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1999 Janelia Publications

Showing 1-10 of 1999 results
12/23/20 | Directed Evolution of a Selective and Sensitive Serotonin Sensor via Machine Learning.
Unger EK, Keller JP, Altermatt M, Liang R, Matsui A, Dong C, Hon OJ, Yao Z, Sun J, Banala S, Flanigan ME, Jaffe DA, Hartanto S, Carlen J, Mizuno GO, Borden PM, Shivange AV, Cameron LP, Sinning S, Underhill SM, Olson DE, Amara SG, Temple Lang D, Rudnick G, Marvin JS, Lavis LD, Lester HA, Alvarez VA, Fisher AJ, Prescher JA, Kash TL, Yarov-Yarovoy V, Gradinaru V, Looger LL, Tian L
Cell. 2020 Dec 23;183(7):1986-2002.e26. doi: 10.1016/j.cell.2020.11.040

Serotonin plays a central role in cognition and is the target of most pharmaceuticals for psychiatric disorders. Existing drugs have limited efficacy; creation of improved versions will require better understanding of serotonergic circuitry, which has been hampered by our inability to monitor serotonin release and transport with high spatial and temporal resolution. We developed and applied a binding-pocket redesign strategy, guided by machine learning, to create a high-performance, soluble, fluorescent serotonin sensor (iSeroSnFR), enabling optical detection of millisecond-scale serotonin transients. We demonstrate that iSeroSnFR can be used to detect serotonin release in freely behaving mice during fear conditioning, social interaction, and sleep/wake transitions. We also developed a robust assay of serotonin transporter function and modulation by drugs. We expect that both machine-learning-guided binding-pocket redesign and iSeroSnFR will have broad utility for the development of other sensors and in vitro and in vivo serotonin detection, respectively.

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12/14/20 | Cis-regulatory variation in the shavenbaby gene underlies intraspecific phenotypic variation, mirroring interspecific divergence in the same trait.
Soverna AF, Rodriguez NC, Korgaonkar A, Hasson E, Stern DL, Frankel N
Evolution. 2020 Dec 14:. doi: 10.1111/evo.14142

Despite considerable progress in recent decades in dissecting the genetic causes of natural morphological variation, there is limited understanding of how variation within species ultimately contributes to species differences. We have studied patterning of the non-sensory hairs, commonly known as "trichomes," on the dorsal cuticle of first-instar larvae of Drosophila. Most Drosophila species produce a dense lawn of dorsal trichomes, but a subset of these trichomes were lost in D. sechellia and D. ezoana due entirely to regulatory evolution of the shavenbaby (svb) gene. Here, we describe intraspecific variation in dorsal trichome patterns of first-instar larvae of D. virilis that is similar to the trichome pattern variation identified previously between species. We found that a single large effect QTL, which includes svb, explains most of the trichome number difference between two D. virilis strains and that svb expression correlates with the trichome difference between strains. This QTL does not explain the entire difference between strains, implying that additional loci contribute to variation in trichome numbers. Thus, the genetic architecture of intraspecific variation exhibits similarities and differences with interspecific variation that may reflect differences in long-term and short-term evolutionary processes.

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12/14/20 | The connectome of the adult mushroom body provides insights into function.
Li F, Lindsey JW, Marin EC, Otto N, Dreher M, Dempsey G, Stark I, Bates AS, Pleijzier MW, Schlegel P, Nern A, Takemura S, Eckstein N, Yang T, Francis A, Braun A, Parekh R, Costa M, Scheffer LK, Aso Y, Jefferis GS, Abbott LF, Litwin-Kumar A, Waddell S, Rubin GM
eLife. 2020 Dec 14;9:. doi: 10.7554/eLife.62576

Making inferences about the computations performed by neuronal circuits from synapse-level connectivity maps is an emerging opportunity in neuroscience. The mushroom body (MB) is well positioned for developing and testing such an approach due to its conserved neuronal architecture, recently completed dense connectome, and extensive prior experimental studies of its roles in learning, memory and activity regulation. Here we identify new components of the MB circuit in , including extensive visual input and MB output neurons (MBONs) with direct connections to descending neurons. We find unexpected structure in sensory inputs, in the transfer of information about different sensory modalities to MBONs, and in the modulation of that transfer by dopaminergic neurons (DANs). We provide insights into the circuitry used to integrate MB outputs, connectivity between the MB and the central complex and inputs to DANs, including feedback from MBONs. Our results provide a foundation for further theoretical and experimental work.

