Sensory circuitry controls cytosolic calcium-mediated phytochrome B phototransduction.

Although Ca2+ has long been recognized as an obligatory intermediate in visual transduction, its role in plant phototransduction remains elusive. Here, we report a Ca2+ signaling that controls photoreceptor phyB nuclear translocation in etiolated seedlings during dark-to-light transition. Red light stimulates acute cytosolic Ca2+ increases via phyB, which are sensed by Ca2+-binding protein kinases, CPK6 and CPK12 (CPK6/12). Upon Ca2+ activation, CPK6/12 in turn directly interact with and phosphorylate photo-activated phyB at Ser80/Ser106 to initiate phyB nuclear import. Non-phosphorylatable mutation, phyBS80A/S106A, abolishes nuclear translocation and fails to complement phyB mutant, which is fully restored by combining phyBS80A/S106A with a nuclear localization signal. We further show that CPK6/12 function specifically in the early phyB-mediated cotyledon expansion, while Ser80/Ser106 phosphorylation generally governs phyB nuclear translocation. Our results uncover a biochemical regulatory loop centered in phyB phototransduction and provide a paradigm for linking ubiquitous Ca2+ increases to specific responses in sensory stimulus processing.

Light links neonatal neurons for learning.

The newborn mouse's retina senses light even before the eye opens, informing the developing brain of the visual world. Without this information, the brain forms fewer connections and the adult mouse learns sluggishly.

Optobiochemistry: Genetically Encoded Control of Protein Activity by Light.

Optobiochemical control of protein activities allows the investigation of protein functions in living cells with high spatiotemporal resolution. Over the last two decades, numerous natural photosensory domains have been characterized and synthetic domains engineered and assembled into photoregulatory systems to control protein function with light. Here, we review the field of optobiochemistry, categorizing photosensory domains by chromophore, describing photoregulatory systems by mechanism of action, and discussing protein classes frequently investigated using optical methods. We also present examples of how spatial or temporal control of proteins in living cells has provided new insights not possible with traditional biochemical or cell biological techniques.

Unexpected pairings.

Being able to precisely turn on or off particular neurons in the brain at will was a major challenge for the neuroscience field, and few could have anticipated that the solution would come from algae. The 2021 Albert Lasker Basic Medical Research Award recognizes the contributions of Peter Hegemann, Dieter Oesterhelt, and Karl Deisseroth for their discovery of light-sensitive microbial proteins that can activate or silence brain cells. Cell editor Nicole Neuman had a conversation with Peter Hegemann about his role in bridging the two fields of microbial phototaxis and neuroscience and his perspective on the nature and future of interdisciplinary science. Excerpts from this conversation are presented below, and the full conversation is available with the article online.

A curious color change.

The field of optogenetics realizes a dream first articulated by Francis Crick in the 1970s: to use light to turn specific neurons on (or off), so as to tease apart brain function and mechanisms. Few could have anticipated that the technical solution to this grand neurobiology challenge would come from basic studies in Archaea and algae. The 2021 Albert Lasker Basic Medical Research Award recognizes the contributions of Dieter Oesterhelt, Peter Hegemann, and Karl Deisseroth for their discovery of microbial light-sensing proteins that can activate or silence individual brain cells and for their use in developing optogenetics, which has revolutionized neuroscience. Cell's Nicole Neuman had a conversation with Dieter Oesterhelt about his startling discovery that Archaea also possess rhodopsins, how this led to many other discoveries and technologies, and his experiences in cultivating scientific talent such as fellow award-winner Peter Hegemann. Excerpts from this conversation are presented below, and the full conversation is available with the article online.

Modularity and robustness of frontal cortical networks.

Neural activity underlying short-term memory is maintained by interconnected networks of brain regions. It remains unknown how brain regions interact to maintain persistent activity while exhibiting robustness to corrupt information in parts of the network. We simultaneously measured activity in large neuronal populations across mouse frontal hemispheres to probe interactions between brain regions. Activity across hemispheres was coordinated to maintain coherent short-term memory. Across mice, we uncovered individual variability in the organization of frontal cortical networks. A modular organization was required for the robustness of persistent activity to perturbations: each hemisphere retained persistent activity during perturbations of the other hemisphere, thus preventing local perturbations from spreading. A dynamic gating mechanism allowed hemispheres to coordinate coherent information while gating out corrupt information. Our results show that robust short-term memory is mediated by redundant modular representations across brain regions. Redundant modular representations naturally emerge in neural network models that learned robust dynamics.

Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity.

Vesicle-inducing protein in plastids 1 (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-remodeling functions. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket on one end of the ring. Inside the ring's lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Using cryo-correlative light and electron microscopy (cryo-CLEM), we observe oligomeric VIPP1 coats encapsulating membrane tubules within the Chlamydomonas chloroplast. Our work provides a structural foundation for understanding how VIPP1 directs thylakoid biogenesis and maintenance.

Mammalian Near-Infrared Image Vision through Injectable and Self-Powered Retinal Nanoantennae.

Mammals cannot see light over 700 nm in wavelength. This limitation is due to the physical thermodynamic properties of the photon-detecting opsins. However, the detection of naturally invisible near-infrared (NIR) light is a desirable ability. To break this limitation, we developed ocular injectable photoreceptor-binding upconversion nanoparticles (pbUCNPs). These nanoparticles anchored on retinal photoreceptors as miniature NIR light transducers to create NIR light image vision with negligible side effects. Based on single-photoreceptor recordings, electroretinograms, cortical recordings, and visual behavioral tests, we demonstrated that mice with these nanoantennae could not only perceive NIR light, but also see NIR light patterns. Excitingly, the injected mice were also able to differentiate sophisticated NIR shape patterns. Moreover, the NIR light pattern vision was ambient-daylight compatible and existed in parallel with native daylight vision. This new method will provide unmatched opportunities for a wide variety of emerging bio-integrated nanodevice designs and applications. VIDEO ABSTRACT.

BMAL1-Driven Tissue Clocks Respond Independently to Light to Maintain Homeostasis.

Circadian rhythms control organismal physiology throughout the day. At the cellular level, clock regulation is established by a self-sustained Bmal1-dependent transcriptional oscillator network. However, it is still unclear how different tissues achieve a synchronized rhythmic physiology. That is, do they respond independently to environmental signals, or require interactions with each other to do so? We show that unexpectedly, light synchronizes the Bmal1-dependent circadian machinery in single tissues in the absence of Bmal1 in all other tissues. Strikingly, light-driven tissue autonomous clocks occur without rhythmic feeding behavior and are lost in constant darkness. Importantly, tissue-autonomous Bmal1 partially sustains homeostasis in otherwise arrhythmic and prematurely aging animals. Our results therefore support a two-branched model for the daily synchronization of tissues: an autonomous response branch, whereby light entrains circadian clocks without any commitment of other Bmal1-dependent clocks, and a memory branch using other Bmal1-dependent clocks to "remember" time in the absence of external cues.

Defining the Independence of the Liver Circadian Clock.

Mammals rely on a network of circadian clocks to control daily systemic metabolism and physiology. The central pacemaker in the suprachiasmatic nucleus (SCN) is considered hierarchically dominant over peripheral clocks, whose degree of independence, or tissue-level autonomy, has never been ascertained in vivo. Using arrhythmic Bmal1-null mice, we generated animals with reconstituted circadian expression of BMAL1 exclusively in the liver (Liver-RE). High-throughput transcriptomics and metabolomics show that the liver has independent circadian functions specific for metabolic processes such as the NAD+ salvage pathway and glycogen turnover. However, although BMAL1 occupies chromatin at most genomic targets in Liver-RE mice, circadian expression is restricted to ∼10% of normally rhythmic transcripts. Finally, rhythmic clock gene expression is lost in Liver-RE mice under constant darkness. Hence, full circadian function in the liver depends on signals emanating from other clocks, and light contributes to tissue-autonomous clock function.

Atoms to Phenotypes: Molecular Design Principles of Cellular Energy Metabolism.

We report a 100-million atom-scale model of an entire cell organelle, a photosynthetic chromatophore vesicle from a purple bacterium, that reveals the cascade of energy conversion steps culminating in the generation of ATP from sunlight. Molecular dynamics simulations of this vesicle elucidate how the integral membrane complexes influence local curvature to tune photoexcitation of pigments. Brownian dynamics of small molecules within the chromatophore probe the mechanisms of directional charge transport under various pH and salinity conditions. Reproducing phenotypic properties from atomistic details, a kinetic model evinces that low-light adaptations of the bacterium emerge as a spontaneous outcome of optimizing the balance between the chromatophore's structural integrity and robust energy conversion. Parallels are drawn with the more universal mitochondrial bioenergetic machinery, from whence molecular-scale insights into the mechanism of cellular aging are inferred. Together, our integrative method and spectroscopic experiments pave the way to first-principles modeling of whole living cells.

Reconstituted Postsynaptic Density as a Molecular Platform for Understanding Synapse Formation and Plasticity.

Synapses are semi-membraneless, protein-dense, sub-micron chemical reaction compartments responsible for signal processing in each and every neuron. Proper formation and dynamic responses to stimulations of synapses, both during development and in adult, are fundamental to functions of mammalian brains, although the molecular basis governing formation and modulation of compartmentalized synaptic assemblies is unclear. Here, we used a biochemical reconstitution approach to show that, both in solution and on supported membrane bilayers, multivalent interaction networks formed by major excitatory postsynaptic density (PSD) scaffold proteins led to formation of PSD-like assemblies via phase separation. The reconstituted PSD-like assemblies can cluster receptors, selectively concentrate enzymes, promote actin bundle formation, and expel inhibitory postsynaptic proteins. Additionally, the condensed phase PSD assemblies have features that are distinct from those in homogeneous solutions and fit for synaptic functions. Thus, we have built a molecular platform for understanding how neuronal synapses are formed and dynamically regulated.

A Suite of Transgenic Driver and Reporter Mouse Lines with Enhanced Brain-Cell-Type Targeting and Functionality.

Modern genetic approaches are powerful in providing access to diverse cell types in the brain and facilitating the study of their function. Here, we report a large set of driver and reporter transgenic mouse lines, including 23 new driver lines targeting a variety of cortical and subcortical cell populations and 26 new reporter lines expressing an array of molecular tools. In particular, we describe the TIGRE2.0 transgenic platform and introduce Cre-dependent reporter lines that enable optical physiology, optogenetics, and sparse labeling of genetically defined cell populations. TIGRE2.0 reporters broke the barrier in transgene expression level of single-copy targeted-insertion transgenesis in a wide range of neuronal types, along with additional advantage of a simplified breeding strategy compared to our first-generation TIGRE lines. These novel transgenic lines greatly expand the repertoire of high-precision genetic tools available to effectively identify, monitor, and manipulate distinct cell types in the mouse brain.

Nuclear Import Receptor Inhibits Phase Separation of FUS through Binding to Multiple Sites.

Liquid-liquid phase separation (LLPS) is believed to underlie formation of biomolecular condensates, cellular compartments that concentrate macromolecules without surrounding membranes. Physical mechanisms that control condensate formation/dissolution are poorly understood. The RNA-binding protein fused in sarcoma (FUS) undergoes LLPS in vitro and associates with condensates in cells. We show that the importin karyopherin-ß2/transportin-1 inhibits LLPS of FUS. This activity depends on tight binding of karyopherin-ß2 to the C-terminal proline-tyrosine nuclear localization signal (PY-NLS) of FUS. Nuclear magnetic resonance (NMR) analyses reveal weak interactions of karyopherin-ß2 with sequence elements and structural domains distributed throughout the entirety of FUS. Biochemical analyses demonstrate that most of these same regions also contribute to LLPS of FUS. The data lead to a model where high-affinity binding of karyopherin-ß2 to the FUS PY-NLS tethers the proteins together, allowing multiple, distributed weak intermolecular contacts to disrupt FUS self-association, blocking LLPS. Karyopherin-ß2 may act analogously to control condensates in diverse cellular contexts.

Corticoamygdala Transfer of Socially Derived Information Gates Observational Learning.

Observational learning is a powerful survival tool allowing individuals to learn about threat-predictive stimuli without directly experiencing the pairing of the predictive cue and punishment. This ability has been linked to the anterior cingulate cortex (ACC) and the basolateral amygdala (BLA). To investigate how information is encoded and transmitted through this circuit, we performed electrophysiological recordings in mice observing a demonstrator mouse undergo associative fear conditioning and found that BLA-projecting ACC (ACC→BLA) neurons preferentially encode socially derived aversive cue information. Inhibition of ACC→BLA alters real-time amygdala representation of the aversive cue during observational conditioning. Selective inhibition of the ACC→BLA projection impaired acquisition, but not expression, of observational fear conditioning. We show that information derived from observation about the aversive value of the cue is transmitted from the ACC to the BLA and that this routing of information is critically instructive for observational fear conditioning. VIDEO ABSTRACT.

Light Affects Mood and Learning through Distinct Retina-Brain Pathways.

