, as a fluorescent marker to identify P. pastoris high-yielding strains easily and rapidly. The use of this fluorescent protein as a fusion partner is exemplified by the production of the antimicrobial peptide NI01, a difficult target to overexpress in its native form. iLOV fluorescence correlated well with protein expression level and copy number of the chromosomally integrated gene. An easy and simple medium-throughput plate-based screen directly following transformation is demonstrated for low complexity screening, while a high-throughput method using fluorescence-activated cell sorting (FACS) allowed for comprehensive library screening. Both codon optimization of the iLOV_NI01 fusion cassettes and different integration strategies into the P. pastoris genome were tested to produce and isolate a high-yielding strain. Checking the genetic stability, process reproducibility and following the purification of the active native peptide are eased by visualization of and efficient cleavage from the iLOV reporter. We show that this system can be used for expression and screening of several different antimicrobial peptides recombinantly produced in P. pastoris., (© 2022 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

The cerebellum is hypothesized to represent timing information important for organizing salient motor events during periodically performed discontinuous movements. To provide functional evidence validating this idea, we measured and manipulated Purkinje cell (PC) activity in the lateral cerebellum of mice trained to volitionally perform periodic bouts of licking for regularly allocated water rewards. Overall, PC simple spiking modulated during task performance, mapping phasic tongue protrusions and retractions, as well as ramping prior to both lick-bout initiation and termination, two important motor events delimiting movement cycles. The ramping onset occurred earlier for the initiation of uncued exploratory licking that anticipated water availability relative to licking that was reactive to water allocation, suggesting that the cerebellum is engaged differently depending on the movement context. In a subpopulation of PCs, climbing-fiber-evoked responses also increased during lick-bout initiation, but not termination, highlighting differences in how cerebellar input pathways represent task-related information. Optogenetic perturbation of PC activity disrupted the behavior by degrading lick-bout rhythmicity in addition to initiating and terminating licking bouts confirming a causative role in movement organization. Together, these results substantiate that the cerebellum contributes to the initiation and timing of repeated motor actions., Competing Interests: MG, BS, JC No competing interests declared, (© 2022, Gaffield et al.)

Duchenne muscular dystrophy (DMD), the most common form of childhood muscular dystrophy, is caused by mutations in the dystrophin gene. In addition to debilitating muscle degeneration, patients display a range of cognitive deficits thought to result from the loss of dystrophin normally expressed in the brain. While the function of dystrophin in muscle tissue is well characterized, its role in the brain is still poorly understood. The highest expression of dystrophin in the mouse brain is in cerebellar Purkinje cells (PCs), where it colocalizes with GABA A receptor clusters. Using ex vivo electrophysiological recordings from connected molecular layer interneuron (MLI)-PC pairs, we investigated changes in inhibitory synaptic transmission caused by dystrophin deficiency. In male mdx mice (which lack long-form dystrophin), we found that responses at MLI-PC pairs were reduced by ∼60% because of both decreased quantal response amplitude and a reduced number of functional vesicle release sites. Using electron microscopy, we found significantly fewer and smaller anatomically defined inhibitory synapses contacting the soma of PCs in mdx mice, suggesting that dystrophin may play a critical role in synapse formation and/or maintenance. Functionally, we found reduced MLI-evoked pauses in PC firing in acute slices. In vivo recordings from awake mdx mice showed increased sensory-evoked simple spike firing in positively modulating PCs, consistent with reduced feedforward inhibition, but no change in negatively modulating PCs. These data suggest that dystrophin deficiency in PCs disrupts inhibitory signaling in the cerebellar circuit and PC firing patterns, potentially contributing to cognitive and motor deficits observed in mdx mice and DMD patients. SIGNIFICANCE STATEMENT Duchenne muscular dystrophy (DMD) is primarily characterized by progressive muscle weakening caused by genetic mutations in the gene for dystrophin. Dystrophin is also normally expressed in the CNS, and DMD patients experience a range of nonprogressive cognitive deficits. The pathophysiology of CNS neurons resulting from loss of dystrophin and the function of dystrophin in neurons are still poorly understood. Using cerebellar PCs as a model, we found that the loss of dystrophin specifically disrupts the number and strength of inhibitory synaptic connections, suggesting that dystrophin participates in formation and/or maintenance of these synapses. This work provides insight into the function of dystrophin in the CNS and establishes neuronal and synaptic dysfunction, which may underlie cognitive deficits in DMD., (Copyright © 2022 the authors.)

Blue-light (BL) phototropin receptors (phot1 and phot2) regulate plant growth by activating NPH3/RPT2-like (NRL) family members. Little is known about roles for BL and phots in regulating plant immunity. We showed previously that Phytophthora infestans RXLR effector Pi02860 targets potato (St)NRL1, promoting its ability to enhance susceptibility by facilitating proteasome-mediated degradation of the immune regulator StSWAP70. This raises the question: do BL and phots negatively regulate immunity? We employed coimmunoprecipitation, virus-induced gene silencing, transient overexpression and targeted mutation to investigate contributions of phots to regulating immunity. Whereas transient overexpression of Stphot1 and Stphot2 enhances P. infestans colonization of Nicotiana benthamiana, silencing endogenous Nbphot1 or Nbphot2 reduces infection. Stphot1, but not Stphot2, suppressed the INF1-triggered cell death (ICD) immune response in a BL- and NRL1-dependent manner. Stphot1, when coexpressed with StNRL1, promotes degradation of StSWAP70, whereas Stphot2 does not. Kinase-dead Stphot1 fails to suppress ICD, enhance P. infestans colonization or promote StSWAP70 degradation. Critically, BL enhances P. infestans infection, which probably involves phots but not other BL receptors such as cryptochromes and F-box proteins ZTL1 and FKF1. We demonstrate that Stphot1 and Stphot2 play different roles in promoting susceptibility, and Stphot1 kinase activity is required for BL- and StNRL1-mediated immune suppression., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