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12/01/20 | The structural basis of Rubisco phase separation in the pyrenoid.
He S, Chou H, Matthies D, Wunder T, Meyer MT, Atkinson N, Martinez-Sanchez A, Jeffrey PD, Port SA, Patena W, He G, Chen VK, Hughson FM, McCormick AJ, Mueller-Cajar O, Engel BD, Yu Z, Jonikas MC
Nature Plants. 2020 Dec 01;6(12):1480-1490. doi: 10.1038/s41477-020-00811-y

Approximately one-third of global CO fixation occurs in a phase-separated algal organelle called the pyrenoid. The existing data suggest that the pyrenoid forms by the phase separation of the CO-fixing enzyme Rubisco with a linker protein; however, the molecular interactions underlying this phase separation remain unknown. Here we present the structural basis of the interactions between Rubisco and its intrinsically disordered linker protein Essential Pyrenoid Component 1 (EPYC1) in the model alga Chlamydomonas reinhardtii. We find that EPYC1 consists of five evenly spaced Rubisco-binding regions that share sequence similarity. Single-particle cryo-electron microscopy of these regions in complex with Rubisco indicates that each Rubisco holoenzyme has eight binding sites for EPYC1, one on each Rubisco small subunit. Interface mutations disrupt binding, phase separation and pyrenoid formation. Cryo-electron tomography supports a model in which EPYC1 and Rubisco form a codependent multivalent network of specific low-affinity bonds, giving the matrix liquid-like properties. Our results advance the structural and functional understanding of the phase separation underlying the pyrenoid, an organelle that plays a fundamental role in the global carbon cycle.

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10/08/21 | Functional architecture of neural circuits for leg proprioception in Drosophila.
Chen C, Agrawal S, Mark B, Mamiya A, Sustar A, Phelps JS, Lee WA, Dickson BJ, Card GM, Tuthill JC
Current Biology. 2021 Oct 08:. doi: 10.1016/j.cub.2021.09.035

To effectively control their bodies, animals rely on feedback from proprioceptive mechanosensory neurons. In the Drosophila leg, different proprioceptor subtypes monitor joint position, movement direction, and vibration. Here, we investigate how these diverse sensory signals are integrated by central proprioceptive circuits. We find that signals for leg joint position and directional movement converge in second-order neurons, revealing pathways for local feedback control of leg posture. Distinct populations of second-order neurons integrate tibia vibration signals across pairs of legs, suggesting a role in detecting external substrate vibration. In each pathway, the flow of sensory information is dynamically gated and sculpted by inhibition. Overall, our results reveal parallel pathways for processing of internal and external mechanosensory signals, which we propose mediate feedback control of leg movement and vibration sensing, respectively. The existence of a functional connectivity map also provides a resource for interpreting connectomic reconstruction of neural circuits for leg proprioception.

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10/06/21 | An open-access volume electron microscopy atlas of whole cells and tissues.
Xu CS, Pang S, Shtengel G, Müller A, Ritter AT, Hoffman HK, Takemura S, Lu Z, Pasolli HA, Iyer N, Chung J, Bennett D, Weigel AV, Freeman M, Van Engelenburg SB, Walther TC, Farese RV, Lippincott-Schwartz J, Mellman I, Solimena M, Hess HF
Nature. 2021 Oct 06:. doi: 10.1038/s41586-021-03992-4

