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7 Janelia Publications

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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
    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:
    03/14/19 | Ultrapotent chemogenetics for research and potential clinical applications.
    Magnus CJ, Lee PH, Bonaventura J, Zemla R, Gomez JL, Ramirez MH, Hu X, Galvan A, Basu J, Michaelides M, Sternson SM
    Science. 2019 Mar 14;364(6436):eaav5282. doi: 10.1126/science.aav5282

    Chemogenetics enables non-invasive chemical control over cell populations in behaving animals. However, existing small molecule agonists show insufficient potency or selectivity. There is also need for chemogenetic systems compatible with both research and human therapeutic applications. We developed a new ion channel-based platform for cell activation and silencing that is controlled by low doses of the anti-smoking drug varenicline. We then synthesized novel sub-nanomolar potency agonists, called uPSEMs, with high selectivity for the chemogenetic receptors. uPSEMs and their receptors were characterized in brains of mice and a rhesus monkey by in vivo electrophysiology, calcium imaging, positron emission tomography, behavioral efficacy testing, and receptor counterscreening. This platform of receptors and selective ultrapotent agonists enables potential research and clinical applications of chemogenetics.

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    06/17/15 | Epigenomic signatures of neuronal diversity in the mammalian brain.
    Mo A, Mukamel EA, Davis FP, Luo C, Henry GL, Picard S, Urich MA, Nery JR, Sejnowski TJ, Lister R, Eddy SR, Ecker JR, Nathans J
    Neuron. 2015 Jun 17;86(6):1369-1384. doi: 10.1016/j.neuron.2015.05.018

    Neuronal diversity is essential for mammalian brain function but poses a challenge to molecular profiling. To address the need for tools that facilitate cell-type-specific epigenomic studies, we developed the first affinity purification approach to isolate nuclei from genetically defined cell types in a mammal. We combine this technique with next-generation sequencing to show that three subtypes of neocortical neurons have highly distinctive epigenomic landscapes. Over 200,000 regions differ in chromatin accessibility and DNA methylation signatures characteristic of gene regulatory regions. By footprinting and motif analyses, these regions are predicted to bind distinct cohorts of neuron subtype-specific transcription factors. Neuronal epigenomes reflect both past and present gene expression, with DNA hyper-methylation at developmentally critical genes appearing as a novel epigenomic signature in mature neurons. Taken together, our findings link the functional and transcriptional complexity of neurons to their underlying epigenomic diversity.

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    10/14/13 | A neuron-based screening platform for optimizing genetically-encoded calcium indicators.
    Wardill TJ, Chen T, Schreiter ER, Hasseman JP, Tsegaye G, Fosque BF, Behnam R, Shields BC, Ramirez M, Kimmel BE, Kerr RA, Jayaraman V, Looger LL, Svoboda K, Kim DS
    PLoS One. 2013;8:e77728. doi: 10.1371/journal.pone.0077728

    Fluorescent protein-based sensors for detecting neuronal activity have been developed largely based on non-neuronal screening systems. However, the dynamics of neuronal state variables (e.g., voltage, calcium, etc.) are typically very rapid compared to those of non-excitable cells. We developed an electrical stimulation and fluorescence imaging platform based on dissociated rat primary neuronal cultures. We describe its use in testing genetically-encoded calcium indicators (GECIs). Efficient neuronal GECI expression was achieved using lentiviruses containing a neuronal-selective gene promoter. Action potentials (APs) and thus neuronal calcium levels were quantitatively controlled by electrical field stimulation, and fluorescence images were recorded. Images were segmented to extract fluorescence signals corresponding to individual GECI-expressing neurons, which improved sensitivity over full-field measurements. We demonstrate the superiority of screening GECIs in neurons compared with solution measurements. Neuronal screening was useful for efficient identification of variants with both improved response kinetics and high signal amplitudes. This platform can be used to screen many types of sensors with cellular resolution under realistic conditions where neuronal state variables are in relevant ranges with respect to timing and amplitude.

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