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13 Janelia Publications

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    05/28/22 | An essential experimental control for functional connectivity mapping with optogenetics.
    David Tadres , Hiroshi M. Shiozaki , Ibrahim Tastekin , David L. Stern , Matthieu Louis
    bioRxiv. 2022 May 28:. doi: 10.1101/2022.05.26.493610

    To establish functional connectivity between two candidate neurons that might form a circuit element, a common approach is to activate an optogenetic tool such as Chrimson in the candidate pre-synaptic neuron and monitor fluorescence of the calcium-sensitive indicator GCaMP in a candidate post-synaptic neuron. While performing such experiments, we found that low levels of leaky Chrimson expression can lead to strong artifactual GCaMP signals in presumptive postsynaptic neurons even when Chrimson is not intentionally expressed in any particular neurons. Withholding all-trans retinal, the chromophore required as a co-factor for Chrimson response to light, eliminates GCaMP signal but does not provide an experimental control for leaky Chrimson expression. Leaky Chrimson expression appears to be an inherent feature of current Chrimson transgenes, since artifactual connectivity was detected with Chrimson transgenes integrated into three different genomic locations (two insertions tested in larvae; a third insertion tested in the adult fly). These false-positive signals may complicate the interpretation of functional connectivity experiments. We illustrate how a no-Gal4 negative control improves interpretability of functional connectivity assays. We also propose a simple but effective procedure to identify experimental conditions that minimize potentially incorrect interpretations caused by leaky Chrimson expression.

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    04/11/22 | BRD2 compartmentalizes the accessible genome.
    Xie L, Dong P, Qi Y, Hsieh TS, English BP, Jung S, Chen X, De Marzio M, Casellas R, Chang HY, Zhang B, Tjian R, Liu Z
    Nature Genetics. 2022 Apr 11;54(4):481-491. doi: 10.1038/s41588-022-01044-9

    Mammalian chromosomes are organized into megabase-sized compartments that are further subdivided into topologically associating domains (TADs). While the formation of TADs is dependent on cohesin, the mechanism behind compartmentalization remains enigmatic. Here, we show that the bromodomain and extraterminal (BET) family scaffold protein BRD2 promotes spatial mixing and compartmentalization of active chromatin after cohesin loss. This activity is independent of transcription but requires BRD2 to recognize acetylated targets through its double bromodomain and interact with binding partners with its low-complexity domain. Notably, genome compartmentalization mediated by BRD2 is antagonized on the one hand by cohesin and on the other hand by the BET homolog protein BRD4, both of which inhibit BRD2 binding to chromatin. Polymer simulation of our data supports a BRD2-cohesin interplay model of nuclear topology, in which genome compartmentalization results from a competition between loop extrusion and chromatin-state-specific affinity interactions.

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    01/22/22 | Visualizing Synaptic Dopamine Efflux with a 2D Nanofilm.
    Chandima Bulumulla , Andrew T. Krasley , Deepika Walpita , Abraham G. Beyene
    bioRxiv. 2022 Jan 22:. doi: 10.1101/2022.01.19.476937

    Chemical neurotransmission constitutes one of the fundamental modalities of communication between neurons. Monitoring release of these chemicals has traditionally been difficult to carry out at spatial and temporal scales relevant to neuron function. To understand chemical neurotransmission more fully, we need to improve the spatial and temporal resolutions of measurements for neurotransmitter release. To address this, we engineered a chemi-sensitive, two-dimensional nanofilm that facilitates subcellular visualization of the release and diffusion of the neurochemical dopamine with synaptic resolution, quantal sensitivity, and simultaneously from hundreds of release sites. Using this technology, we were able to monitor the spatiotemporal dynamics of dopamine release in dendritic processes, a poorly understood phenomenon. We found that dopamine release is broadcast from a subset of dendritic processes as hotspots that have a mean spatial spread of ≈3.2 µm (full width at half maximum) and are observed with a mean spatial frequency of 1 hotspot per ≈7.5 µm of dendritic length. Major dendrites of dopamine neurons and fine dendritic processes, as well as dendritic arbors and dendrites with no apparent varicose morphology participated in dopamine release. Remarkably, these release hotspots colocalized with Bassoon, suggesting that Bassoon may contribute to organizing active zones in dendrites, similar to its role in axon terminals.