Understanding cellular architecture is essential for understanding biology. Electron microscopy (EM) uniquely visualizes cellular structures with nanometre resolution. However, traditional methods, such as thin-section EM or EM tomography, have limitations in that they visualize only a single slice or a relatively small volume of the cell, respectively. Focused ion beam-scanning electron microscopy (FIB-SEM) has demonstrated the ability to image small volumes of cellular samples with 4-nm isotropic voxels. Owing to advances in the precision and stability of FIB milling, together with enhanced signal detection and faster SEM scanning, we have increased the volume that can be imaged with 4-nm voxels by two orders of magnitude. Here we present a volume EM atlas at such resolution comprising ten three-dimensional datasets for whole cells and tissues, including cancer cells, immune cells, mouse pancreatic islets and Drosophila neural tissues. These open access data (via OpenOrganelle) represent the foundation of a field of high-resolution whole-cell volume EM and subsequent analyses, and we invite researchers to explore this atlas and pose questions.

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10/06/21 | Biosensors based on peptide exposure show single molecule conformations in live cells.
Liu B, Stone OJ, Pablo M, Herron JC, Nogueira AT, Dagliyan O, Grimm JB, Lavis LD, Elston TC, Hahn KM
Cell. 2021 Oct 06:. doi: 10.1016/j.cell.2021.09.026

We describe an approach to study the conformation of individual proteins during single particle tracking (SPT) in living cells. "Binder/tag" is based on incorporation of a 7-mer peptide (the tag) into a protein where its solvent exposure is controlled by protein conformation. Only upon exposure can the peptide specifically interact with a reporter protein (the binder). Thus, simple fluorescence localization reflects protein conformation. Through direct excitation of bright dyes, the trajectory and conformation of individual proteins can be followed. Simple protein engineering provides highly specific biosensors suitable for SPT and FRET. We describe tagSrc, tagFyn, tagSyk, tagFAK, and an orthogonal binder/tag pair. SPT showed slowly diffusing islands of activated Src within Src clusters and dynamics of activation in adhesions. Quantitative analysis and stochastic modeling revealed in vivo Src kinetics. The simplicity of binder/tag can provide access to diverse proteins.

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10/06/21 | Whole-cell organelle segmentation in volume electron microscopy.
Heinrich L, Bennett D, Ackerman D, Park W, Bogovic J, Eckstein N, Petruncio A, Clements J, Pang S, Xu CS, Funke J, Korff W, Hess HF, Lippincott-Schwartz J, Saalfeld S, Weigel AV, COSEM Project Team
Nature. 2021 Oct 06:. doi: 10.1038/s41586-021-03977-3

Cells contain hundreds of organelles and macromolecular assemblies. Obtaining a complete understanding of their intricate organization requires the nanometre-level, three-dimensional reconstruction of whole cells, which is only feasible with robust and scalable automatic methods. Here, to support the development of such methods, we annotated up to 35 different cellular organelle classes-ranging from endoplasmic reticulum to microtubules to ribosomes-in diverse sample volumes from multiple cell types imaged at a near-isotropic resolution of 4 nm per voxel with focused ion beam scanning electron microscopy (FIB-SEM). We trained deep learning architectures to segment these structures in 4 nm and 8 nm per voxel FIB-SEM volumes, validated their performance and showed that automatic reconstructions can be used to directly quantify previously inaccessible metrics including spatial interactions between cellular components. We also show that such reconstructions can be used to automatically register light and electron microscopy images for correlative studies. We have created an open data and open-source web repository, 'OpenOrganelle', to share the data, computer code and trained models, which will enable scientists everywhere to query and further improve automatic reconstruction of these datasets.

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10/04/21 | An adaptive optics module for deep tissue multiphoton imaging in vivo
Cristina Rodríguez , Anderson Chen , José A. Rivera , Manuel A. Mohr , Yajie liang , Wenzhi Sun , Daniel E. Milkie , Thomas G. Bifano , Xiaoke Chen , Na Ji
Nature Methods. 2021 Oct 04:1259-64. doi: 10.1038/s41592-021-01279-0

Understanding complex biological systems requires visualizing structures and processes deep within living organisms. We developed a compact adaptive optics module and incorporated it into two- and three-photon fluorescence microscopes, to measure and correct tissue-induced aberrations. We resolved synaptic structures in deep cortical and subcortical areas of the mouse brain, and demonstrated high-resolution imaging of neuronal structures and somatosensory-evoked calcium responses in the mouse spinal cord at unprecedented depths in vivo.