Light exerts a range of powerful biological effects beyond image vision, including mood and learning regulation. While the source of photic information affecting mood and cognitive functions is well established, viz. intrinsically photosensitive retinal ganglion cells (ipRGCs), the central mediators are unknown. Here, we reveal that the direct effects of light on learning and mood utilize distinct ipRGC output streams. ipRGCs that project to the suprachiasmatic nucleus (SCN) mediate the effects of light on learning, independently of the SCN's pacemaker function. Mood regulation by light, on the other hand, requires an SCN-independent pathway linking ipRGCs to a previously unrecognized thalamic region, termed perihabenular nucleus (PHb). The PHb is integrated in a distinctive circuitry with mood-regulating centers and is both necessary and sufficient for driving the effects of light on affective behavior. Together, these results provide new insights into the neural basis required for light to influence mood and learning.

Reducing Pericyte-Derived Scarring Promotes Recovery after Spinal Cord Injury.

CNS injury often severs axons. Scar tissue that forms locally at the lesion site is thought to block axonal regeneration, resulting in permanent functional deficits. We report that inhibiting the generation of progeny by a subclass of pericytes led to decreased fibrosis and extracellular matrix deposition after spinal cord injury in mice. Regeneration of raphespinal and corticospinal tract axons was enhanced and sensorimotor function recovery improved following spinal cord injury in animals with attenuated pericyte-derived scarring. Using optogenetic stimulation, we demonstrate that regenerated corticospinal tract axons integrated into the local spinal cord circuitry below the lesion site. The number of regenerated axons correlated with improved sensorimotor function recovery. In conclusion, attenuation of pericyte-derived fibrosis represents a promising therapeutic approach to facilitate recovery following CNS injury.

Microbial Rhodopsins: Diversity, Mechanisms, and Optogenetic Applications.

Microbial rhodopsins are a family of photoactive retinylidene proteins widespread throughout the microbial world. They are notable for their diversity of function, using variations of a shared seven-transmembrane helix design and similar photochemical reactions to carry out distinctly different light-driven energy and sensory transduction processes. Their study has contributed to our understanding of how evolution modifies protein scaffolds to create new protein chemistry, and their use as tools to control membrane potential with light is fundamental to optogenetics for research and clinical applications. We review the currently known functions and present more in-depth assessment of three functionally and structurally distinct types discovered over the past two years: (a) anion channelrhodopsins (ACRs) from cryptophyte algae, which enable efficient optogenetic neural suppression; (b) cryptophyte cation channelrhodopsins (CCRs), structurally distinct from the green algae CCRs used extensively for neural activation and from cryptophyte ACRs; and

A New Facet of Vitamin B12: Gene Regulation by Cobalamin-Based Photoreceptors.

Living organisms sense and respond to light, a crucial environmental factor, using photoreceptors, which rely on bound chromophores such as retinal, flavins, or linear tetrapyrroles for light sensing. The discovery of photoreceptors that sense light using 5'-deoxyadenosylcobalamin, a form of vitamin B12 that is best known as an enzyme cofactor, has expanded the number of known photoreceptor families and unveiled a new biological role of this vitamin. The prototype of these B12-dependent photoreceptors, the transcriptional repressor CarH, is widespread in bacteria and mediates light-dependent gene regulation in a photoprotective cellular response. CarH activity as a transcription factor relies on the modulation of its oligomeric state by 5'-deoxyadenosylcobalamin and light. This review surveys current knowledge about these B12-dependent photoreceptors, their distribution and mode of action, and the structural and photochemical basis of how they orchestrate signal transduction and control gene expression.

Remodeling of light and dark zone follicular dendritic cells governs germinal center responses.

Efficient generation of germinal center (GC) responses requires directed movement of B cells between distinct microenvironments underpinned by specialized B cell-interacting reticular cells (BRCs). How BRCs are reprogrammed to cater to the developing GC remains unclear, and studying this process is largely hindered by incomplete resolution of the cellular composition of the B cell follicle. Here we used genetic targeting of Cxcl13-expressing cells to define the molecular identity of the BRC landscape. Single-cell transcriptomic analysis revealed that BRC subset specification was predetermined in the primary B cell follicle. Further topological remodeling of light and dark zone follicular dendritic cells required CXCL12-dependent crosstalk with B cells and dictated GC output by retaining B cells in the follicle and steering their interaction with follicular helper T cells. Together, our results reveal that poised BRC-defined microenvironments establish a feed-forward system that determines the efficacy of the GC reaction.