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    09/28/21 | A serotonergic axon-cilium synapse drives nuclear signaling to maintain chromatin accessibility
    Shu-Hsien Sheu , Srigokul Upadhyayula , Vincent Dupuy , Song Pang , Andrew L. Lemire , Deepika Walpita , H. Amalia Pasolli , Fei Deng , Jinxia Wan , Lihua Wang , Justin Houser , Silvia Sanchez-Martinez , Sebastian E. Brauchi , Sambashiva Banala , Melanie Freeman , C. Shan Xu , Tom Kirchhausen , Harald F. Hess , Luke Lavis , Yu-Long Li , Séverine Chaumont-Dubel , David E. Clapham
    bioRxiv. 2021 Sep 28:

    Chemical synapses between axons and dendrites mediate much of the brain’s intercellular communication. Here we describe a new kind of synapse – the axo-ciliary synapse - between axons and primary cilia. By employing enhanced focused ion beam – scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between the serotonergic axons arising from the brainstem, and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, 5-hydroxytryptamine receptor 6 (HTR6), whose mutation is associated with learning and memory defects. Using a newly developed cilia-targeted serotonin sensor, we show that optogenetic stimulation of serotonergic axons results in serotonin release onto cilia. Ciliary HTR6 stimulation activates a non-canonical Gαq/11-RhoA pathway. Ablation of this pathway results in nuclear actin and chromatin accessibility changes in CA1 pyramidal neurons. Axo-ciliary synapses serve as a distinct mechanism for neuromodulators to program neuron transcription through privileged access to the nuclear compartment.

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    04/01/21 | The HaloTag as a general scaffold for far-red tunable chemigenetic indicators.
    Deo C, Abdelfattah AS, Bhargava HK, Berro AJ, Falco N, Farrants H, Moeyaert B, Chupanova M, Lavis LD, Schreiter ER
    Nature Chemical Biology. 2021 Apr 01:. doi: 10.1038/s41589-021-00775-w

    Functional imaging using fluorescent indicators has revolutionized biology, but additional sensor scaffolds are needed to access properties such as bright, far-red emission. Here, we introduce a new platform for 'chemigenetic' fluorescent indicators, utilizing the self-labeling HaloTag protein conjugated to environmentally sensitive synthetic fluorophores. We solve a crystal structure of HaloTag bound to a rhodamine dye ligand to guide engineering efforts to modulate the dye environment. We show that fusion of HaloTag with protein sensor domains that undergo conformational changes near the bound dye results in large and rapid changes in fluorescence output. This generalizable approach affords bright, far-red calcium and voltage sensors with highly tunable photophysical and chemical properties, which can reliably detect single action potentials in cultured neurons.

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    01/01/21 | Neural circuit mechanisms of sexual receptivity in Drosophila females.
    Wang K, Wang F, Forknall N, Yang T, Patrick C, Parekh R, Dickson BJ
    Nature. 2021 Jan 01;589(7843):577-81. doi: 10.1038/s41586-020-2972-7

    Choosing a mate is one of the most consequential decisions a female will make during her lifetime. A female fly signals her willingness to mate by opening her vaginal plates, allowing a courting male to copulate. Vaginal plate opening (VPO) occurs in response to the male courtship song and is dependent on the mating status of the female. How these exteroceptive (song) and interoceptive (mating status) inputs are integrated to regulate VPO remains unknown. Here we characterize the neural circuitry that implements mating decisions in the brain of female Drosophila melanogaster. We show that VPO is controlled by a pair of female-specific descending neurons (vpoDNs). The vpoDNs receive excitatory input from auditory neurons (vpoENs), which are tuned to specific features of the D. melanogaster song, and from pC1 neurons, which encode the mating status of the female. The song responses of vpoDNs, but not vpoENs, are attenuated upon mating, accounting for the reduced receptivity of mated females. This modulation is mediated by pC1 neurons. The vpoDNs thus directly integrate the external and internal signals that control the mating decisions of Drosophila females.