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10/01/21 | QUAREP-LiMi: A community-driven initiative to establish guidelines for quality assessment and reproducibility for instruments and images in light microscopy
Glyn Nelson , Ulrike Boehm , Steve Bagley , Peter Bajcsy , Johanna Bischof , Claire M Brown , Aurelien Dauphin , Ian M Dobbie , John E Eriksson , Orestis Faklaris , Julia Fernandez-Rodriguez , Alexia Ferrand , Ali Gheisari , Hella Hartmann , Christian Kukat , Alex Laude , Miso Mitkovski , Sebastian Munck , Alison J North , Tobias M Rasse , Ute Resch-Genger , Lucas C Schuetz , Arne Seitz , Caterina Strambio-De-Castillia , Jason R Swedlow , Ioannis Alexopoulos , Karin Aumayr , Sergiy Avilov , Gert-Jan Bakker , Rodrigo R Bammann , Andrea Bassi , Hannes Beckert , Sebastian Beer , Yury Belyaev , Jakob Bierwagen , Konstantin A Birngruber , Manel Bosch , Juergen Breitlow , Lisa A Cameron , Joe Chalfoun , James J Chambers , Chieh-Li Chen , Eduardo Conde-Sousa , Alexander D Corbett , Fabrice P Cordelieres , Elaine Del Nery , Ralf Dietzel , Frank Eismann , Elnaz Fazeli , Andreas Felscher , Hans Fried , Nathalie Gaudreault , Wah Ing Goh , Thomas Guilbert , Roland Hadleigh , Peter Hemmerich , Gerhard A Holst , Michelle S Itano , Claudia B Jaffe , Helena K Jambor , Stuart C Jarvis , Antje Keppler , David Kirchenbuechler , Marcel Kirchner , Norio Kobayashi , Gabriel Krens , Susanne Kunis , Judith Lacoste , Marco Marcell , Gabriel G Martins , Daniel J Metcalf , Claire A Mitchell , Joshua Moore , Tobias Mueller , Michael S Nelson , Stephen Ogg , Shuichi Onami , Alexandra L Palmer , Perrine Paul-Gilloteaux , Jaime A Pimentel , Laure Plantard , Santosh Podder , Elton Rexhepaj , Arnaud Royon , Markku A Saari , Damien Schapman , Vincent Schoonderwoert , Britta Schroth-Diez , Stanley Schwartz , Michael Shaw , Martin Spitaler , Martin T Stoeckl , Damir Sudar , Jeremie Teillon , Stefan Terjung , Roland Thuenauer , Christian D Wilms , Graham D Wright , Roland Nitschke , Laurent Gelman
Journal of Microscopy. 2021 Oct 01;284(1):56-73

In April 2020, the QUality Assessment and REProducibility for Instruments and Images in Light Microscopy (QUAREP-LiMi) initiative was formed. This initiative comprises imaging scientists from academia and industry who share a common interest in achieving a better understanding of the performance and limitations of microscopes and improved quality control (QC) in light microscopy. The ultimate goal of the QUAREP-LiMi initiative is to establish a set of common QC standards, guidelines, metadata models, and tools, including detailed protocols, with the ultimate aim of improving reproducible advances in scientific research. This White Paper 1) summarizes the major obstacles identified in the field that motivated the launch of the QUAREP-LiMi initiative; 2) identifies the urgent need to address these obstacles in a grassroots manner, through a community of stakeholders including, researchers, imaging scientists, bioimage analysts, bioimage informatics developers, corporate partners, funding agencies, standards organizations, scientific publishers, and observers of such; 3) outlines the current actions of the QUAREP-LiMi initiative, and 4) proposes future steps that can be taken to improve the dissemination and acceptance of the proposed guidelines to manage QC. To summarize, the principal goal of the QUAREP-LiMi initiative is to improve the overall quality and reproducibility of light microscope image data by introducing broadly accepted standard practices and accurately captured image data metrics.

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