Polarity underlies all directional growth responses in plants including growth towards the light (phototropism). The plasma-membrane associated protein, NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) is a key determinant of phototropic growth which is regulated by phototropin (phot) AGC kinases. Here we demonstrate that NPH3 is directly phosphorylated by phot1 within a conserved C-terminal consensus sequence (RxS) that is necessary to promote phototropism and petiole positioning in Arabidopsis. RxS phosphorylation also triggers 14-3-3 binding combined with changes in NPH3 phosphorylation and localisation status. Mutants of NPH3 that are unable to bind or constitutively bind 14-3-3 s show compromised functionality consistent with a model where phototropic curvature is established by signalling outputs arising from a gradient of NPH3 RxS phosphorylation across the stem. Our findings therefore establish that NPH3/RPT2-Like (NRL) proteins are phosphorylation targets for plant AGC kinases. Moreover, RxS phosphorylation is conserved in other members of the NRL family, suggesting a common mechanism of regulating plant growth to the prevailing light environment., (© 2021. The Author(s).)

Ferns appear in the fossil record some 200 Myr before angiosperms. However, as angiosperm-dominated forest canopies emerged in the Cretaceous period there was an explosive diversification of modern (leptosporangiate) ferns, which thrived in low, blue-enhanced light beneath angiosperm canopies. A mechanistic explanation for this transformative event in the diversification of ferns has remained elusive. We used physiological assays, transcriptome analysis and evolutionary bioinformatics to investigate a potential connection between the evolution of enhanced stomatal sensitivity to blue light in modern ferns and the rise of angiosperm-dominated forests in the geological record. We demonstrate that members of the largest subclade of leptosporangiate ferns, Polypodiales, have significantly faster stomatal response to blue light than more ancient fern lineages and a representative angiosperm. We link this higher sensitivity to levels of differentially expressed genes in blue-light signaling, particularly in the cryptochrome (CRY) signaling pathway. Moreover, CRYs of the Polypodiales examined show gene duplication events between 212.9-196.9 and 164.4-151.8 Ma, when angiosperms were emerging, which are lacking in other major clades of extant land plants. These findings suggest that evolution of stomatal blue-light sensitivity helped modern ferns exploit the shady habitat beneath angiosperm forest canopies, fueling their Cretaceous hyperdiversification., (© 2020 The Authors New Phytologist © 2020 New Phytologist Foundation.)

The signals in cerebellar Purkinje cells sufficient to instruct motor learning have not been systematically determined. Therefore, we applied optogenetics in mice to autonomously excite Purkinje cells and measured the effect of this activity on plasticity induction and adaptive behavior. Ex vivo, excitation of channelrhodopsin-2-expressing Purkinje cells elicits dendritic Ca 2+ transients with high-intensity stimuli initiating dendritic spiking that additionally contributes to the Ca 2+ response. Channelrhodopsin-2-evoked Ca 2+ transients potentiate co-active parallel fiber synapses; depression occurs when Ca 2+ responses were enhanced by dendritic spiking. In vivo, optogenetic Purkinje cell activation drives an adaptive decrease in vestibulo-ocular reflex gain when vestibular stimuli are paired with relatively small-magnitude Purkinje cell Ca 2+ responses. In contrast, pairing with large-magnitude Ca 2+ responses increases vestibulo-ocular reflex gain. Optogenetically induced plasticity and motor adaptation are dependent on endocannabinoid signaling, indicating engagement of this pathway downstream of Purkinje cell Ca 2+ elevation. Our results establish a causal relationship among Purkinje cell Ca 2+ signal size, opposite-polarity plasticity induction, and bidirectional motor learning.

Electron microscopy (EM) enables high-resolution visualization of protein distributions in biological tissues. For detection, gold nanoparticles are typically used as an electron-dense marker for immunohistochemically labeled proteins. Manual annotation of gold particle labels is laborious and time consuming, as gold particle counts can exceed 100,000 across hundreds of image segments to obtain conclusive data sets. To automate this process, we developed Gold Digger, a software tool that uses a modified pix2pix deep learning network capable of detecting and annotating colloidal gold particles in biological EM images obtained from both freeze-fracture replicas and plastic sections prepared with the post-embedding method. Gold Digger performs at near-human-level accuracy, can handle large images, and includes a user-friendly tool with a graphical interface for proof reading outputs by users. Manual error correction also helps for continued re-training of the network to improve annotation accuracy over time. Gold Digger thus enables rapid high-throughput analysis of immunogold-labeled EM data and is freely available to the research community.

The last two decades have witnessed the emergence of optogenetics; a field that has given researchers the ability to use light to control biological processes at high spatiotemporal and quantitative resolutions, in a reversible manner with minimal side-effects. Optogenetics has revolutionized the neurosciences, increased our understanding of cellular signalling and metabolic networks and resulted in variety of applications in biotechnology and biomedicine. However, implementing optogenetics in plants has been less straightforward, given their dependency on light for their life cycle. Here, we highlight some of the widely used technologies in microorganisms and animal systems derived from plant photoreceptor proteins and discuss strategies recently implemented to overcome the challenges for using optogenetics in plants., (© 2020 The Authors New Phytologist © 2020 New Phytologist Foundation.)

Phototropins (phot1 and phot2) are plant blue light receptor kinases that function to mediate phototropism, chloroplast movement, leaf flattening, and stomatal opening in Arabidopsis. Considerable progress has been made in understanding the mechanisms associated with phototropin receptor activation by light. However, the identities of phototropin signaling components are less well understood by comparison. In this study, we specifically searched for protein kinases that interact with phototropins by using an in vitro screening method (AlphaScreen) to profile interactions against an Arabidopsis protein kinase library. We found that CBL-interacting protein kinase 23 (CIPK23) interacts with both phot1 and phot2. Although these interactions were verified by in vitro pull-down and in vivo bimolecular fluorescence complementation assays, CIPK23 was not phosphorylated by phot1, as least in vitro. Mutants lacking CIPK23 were found to exhibit impaired stomatal opening in response to blue light but no deficits in other phototropin-mediated responses. We further found that blue light activation of inward-rectifying K + (K + in ) channels was impaired in the guard cells of cipk23 mutants, whereas activation of the plasma membrane H + -ATPase was not. The blue light activation of K + in channels was also impaired in the mutant of BLUS1, which is one of the phototropin substrates in guard cells. We therefore conclude that CIPK23 promotes stomatal opening through activation of K + in channels most likely in concert with BLUS1, but through a mechanism other than activation of the H + -ATPase. The role of CIPK23 as a newly identified component of phototropin signaling in stomatal guard cells is discussed., (© 2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

The ability to enhance photosynthetic capacity remains a recognized bottleneck to improving plant productivity. Phototropin blue light receptors (phot1 and phot2) optimize photosynthetic efficiency in Arabidopsis thaliana by coordinating multiple light-capturing processes. In this study, we explore the potential of using protein engineering to improve photoreceptor performance and thereby plant growth. We demonstrate that targeted mutagenesis can decrease or increase the photocycle lifetime of Arabidopsis phototropins in vitro and show that these variants can be used to reduce or extend the duration of photoreceptor activation in planta Our findings show that slowing the phototropin photocycle enhanced several light-capturing responses, while accelerating it reduced phototropin's sensitivity for chloroplast accumulation movement. Moreover, plants engineered to have a slow-photocycling variant of phot1 or phot2 displayed increased biomass production under low-light conditions as a consequence of their improved sensitivity. Together, these findings demonstrate the feasibility of engineering photoreceptors to manipulate plant growth and offer additional opportunities to enhance photosynthetic competence, particularly under suboptimal light regimes., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Phototropin (phot) receptor kinases play important roles in promoting plant growth by controlling light-capturing processes, such as phototropism. Phototropism is mediated through the action of NON-PHOTOTROPIC HYPOCOTYL3 (NPH3), which is dephosphorylated following phot activation. However, the functional significance of this early signaling event remains unclear. Here, we show that the onset of phototropism in dark-grown (etiolated) seedlings of Arabidopsis ( Arabidopsis thaliana ) and tomato ( Solanum lycopersicum ) is enhanced by greening (deetiolation). Red and blue light were equally effective in promoting phototropism in Arabidopsis, consistent with our observations that deetiolation by phytochrome or cryptochrome was sufficient to enhance phototropism. Increased responsiveness did not result from an enhanced sensitivity to the phytohormone auxin, nor does it involve the phot-interacting protein, ROOT PHOTOTROPISM2. Instead, deetiolated seedlings showed attenuated levels of NPH3 dephosphorylation and diminished relocalization of NPH3 from the plasma membrane during phototropism. Likewise, etiolated seedlings that lack the PHYTOCHROME-INTERACTING FACTORS (PIFs) PIF1, PIF3, PIF4, and PIF5 displayed reduced NPH3 dephosphorylation and enhanced phototropism, consistent with their constitutive photomorphogenic phenotype in darkness. Phototropic enhancement could also be achieved in etiolated seedlings by lowering the light intensity to diminish NPH3 dephosphorylation. Thus, phototropism is enhanced following deetiolation through the modulation of a phosphorylation rheostat, which in turn sustains the activity of NPH3. We propose that this dynamic mode of regulation enables young seedlings to maximize their establishment under changing light conditions, depending on their photoautotrophic capacity., (© 2019 The author(s). All Rights Reserved.)

The brain must make sense of external stimuli to generate relevant behavior. We used a combination of in vivo approaches to investigate how the cerebellum processes sensory-related information. We found that the inferior olive encodes contexts of sensory-associated external cues in a graded manner, apparent in the presynaptic activity of their axonal projections (climbing fibers) in the cerebellar cortex. Individual climbing fibers were broadly responsive to different sensory modalities but relayed sensory-related information to the cortex in a lobule-dependent manner. Purkinje cell dendrites faithfully transformed this climbing fiber activity into dendrite-wide Ca 2+ signals without a direct contribution from the mossy fiber pathway. These results demonstrate that the size of climbing-fiber-evoked Ca 2+ signals in Purkinje cell dendrites is largely determined by the firing level of climbing fibers. This coding scheme emphasizes the overwhelming role of the inferior olive in generating salient signals useful for instructing plasticity and learning., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Stomata serve dual and often conflicting roles, facilitating carbon dioxide influx into the plant leaf for photosynthesis and restricting water efflux via transpiration. Strategies for reducing transpiration without incurring a cost for photosynthesis must circumvent this inherent coupling of carbon dioxide and water vapor diffusion. We expressed the synthetic, light-gated K + channel BLINK1 in guard cells surrounding stomatal pores in Arabidopsis to enhance the solute fluxes that drive stomatal aperture. BLINK1 introduced a K + conductance and accelerated both stomatal opening under light exposure and closing after irradiation. Integrated over the growth period, BLINK1 drove a 2.2-fold increase in biomass in fluctuating light without cost in water use by the plant. Thus, we demonstrate the potential of enhancing stomatal kinetics to improve water use efficiency without penalty in carbon fixation., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

UVR8 is a plant photoreceptor protein that regulates photomorphogenic and protective responses to UV light. The inactive, homodimeric state absorbs UV-B light, resulting in dissociation into monomers, which are considered to be the active state and comprise a β-propeller core domain and intrinsically disordered N- and C-terminal tails. The C terminus is required for functional binding to signaling partner COP1. To date, however, structural studies have only been conducted with the core domain where the terminal tails have been truncated. Here, we report structural investigations of full-length UVR8 using native ion mobility mass spectrometry adapted for photoactivation. We show that, while truncated UVR8 photoconverts from a single conformation of dimers to a single monomer conformation, the full-length protein exists in numerous conformational families. The full-length dimer adopts both a compact state and an extended state where the C terminus is primed for activation. In the monomer the extended C terminus destabilizes the core domain to produce highly extended yet stable conformations, which we propose are the fully active states that bind COP1. Our results reveal the conformational diversity of full-length UVR8. We also demonstrate the potential power of native mass spectrometry to probe functionally important structural dynamics of photoreceptor proteins throughout nature., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

In addition to cognitive impairments, neurodevelopmental disorders often result in sensory processing deficits. However, the biological mechanisms that underlie impaired sensory processing associated with neurodevelopmental disorders are generally understudied and poorly understood. We found that SYNGAP1 haploinsufficiency in humans, which causes a sporadic neurodevelopmental disorder defined by cognitive impairment, autistic features, and epilepsy, also leads to deficits in tactile-related sensory processing. In vivo neurophysiological analysis in Syngap1 mouse models revealed that upper-lamina neurons in somatosensory cortex weakly encode information related to touch. This was caused by reduced synaptic connectivity and impaired intrinsic excitability within upper-lamina somatosensory cortex neurons. These results were unexpected, given that Syngap1 heterozygosity is known to cause circuit hyperexcitability in brain areas more directly linked to cognitive functions. Thus, Syngap1 heterozygosity causes a range of circuit-specific pathologies, including reduced activity within cortical neurons required for touch processing, which may contribute to sensory phenotypes observed in patients.

Purkinje cell dendrites convert excitatory climbing fiber input into signals that instruct plasticity and motor learning. Modulation of instructive signaling may increase the range in which learning is encoded, yet the mechanisms that allow for this are poorly understood. We found that optogenetic activation of molecular layer interneurons (MLIs) that inhibit Purkinje cells suppressed climbing-fiber-evoked dendritic Ca 2+ spiking. Inhibitory suppression of Ca 2+ spiking depended on the level of MLI activation and influenced the induction of associative synaptic plasticity, converting climbing-fiber-mediated potentiation of parallel fiber-evoked responses into depression. In awake mice, optogenetic activation of floccular climbing fibers in association with head rotation produced an adaptive increase in the vestibulo-ocular reflex (VOR). However, when climbing fibers were co-activated with MLIs, adaptation occurred in the opposite direction, decreasing the VOR. Thus, MLIs can direct a continuous spectrum of plasticity and learning through their influence on Purkinje cell dendritic Ca 2+ signaling., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Motor learning involves neural circuit modifications in the cerebellar cortex, likely through re-weighting of parallel fiber inputs onto Purkinje cells (PCs). Climbing fibers instruct these synaptic modifications when they excite PCs in conjunction with parallel fiber activity, a pairing that enhances climbing fiber-evoked Ca 2+ signaling in PC dendrites. In vivo, climbing fibers spike continuously, including during movements when parallel fibers are simultaneously conveying sensorimotor information to PCs. Whether parallel fiber activity enhances climbing fiber Ca 2+ signaling during motor behaviors is unknown. In mice, we found that inhibitory molecular layer interneurons (MLIs), activated by parallel fibers during practiced movements, suppressed parallel fiber enhancement of climbing fiber Ca 2+ signaling in PCs. Similar results were obtained in acute slices for brief parallel fiber stimuli. Interestingly, more prolonged parallel fiber excitation revealed latent supralinear Ca 2+ signaling. Therefore, the balance of parallel fiber and MLI input onto PCs regulates concomitant climbing fiber Ca 2+ signaling., Competing Interests: MG, MR, SA, HH, JC No competing interests declared, (© 2018, Gaffield et al.)

The geomagnetic field (GMF) is an environmental element whose instability affects plant growth and development. Despite known plant responses to GMF direction and intensity, the mechanism of magnetoreception in plants is still not known. Magnetic field variations affect many light-dependent plant processes, suggesting that the magnetoreception could require light. The objective of this work was to comprehensively investigate the influence of GMF on Arabidopsis thaliana (Col-0) photoreceptor signaling. Wild-type Arabidopsis seedlings and photoreceptor-deficient mutants (cry1cry2, phot1, phyA and phyAphyB) were exposed to near null magnetic field (NNMF, ≤40 nT) and GMF (~43 μT) under darkness and different light wavelengths. The GMF did not alter skotomorphogenic or photomorphogenic seedling development but had a significant impact on gene expression pathways downstream of cryptochrome and phytochrome photoactivation. GMF-induced changes in gene expression observed under blue light were partially associated with an alteration of cryptochrome activation. GMF impacts on phytochrome-regulated gene expression could be attributed to alterations in phytochrome protein abundance that were also dependent on the presence of cry1, cry2 and phot1. Moreover, the GMF was found to impact photomorphogenic-promoting gene expression in etiolated seedlings, indicating the existence of a light-independent magnetoreception mechanism. In conclusion, our data shows that magnetoreception alters photoreceptor signaling in Arabidopsis, but it does not necessarily depend on light., (Copyright © 2018 Elsevier B.V. All rights reserved.)

LOV-domains are ubiquitous photosensory proteins that are commonly re-engineered to serve as powerful and versatile fluorescent proteins and optogenetic tools. The photoactive, flavin chromophore, however, is excited using short wavelengths of light in the blue and UV regions, which have limited penetration into biological samples and can cause photodamage. Here, we have used non-linear spectroscopy and microscopy of the fluorescent protein, iLOV, to reveal that functional variants of LOV can be activated to great effect by two non-resonant photons of lower energy, near infrared light, not only in solution but also in biological samples. The two photon cross section of iLOV has a significantly blue-shifted S0 → S1 transition compared with the one photon absorption spectrum, suggesting preferential population of excited vibronic states. It is highly likely, therefore, that the two photon absorption wavelength of engineered, LOV-based tools is tuneable. We also demonstrate for the first time two photon imaging using iLOV in human epithelial kidney cells. Consequently, two photon absorption by engineered, flavin-based bio-molecular tools can enable non-invasive activation with high depth resolution and the potential for not only improved image clarity but also enhanced spatiotemporal control for optogenetic applications.

Phototropin (phot)-mediated signaling initiated by blue light (BL) plays a critical role in optimizing photosynthetic light capture at the plasma membrane (PM) in plants. However, the mechanisms underlying the regulation of phot activity at the PM in response to BL remain largely unclear. In this study, by single-particle tracking and stepwise photobleaching analysis of phot1-GFP proteins we demonstrated that in the dark phot1 proteins remain in an inactive state and mostly exist as monomers. Dimerization and the diffusion rate of phot1-GFP increased in a dose-dependent manner in response to BL. In contrast, BL did not affect the lateral diffusion of kinase-inactive phot1 D806N -GFP but did enhance its dimerization, suggesting that phot1 dimerization is independent of phosphorylation. Förster resonance energy transfer-fluorescence lifetime imaging microscopy analysis revealed that the interaction between phot1-GFP and a marker of sterol-rich lipid environments, AtRem1.3-mCherry, was enhanced with increased time of BL treatment. However, this BL-dependent interaction was not obvious in plants co-expressing phot1 D806N -GFP and AtRem1.3-mCherry, indicating that BL facilitates the translocation of functional phot1-GFP into AtRem1.3-labeled microdomains to activate phot-mediated signaling. Conversely, sterol depletion attenuated phot1-GFP dynamics, dimerization, and phosphorylation. Taken together, these results indicate that membrane microdomains act as organizing platforms essential for the proper function of activated phot1 at the PM., (Copyright © 2018 The Author. Published by Elsevier Inc. All rights reserved.)

Protein kinases (PKs) control many aspects of plant physiology by regulating signaling networks through protein phosphorylation. Phototropins (phots) are plasma membrane-associated serine/threonine PKs that control a range of physiological processes that collectively serve to optimize photosynthetic efficiency in plants. These include phototropism, leaf positioning and flattening, chloroplast movement, and stomatal opening. Despite their identification over two decades ago, only a handful of substrates have been identified for these PKs. Progress in this area has been hampered by the lack of a convenient means to confirm the identity of potential substrate candidates. Here we demonstrate that the kinase domain of Arabidopsis phot1 and phot2 can be successfully engineered to accommodate non-natural ATP analogues by substituting the bulky gatekeeper residue threonine for glycine. This approach circumvents the need for radioactivity to track phot kinase activity and follow light-induced receptor autophosphorylation in vitro by incorporating thiophosphate from N 6 -benzyl-ATPγS. Consequently, thiophosphorylation of phot substrate candidates can be readily monitored when added or co-expressed with phots in vitro Furthermore, gatekeeper-modified phot1 retained its functionality and its ability to accommodate N 6 -benzyl-ATPγS as a phosphodonor when expressed in Arabidopsis We therefore anticipate that this chemical genetic approach will provide new opportunities for labeling and identifying substrates for phots and other related AGC kinases under in vitro and near-native in vivo conditions., (© 2018 Schnabel et al.)

Salmonella invasion protein A (SipA) is a dual-function effector protein that plays roles in both actin polymerization and caspase-3 activation in intestinal epithelial cells. To date its function in other cell types has remained largely unknown despite its expression in multiple cell types and its extracellular secretion during infection. Here we show that in macrophages SipA induces increased caspase-3 activation early in infection. This activation required a threshold level of SipA linked to multiplicity of infection and may be a limiting factor controlling bacterial numbers in infected macrophages. In polymorphonuclear leukocytes, SipA or other Salmonella pathogenicity island 1 effectors had no effect on induction of caspase-3 activation either alone or in the presence of whole bacteria. Tagging of SipA with the small fluorescent phiLOV tag, which can pass through the type three secretion system, allowed visualization and quantification of caspase-3 activation by SipA-phiLOV in macrophages. Additionally, SipA-phiLOV activation of caspase-3 could be tracked in the intestine through multiphoton laser scanning microscopy in an ex vivo intestinal model. This allowed visualization of areas where the intestinal epithelium had been compromised and demonstrated the potential use of this fluorescent tag for in vivo tracking of individual effectors., (Copyright © 2017 McIntosh et al.)

Phototropins (phots) are plasma membrane-associated serine/threonine kinases that coordinate a range of processes linked to optimizing photosynthetic efficiency in plants. These photoreceptors contain two light-, oxygen-, or voltage-sensing (LOV) domains within their N terminus, with each binding one molecule of flavin mononucleotide as a UV/blue light-absorbing chromophore. Although phots contain two LOV domains, light-induced activation of the C-terminal kinase domain and subsequent receptor autophosphorylation is controlled primarily by the A'α-LOV2-Jα photosensory module. Mutations that disrupt interactions between the LOV2 core and its flanking helical segments can uncouple this mode of light regulation. However, the impact of these mutations on phot function in Arabidopsis has not been explored. Here we report that histidine substitution of Arg-472 located within the A'α-helix of Arabidopsis phot1 constitutively activates phot1 kinase activity in vitro without affecting LOV2 photochemistry. Expression analysis of phot1 R472H in the phot-deficient mutant confirmed that it is autophosphorylated in darkness in vivo but unable to initiate phot1 signaling in the absence of light. Instead, we found that phot1 R472H is poorly functional under low-light conditions but can restore phototropism, chloroplast accumulation, stomatal opening, and leaf positioning and expansion at higher light intensities. Our findings suggest that Arabidopsis can adapt to the elevated phosphorylation status of the phot1 R472H mutant in part by reducing its stability, whereas the activity of the mutant under high-light conditions can be attributed to additional increases in LOV2-mediated photoreceptor autophosphorylation., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The cerebellar system helps modulate and fine-tune motor action. Purkinje cells (PCs) provide the sole output of the cerebellar cortex, therefore, any cerebellar involvement in motor activity must be driven by changes in PC firing rates. Several different cell types influence PC activity including excitatory input from parallel fibers and inhibition from molecular layer interneurons (MLIs). Similar to PCs, MLI activity is driven by parallel fibers, therefore, MLIs provide feed-forward inhibition onto PCs. To aid in the experimental assessment of how molecular layer inhibition contributes to cerebellar function and motor behavior, we characterized a new knock-in mouse line with Cre recombinase expression under control of endogenous c-kit transcriptional machinery. Using these engineered c-Kit mice, we were able to obtain high levels of conditional MLI transduction in adult mice using Cre-dependent viral vectors without any PC or granule cell labeling. We then used the mouse line to target MLIs for activity perturbation in vitro using opto- and chemogenetics.