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    08/20/20 | Rational design of bioavailable photosensitizers for manipulation and imaging of biological systems.
    Binns TC, Ayala AX, Grimm JB, Tkachuk AN, Castillon GA, Phan S, Zhang L, Brown TA, Liu Z, Adams SR, Ellisman MH, Koyama M, Lavis LD
    Cell Chemical Biology. 2020 Aug 20;27(8):1063-72. doi: 10.1016/j.chembiol.2020.07.001

    Light-mediated chemical reactions are powerful methods for manipulating and interrogating biological systems. Photosensitizers, compounds that generate reactive oxygen species upon excitation with light, can be utilized for numerous biological experiments, but the repertoire of bioavailable photosensitizers is limited. Here, we describe the synthesis, characterization, and utility of two photosensitizers based upon the widely used rhodamine scaffold and demonstrate their efficacy for chromophore-assisted light inactivation, cell ablation in culture and in vivo, and photopolymerization of diaminobenzidine for electron microscopy. These chemical tools will facilitate a broad range of applications spanning from targeted destruction of proteins to high-resolution imaging.

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    07/10/20 | A general approach to engineer positive-going eFRET voltage indicators
    Abdelfattah AS, Valenti R, Zheng J, Wong A, Podgorski K, Koyama M, Kim DS, Schreiter ER, Project Team GENIE
    Nature Communications. 2020 Jul 10;11(1):

    We engineered electrochromic fluorescence resonance energy transfer (eFRET) genetically encoded voltage indicators (GEVIs) with “positive-going” fluorescence response to membrane depolarization through rational manipulation of the native proton transport pathway in microbial rhodopsins. We transformed the state-of-the-art eFRET GEVI Voltron into Positron, with kinetics and sensitivity equivalent to Voltron but flipped fluorescence signal polarity. We further applied this general approach to GEVIs containing different voltage sensitive rhodopsin domains and various fluorescent dye and fluorescent protein reporters.

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    03/02/20 | Neural circuitry linking mating and egg laying in Drosophila females.
    Wang F, Wang K, Forknall N, Patrick C, Yang T, Parekh R, Bock D, Dickson BJ
    Nature. 2020 Mar 02;579(7797):101-105. doi: 10.1038/s41586-020-2055-9

    Mating and egg laying are tightly cooordinated events in the reproductive life of all oviparous females. Oviposition is typically rare in virgin females but is initiated after copulation. Here we identify the neural circuitry that links egg laying to mating status in Drosophila melanogaster. Activation of female-specific oviposition descending neurons (oviDNs) is necessary and sufficient for egg laying, and is equally potent in virgin and mated females. After mating, sex peptide-a protein from the male seminal fluid-triggers many behavioural and physiological changes in the female, including the onset of egg laying. Sex peptide is detected by sensory neurons in the uterus, and silences these neurons and their postsynaptic ascending neurons in the abdominal ganglion. We show that these abdominal ganglion neurons directly activate the female-specific pC1 neurons. GABAergic (γ-aminobutyric-acid-releasing) oviposition inhibitory neurons (oviINs) mediate feed-forward inhibition from pC1 neurons to both oviDNs and their major excitatory input, the oviposition excitatory neurons (oviENs). By attenuating the abdominal ganglion inputs to pC1 neurons and oviINs, sex peptide disinhibits oviDNs to enable egg laying after mating. This circuitry thus coordinates the two key events in female reproduction: mating and egg laying.

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    01/09/20 | Bright and tunable far-red chemigenetic indicators.
    Deo C, Abdelfattah AS, Bhargava HK, Berro AJ, Falco N, Moeyaert B, Chupanova M, Lavis LD, Schreiter ER
    bioRxiv. 2020 Jan 9: