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4 Janelia Publications

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    03/26/22 | Transverse endoplasmic reticulum expansion in hereditary spastic paraplegia corticospinal axons.
    Zhu P, Hung H, Batchenkova N, Nixon-Abell J, Henderson J, Zheng P, Renvoisé B, Pang S, Xu CS, Saalfeld S, Funke J, Xie Y, Svara F, Hess HF, Blackstone C
    Human Molecular Genetics. 2022 Mar 26:. doi: 10.1093/hmg/ddac072

    Hereditary spastic paraplegias (HSPs) comprise a large group of inherited neurologic disorders affecting the longest corticospinal axons (SPG1-86 plus others), with shared manifestations of lower extremity spasticity and gait impairment. Common autosomal dominant HSPs are caused by mutations in genes encoding the microtubule-severing ATPase spastin (SPAST; SPG4), the membrane-bound GTPase atlastin-1 (ATL1; SPG3A), and the reticulon-like, microtubule-binding protein REEP1 (REEP1; SPG31). These proteins bind one another and function in shaping the tubular endoplasmic reticulum (ER) network. Typically, mouse models of HSPs have mild, later-onset phenotypes, possibly reflecting far shorter lengths of their corticospinal axons relative to humans. Here, we have generated a robust, double mutant mouse model of HSP in which atlastin-1 is genetically modified with a K80A knock-in (KI) missense change that abolishes its GTPase activity, while its binding partner Reep1 is knocked out. Atl1KI/KI/Reep1-/- mice exhibit early-onset and rapidly progressive declines in several motor function tests. Also, ER in mutant corticospinal axons dramatically expands transversely and periodically in a mutation dosage-dependent manner to create a ladder-like appearance, based on reconstructions of focused ion beam-scanning electron microscopy datasets using machine learning-based auto-segmentation. In lockstep with changes in ER morphology, axonal mitochondria are fragmented and proportions of hypophosphorylated neurofilament H and M subunits are dramatically increased in Atl1KI/KI/Reep1-/- spinal cord. Co-occurrence of these findings links ER morphology changes to alterations in mitochondrial morphology and cytoskeletal organization. Atl1KI/KI/Reep1-/- mice represent an early-onset rodent HSP model with robust behavioral and cellular readouts for testing novel therapies.

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    11/01/21 | 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 Nov 01;599(7883):141-46. 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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    09/03/20 | A connectome of the adult drosophila central brain.
    Xu CS, Januszewski M, Lu Z, Takemura S, Hayworth KJ, Huang G, Shinomiya K, Maitin-Shepard J, Ackerman D, Berg S, Blakely T, Bogovic J, Clements J, Dolafi T, Hubbard P, Kainmueller D, Katz W, Kawase T, Khairy KA, Leavitt L, Li PH, Lindsey L, Neubarth N, Olbris DJ, Otsuna H, Troutman ET, Umayam L, Zhao T, Ito M, Goldammer J, Wolff T, Svirskas R, Schlegel P, Neace ER, Knecht CJ, Alvarado CX, Bailey DA, Ballinger S, Borycz JA, Canino BS
    eLife. 2020 Sep 03:. doi:

    The neural circuits responsible for behavior remain largely unknown. Previous efforts have reconstructed the complete circuits of small animals, with hundreds of neurons, and selected circuits for larger animals. Here we (the FlyEM project at Janelia and collaborators at Google) summarize new methods and present the complete circuitry of a large fraction of the brain of a much more complex animal, the fruit fly Drosophila melanogaster. Improved methods include new procedures to prepare, image, align, segment, find synapses, and proofread such large data sets; new methods that define cell types based on connectivity in addition to morphology; and new methods to simplify access to a large and evolving data set. From the resulting data we derive a better definition of computational compartments and their connections; an exhaustive atlas of cell examples and types, many of them novel; detailed circuits for most of the central brain; and exploration of the statistics and structure of different brain compartments, and the brain as a whole. We make the data public, with a web site and resources specifically designed to make it easy to explore, for all levels of expertise from the expert to the merely curious. The public availability of these data, and the simplified means to access it, dramatically reduces the effort needed to answer typical circuit questions, such as the identity of upstream and downstream neural partners, the circuitry of brain regions, and to link the neurons defined by our analysis with genetic reagents that can be used to study their functions.

    Note: In the next few weeks, we will release a series of papers with more involved discussions. One paper will detail the hemibrain reconstruction with more extensive analysis and interpretation made possible by this dense connectome. Another paper will explore the central complex, a brain region involved in navigation, motor control, and sleep. A final paper will present insights from the mushroom body, a center of multimodal associative learning in the fly brain.

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    01/18/19 | Cortical column and whole-brain imaging with molecular contrast and nanoscale resolution.
    Gao R, Asano SM, Upadhyayula S, Pisarev I, Milkie DE, Liu T, Singh V, Graves AR, Huynh GH, Zhao Y, Bogovic JA, Colonell J, Ott CM, Zugates CT, Tappan S, Rodriguez A, Mosaliganti KR, Sheu S, Pasolli HA, et al
    Science (New York, N.Y.). 2019 Jan 18;363(6424):eaau8302. doi: 10.1126/science.aau8302

    Optical and electron microscopy have made tremendous inroads toward understanding the complexity of the brain. However, optical microscopy offers insufficient resolution to reveal subcellular details, and electron microscopy lacks the throughput and molecular contrast to visualize specific molecular constituents over millimeter-scale or larger dimensions. We combined expansion microscopy and lattice light-sheet microscopy to image the nanoscale spatial relationships between proteins across the thickness of the mouse cortex or the entire Drosophila brain. These included synaptic proteins at dendritic spines, myelination along axons, and presynaptic densities at dopaminergic neurons in every fly brain region. The technology should enable statistically rich, large-scale studies of neural development, sexual dimorphism, degree of stereotypy, and structural correlations to behavior or neural activity, all with molecular contrast.

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