Inhibition from molecular layer interneurons (MLIs) is thought to play an important role in cerebellar function by sharpening the precision of Purkinje cell spike output. Yet the coding features of MLIs during behavior are poorly understood. To study MLI activity, we used in vivo Ca 2+ imaging in head-fixed mice during the performance of a rhythmic motor behavior, licking during water consumption. MLIs were robustly active during lick-related movement across a lobule-specific region of the cerebellum showing high temporal correspondence within their population. Average MLI Ca 2+ activity strongly correlated with movement rate but not to the intentional, or unexpected, adjustment of lick position or to sensory feedback that varied with task condition. Chemogenetic suppression of MLI output reduced lick rate and altered tongue movements, indicating that activity of these interneurons not only encodes temporal aspects of movement kinematics but also influences motor outcome pointing to an integral role in online control of rhythmic behavior. SIGNIFICANCE STATEMENT The cerebellum helps fine-tune coordinated motor actions via signaling from projection neurons called Purkinje cells. Molecular layer interneurons (MLIs) provide powerful inhibition onto Purkinje cells, but little is understood about how this inhibitory circuit is engaged during behavior or what type of information is transmitted through these neurons. Our work establishes that MLIs in the lateral cerebellum are broadly activated during movement with calcium activity corresponding to movement rate. We also show that suppression of MLI output slows and disorganizes the precise movement pattern. Therefore, MLIs are an important circuit element in the cerebellum allowing for accurate motor control., (Copyright © 2017 the authors 0270-6474/17/374751-15$15.00/0.)

In many neurons, subthreshold depolarization in the soma can transiently increase action-potential (AP)-evoked neurotransmission via analog-to-digital facilitation. The mechanisms underlying this form of short-term synaptic plasticity are unclear, in part, due to the relative inaccessibility of the axon to direct physiological interrogation. Using voltage imaging and patch-clamp recording from presynaptic boutons of cerebellar stellate interneurons, we observed that depolarizing somatic potentials readily spread into the axon, resulting in AP broadening, increased spike-evoked Ca 2+ entry, and enhanced neurotransmission strength. K v 3 channels, which drive AP repolarization, rapidly inactivated upon incorporation of K v 3.4 subunits. This leads to fast susceptibility to depolarization-induced spike broadening and analog facilitation independent of Ca 2+ -dependent protein kinase C signaling. The spread of depolarization into the axon was attenuated by hyperpolarization-activated currents (I h currents) in the maturing cerebellum, precluding analog facilitation. These results suggest that analog-to-digital facilitation is tempered by development or experience in stellate cells., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Phototropin (phot1) is a blue light-activated plasma membrane-associated kinase that acts as the principal photoreceptor for shoot phototropism in Arabidopsis in conjunction with the signalling component Non-Phototropic Hypocotyl 3 (NPH3). PHOT1 is uniformly expressed throughout the Arabidopsis hypocotyl, yet decapitation experiments have localized the site of light perception to the upper hypocotyl. This prompted us to investigate in more detail the functional role of the hypocotyl apex, and the regions surrounding it, in establishing phototropism. We used a non-invasive approach where PHOT1-GFP (P1-GFP) expression was targeted to the hypocotyl apex of the phot-deficient mutant using the promoters of CUP-SHAPED COTYLEDON 3 (CUC3) and AINTEGUMENTA (ANT). Expression of CUC3::P1-GFP was clearly visible at the hypocotyl apex, with weaker expression in the cotyledons, whereas ANT::P1-GFP was specifically targeted to the developing leaves. Both lines showed impaired curvature to 0.005 μmol m -2  sec -1 unilateral blue light, indicating that regions below the apical meristem are necessary for phototropism. Curvature was however apparent at higher fluence rates. Moreover, CUC3::P1-GFP partially or fully complemented petiole positioning, leaf flattening and chloroplast accumulation, but not stomatal opening. Yet, tissue analysis of NPH3 de-phosphorylation showed that CUC3::P1-GFP and ANT::P1-GFP mis-express very low levels of phot1 that likely account for this responsiveness. Our spatial targeting approach therefore excludes the hypocotyl apex as the site for light perception for phototropism and shows that phot1-mediated NPH3 de-phosphorylation is tissue autonomous and occurs more prominently in the basal hypocotyl., (© 2016 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

UV RESISTANCE LOCUS8 (UVR8) is a photoreceptor for ultraviolet-B (UV-B) light that initiates photomorphogenic responses in plants. UV-B photoreception causes rapid dissociation of dimeric UVR8 into monomers that interact with CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) to initiate signal transduction. Experiments with purified UVR8 show that the dimer is maintained by salt-bridge interactions between specific charged amino acids across the dimer interface. However, little is known about the importance of these charged amino acids in determining dimer/monomer status and UVR8 function in plants. Here we evaluate the use of different methods to examine dimer/monomer status of UVR8 and show that mutations of several salt-bridge amino acids affect dimer/monomer status, interaction with COP1 and photoreceptor function of UVR8 in vivo. In particular, the salt-bridges formed between arginine 286 and aspartates 96 and 107 are key to dimer formation. Mutation of arginine 286 to alanine impairs dimer formation, interaction with COP1 and function in vivo, whereas mutation to lysine gives a weakened dimer that is functional in vivo, indicating the importance of the positive charge of the arginine/lysine residue for dimer formation. Notably, a UVR8 mutant in which aspartates 96 and 107 are conservatively mutated to asparagine is strongly impaired in dimer formation but mediates UV-B responses in vivo with a similar dose-response relationship to wild-type. The UV-B responsiveness of this mutant does not correlate with dimer formation and monomerisation, indicating that monomeric UVR8 has the potential for UV-B photoreception, initiating signal transduction and responses in plants., (© 2016 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

In axons, an action potential (AP) is thought to be broadcast as an unwavering binary pulse over its arbor, driving neurotransmission uniformly at release sites. Yet by recording from axons of cerebellar stellate cell (SC) interneurons, we show that AP width varies between presynaptic bouton sites, even within the same axon branch. The varicose geometry of SC boutons alone does not impose differences in spike duration. Rather, axonal patching revealed heterogeneous peak conductance densities of currents mediated mainly by fast-activating Kv3-type potassium channels, with clustered hotspots at boutons and restricted expression at adjoining shafts. Blockade of Kv channels at individual boutons indicates that currents immediately local to a release site direct spike repolarization at that location. Thus, the clustered arrangement and variable expression density of Kv3 channels at boutons are key determinants underlying compartmentalized control of AP width in a near synapse-by-synapse manner, multiplying the signaling capacity of these structures., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Bacterial type III secretion system (T3SS) effector proteins are critical determinants of infection for many animal and plant pathogens. However, monitoring of the translocation and delivery of these important virulence determinants has proved to be technically challenging. Here, we used a genetically engineered LOV (light-oxygen-voltage) sensing domain derivative to monitor the expression, translocation, and localization of bacterial T3SS effectors. We found the Escherichia coli O157:H7 bacterial effector fusion Tir-LOV was functional following its translocation and localized to the host cell membrane in discrete foci, demonstrating that LOV-based reporters can be used to visualize the effector translocation with minimal manipulation and interference. Further evidence for the versatility of the reporter was demonstrated by fusing LOV to the C terminus of the Shigella flexneri effector IpaB. IpaB-LOV localized preferentially at bacterial poles before translocation. We observed the rapid translocation of IpaB-LOV in a T3SS-dependent manner into host cells, where it localized at the bacterial entry site within membrane ruffles., (Copyright © 2016 Gawthorne et al.)

The uses of fluorescent reporters derived from green fluorescent protein have proved invaluable for the visualisation of biological processes in bacteria grown under aerobic conditions. However, their requirement for oxygen has limited their application in obligate anaerobes such as Clostridium difficile. Fluorescent proteins derived from Light, Oxygen or Voltage sensing (LOV) domains have been shown to bridge this limitation, but their utility as translational fusions to monitor protein expression and localisation in a strict anaerobic bacterium has not been reported. Here we demonstrate the utility of phiLOV in three species of Clostridium and its application as a marker of real-time protein translation and dynamics through genetic fusion with the cell division protein, FtsZ. Time lapse microscopy of dividing cells suggests that Z ring assembly arises through the extension of the FtsZ arc starting from one point on the circumference. Furthermore, through incorporation of phiLOV into the flagella subunit, FliC, we show the potential of bacterial LOV-based fusion proteins to be successfully exported to the extracellular environment.

Hypocotyl phototropism of etiolated Arabidopsis seedlings is primarily mediated by the blue-light receptor kinase phototropin 1 (phot1). Phot1-mediated curvature to continuous unilateral blue light irradiation (0.5 μmol m(-2) s(-1)) is enhanced by overhead pre-treatment with red light (20 μmol m(-2) s(-1) for 15 min) through the action of phytochrome (phyA). Here, we show that pre-treatment with blue light is equally as effective in eliciting phototropic enhancement and is dependent on phyA. Although blue light pre-treatment was sufficient to activate early phot1 signaling events, phot1 autophosphorylation in vivo was not found to be saturated, as assessed by subsequently measuring phot1 kinase activity in vitro. However, enhancement effects by red and blue light pre-treatment were not observed at higher intensities of phototropic stimulation (10 μmol m(-2) s(-1)). Phototropic enhancement by red and blue light pre-treatments to 0.5 μmol m(-2) s(-1) unilateral blue light irradiation was also lacking in transgenic Arabidopsis where PHOT1 expression was restricted to the epidermis. Together, these findings indicate that phyA-mediated effects on phot1 signaling are restricted to low intensities of phototropic stimulation and originate from tissues other than the epidermis.

Purkinje cells (PCs) are a major site of information integration and plasticity in the cerebellum, a brain region involved in motor task refinement. Thus PCs provide an ideal location for studying the mechanisms necessary for cerebellum-dependent motor learning. Increasingly, sophisticated behavior tasks, used in combination with genetic reporters and effectors of activity, have opened up the possibility of studying cerebellar circuits during voluntary movement at an unprecedented level of quantitation. However, current methods used to monitor PC activity do not take full advantage of these advances. For example, single-unit or multiunit electrode recordings, which provide excellent temporal information regarding electrical activity, only monitor a small population of cells and can be quite invasive. Bolus loading of cell-permeant calcium (Ca(2+)) indicators is short-lived, requiring same-day imaging immediately after surgery and/or indicator injection. Genetically encoded Ca(2+) indicators (GECIs) overcome many of these limits and have garnered considerable use in many neuron types but only limited use in PCs. Here we employed these indicators to monitor Ca(2+) activity in PCs over several weeks. We could repeatedly image from the same cerebellar regions across multiple days and observed stable activity. We used chronic imaging to monitor PC activity in crus II, an area previously linked to licking behavior, and identified a region of increased activity at the onset of licking. We then monitored this same region after training tasks to initiate voluntary licking behavior in response to different sensory stimuli. In all cases, PC Ca(2+) activity increased at the onset of rhythmic licking., (Copyright © 2016 the American Physiological Society.)

Phototropins (phots) regulate a range of adaptive processes in plants that serve to optimize photosynthetic efficiency and promote growth. Light sensing by Arabidopsis thaliana phots is predominantly mediated by the Light, Oxygen and Voltage sensing 2 (LOV2) flavin-binding motif located within the N-terminus of the photoreceptor. Here we characterize the photochemical and biochemical properties of phot from the marine picoalga Ostreococcus tauri phototropin (Otphot) and examine its ability to replace phot-mediated function in Arabidopsis. Photochemical properties of Otphot rely on both LOV1 and LOV2. Yet, biochemical analysis indicates that light-dependent receptor autophosphorylation is primarily dependent on LOV2. As found for Arabidopsis phots, Otphot associates with the plasma membrane and partially internalizes, albeit to a limited extent, in response to blue-light irradiation. Otphot is able to elicit a number of phot-regulated processes in Arabidopsis, including petiole positioning, leaf expansion, stomatal opening and chloroplast accumulation movement. However, Otphot is unable to restore phototropism and chloroplast avoidance movement. Consistent with its lack of phototropic function in Arabidopsis, Otphot does not associate with or trigger dephosphorylation of the phototropic signalling component Non-Phototropic Hypocotyl 3 (NPH3). Taken together, these findings indicate that the mechanism of action of plant and evolutionarily distant algal phots is less well conserved than previously thought., (© 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.)

These updated guidelines on the management of variceal haemorrhage have been commissioned by the Clinical Services and Standards Committee (CSSC) of the British Society of Gastroenterology (BSG) under the auspices of the liver section of the BSG. The original guidelines which this document supersedes were written in 2000 and have undergone extensive revision by 13 members of the Guidelines Development Group (GDG). The GDG comprises elected members of the BSG liver section, representation from British Association for the Study of the Liver (BASL) and Liver QuEST, a nursing representative and a patient representative. The quality of evidence and grading of recommendations was appraised using the AGREE II tool.The nature of variceal haemorrhage in cirrhotic patients with its complex range of complications makes rigid guidelines inappropriate. These guidelines deal specifically with the management of varices in patients with cirrhosis under the following subheadings: (1) primary prophylaxis; (2) acute variceal haemorrhage; (3) secondary prophylaxis of variceal haemorrhage; and (4) gastric varices. They are not designed to deal with (1) the management of the underlying liver disease; (2) the management of variceal haemorrhage in children; or (3) variceal haemorrhage from other aetiological conditions., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Chromophore-binding domains from plant and bacterial photoreceptor proteins have recently gathered increasing attention as new sources of genetically encoded fluorescent proteins (FPs). In particular, FPs based on the flavin-binding LOV (light, oxygen, or voltage sensing) domain offer advantages over green fluorescent protein (GFP) owing to their smaller size, pH and thermal stability, utility under anaerobic conditions and their ability to generate reactive oxygen species. This review focuses on the potential applications of this emerging class of fluorescent reporters, discusses the advantages and limitations of LOV-based FPs, whilst offering insights regarding the further development of this technology for bioimaging and photodynamic therapy., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

UVR8 is a novel UV-B photoreceptor that regulates a range of plant responses and is already used as a versatile optogenetic tool. Instead of an exogenous chromophore, UVR8 uniquely employs tryptophan side chains to accomplish UV-B photoreception. UV-B absorption by homodimeric UVR8 induces monomerization and hence signaling, but the underlying photodynamic mechanisms are not known. Here, by using a combination of time-resolved fluorescence and absorption spectroscopy from femto- to microseconds, we provide the first experimental evidence for the UVR8 molecular signaling mechanism. The results indicate that tryptophan residues at the dimer interface engage in photoinduced proton coupled electron transfer reactions that induce monomerization.

The present palette of opsin-based optogenetic tools lacks a light-gated potassium (K(+)) channel desirable for silencing of excitable cells. Here, we describe the construction of a blue-light-induced K(+) channel 1 (BLINK1) engineered by fusing the plant LOV2-Jα photosensory module to the small viral K(+) channel Kcv. BLINK1 exhibits biophysical features of Kcv, including K(+) selectivity and high single-channel conductance but reversibly photoactivates in blue light. Opening of BLINK1 channels hyperpolarizes the cell to the K(+) equilibrium potential. Ectopic expression of BLINK1 reversibly inhibits the escape response in light-exposed zebrafish larvae. BLINK1 therefore provides a single-component optogenetic tool that can establish prolonged, physiological hyperpolarization of cells at low light intensities., (Copyright © 2015, American Association for the Advancement of Science.)

Plants are photoautotrophic sessile organisms that use environmental cues to optimize multiple facets of growth and development. A classic example is phototropism - in shoots this is typically positive, leading to growth towards the light, while roots frequently show negative phototropism triggering growth away from the light. Shoot phototropism optimizes light capture of leaves in low light environments and hence increases photosynthetic productivity. Phototropins are plasma-membrane-associated UV-A/blue-light activated kinases that trigger phototropic growth. Light perception liberates their protein kinase domain from the inhibitory action of the amino-terminal photosensory portion of the photoreceptor. Following a series of still poorly understood events, phototropin activation leads to the formation of a gradient of the growth hormone auxin across the photo-stimulated stem. The greater auxin concentration on the shaded compared with the lit side of the stem enables growth reorientation towards the light. In this Minireview, we briefly summarize the signaling steps starting from photoreceptor activation until the establishment of a lateral auxin gradient, ultimately leading to phototropic growth in shoots., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

UVR8 is a recently discovered ultraviolet-B (UV-B) photoreceptor protein identified in plants and algae. In the dark state, UVR8 exists as a homodimer, whereas UV-B irradiation induces UVR8 monomerization and initiation of signaling. Although the biological functions of UVR8 have been studied, the fundamental reaction mechanism and associated kinetics have not yet been fully elucidated. Here, we used the transient grating method to determine the reaction dynamics of UVR8 monomerization based on its diffusion coefficient. We found that the UVR8 photodissociation reaction proceeds in three stages: (i) photoexcitation of cross-dimer tryptophan (Trp) pyramids; (ii) an initial conformational change with a time constant of 50 ms; and (iii) dimer dissociation with a time constant of 200 ms. We identified W285 as the key Trp residue responsible for initiating this photoreaction. Although the C-terminus of UVR8 is essential for biological interactions and signaling via downstream components such as COP1, no obvious differences were detected between the photoreactions of wild-type UVR8 (amino acids 1-440) and a mutant lacking the C-terminus (amino acids 1-383). This similarity indicates that the conformational change associated with stage ii cannot primarily be attributed to this region. A UV-B-driven conformational change with a time constant of 50 ms was also detected in the monomeric mutants of UVR8. Dimer recovery following monomerization, as measured by circular dichroism spectroscopy, was decreased under oxygen-purged conditions, suggesting that redox reactivity is a key factor contributing to the UVR8 oligomeric state.