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13 Janelia Publications

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    08/25/22 | In situ single particle classification reveals distinct 60S maturation intermediates in cells.
    Bronwyn A. Lucas , Kexin Zhang , Sarah Loerch , Nikolaus Grigorieff
    eLife. 2022 Aug 25:. doi: 10.7554/eLife.79272

    Electron cryo-microscopy (cryo-EM) can generate high-resolution views of cells with faithful preservation of molecular structure. In situ cryo-EM, therefore, has enormous potential to reveal the atomic details of biological processes in their native context. However, in practice, the utility of in situ cryo-EM is limited by the difficulty of reliably locating and confidently identifying molecular targets (particles) and their conformational states in the crowded cellular environment. We recently showed that 2DTM, a fine-grained template-based search applied to cryo-EM micrographs, can localize particles in two-dimensional views of cells with high precision. Here we demonstrate that the signal-to-noise ratio (SNR) observed with 2DTM can be used to differentiate related complexes in focused ion beam (FIB)-milled cell sections. We apply this method in two contexts to locate and classify related intermediate states of 60S ribosome biogenesis in the Saccharomyces cerevisiae cell nucleus. In the first, we separate the nuclear pre-60S population from the cytoplasmic mature 60S population, using the subcellular localization to validate assignment. In the second, we show that relative 2DTM SNRs can be used to separate mixed populations of nuclear pre-60S that are not visually separable. We use a maximum likelihood approach to define the probability of each particle belonging to each class, thereby establishing a statistic to describe the confidence of our classification. Without the need to generate 3D reconstructions, 2DTM can be applied even when only a few target particles exist in a cell.

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    08/24/22 | A single-cell transcriptomic atlas of complete insect nervous systems across multiple life stages.
    Corrales M, Cocanougher BT, Kohn AB, Wittenbach JD, Long XS, Lemire A, Cardona A, Singer RH, Moroz LL, Zlatic M
    Neural Development. 2022 Aug 24;17(1):8. doi: 10.1186/s13064-022-00164-6

    Molecular profiles of neurons influence neural development and function but bridging the gap between genes, circuits, and behavior has been very difficult. Here we used single cell RNAseq to generate a complete gene expression atlas of the Drosophila larval central nervous system composed of 131,077 single cells across three developmental stages (1 h, 24 h and 48 h after hatching). We identify 67 distinct cell clusters based on the patterns of gene expression. These include 31 functional mature larval neuron clusters, 1 ring gland cluster, 8 glial clusters, 6 neural precursor clusters, and 13 developing immature adult neuron clusters. Some clusters are present across all stages of larval development, while others are stage specific (such as developing adult neurons). We identify genes that are differentially expressed in each cluster, as well as genes that are differentially expressed at distinct stages of larval life. These differentially expressed genes provide promising candidates for regulating the function of specific neuronal and glial types in the larval nervous system, or the specification and differentiation of adult neurons. The cell transcriptome Atlas of the Drosophila larval nervous system is a valuable resource for developmental biology and systems neuroscience and provides a basis for elucidating how genes regulate neural development and function.

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    08/23/22 | 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 Aug 23;31(16):2779-2795. 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, whereas 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, on the basis of 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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    08/22/22 | Visual projection neuron convergence and compensation in developing sensorimotor circuits in the Drosophila optic glomeruli
    Brennan W. McFarland , HyoJong Jang , Natalie Smolin , Tanja A. Godenschwege , Aljoscha Nern , Yerbol Z. Kurmangaliyev , Catherine R. von Reyn

    Visual features detected by the early visual system must be combined into higher order representations to guide behavioral decision. Although key developmental mechanisms that enable the separation of visual feature channels in early visual circuits have been discovered, relatively little is known about the mechanisms that underlie their convergence in later stages of visual processing. Here we explore the development of a functionally well-characterized sensorimotor circuit in Drosophila melanogaster, the convergence of visual projection neurons (VPNs) onto the dendrites of a large descending neuron called the giant fiber (GF). We find two VPNs encoding different visual features that target the same giant fiber dendrite establish their territories on the dendrite, in part, through sequential axon arrival during development prior to synaptogenesis. Physical occupancy is important to maintain territories, as we find the ablation of one VPN results in expanded dendrite territory of the remaining VPN, and that this compensation enables the GF to remain responsive to ethologically relevant visual stimuli. Our data highlight temporal mechanisms for visual feature convergence and promote the GF circuit, and the Drosophila optic glomeruli where VPN to GF connectivity resides, as an ideal developmental model for investigating complex wiring programs and plasticity in visual feature convergence.

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    08/19/22 | Flexible control of behavioral variability mediated by an internal representation of head direction
    Chuntao Dan , Brad K. Hulse , Vivek Jayaraman , Ann M. Hermundstad
    bioRxiv. 2022 Aug 19:. doi: 10.1101/2021.08.18.456004

    Internal representations are thought to support the generation of flexible, long-timescale behavioral patterns in both animals and artificial agents. Here, we present a novel conceptual framework for how Drosophila use their internal representation of head direction to maintain preferred headings in their surroundings, and how they learn to modify these preferences in the presence of selective thermal reinforcement. To develop the framework, we analyzed flies’ behavior in a classical operant visual learning paradigm and found that they use stochastically generated fixations and directed turns to express their heading preferences. Symmetries in the visual scene used in the paradigm allowed us to expose how flies’ probabilistic behavior in this setting is tethered to their head direction representation. We describe how flies’ ability to quickly adapt their behavior to the rules of their environment may rest on a behavioral policy whose parameters are flexible but whose form is genetically encoded in the structure of their circuits. Many of the mechanisms we outline may also be relevant for rapidly adaptive behavior driven by internal representations in other animals, including mammals.

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    08/17/22 | Homeodomain proteins hierarchically specify neuronal diversity and synaptic connectivity
    Chundi Xu , Tyler B. Ramos , Ed M. Rogers , Michael B. Reiser , Chris Q. Doe
    bioRxiv. 2022 Aug 17:. doi: 10.1101/2021.10.01.462699

    The brain generates diverse neuron types which express unique homeodomain transcription factors (TFs) and assemble into precise neural circuits. Yet a mechanistic framework is lacking for how homeodomain TFs specify both neuronal fate and synaptic connectivity. We use Drosophila lamina neurons (L1-L5) to show the homeodomain TF Brain-specific homeobox (Bsh) is initiated in lamina precursor cells (LPCs) where it specifies L4/L5 fate and suppresses homeodomain TF Zfh1 to prevent L1/L3 fate. Subsequently, Bsh activates the homeodomain TF Apterous (Ap) in L4 in a feedforward loop to express the synapse recognition molecule DIP-β, in part by Bsh direct binding a DIP-β intron. Thus, homeodomain TFs function hierarchically: primary homeodomain TF (Bsh) first specifies neuronal fate, and subsequently acts with secondary homeodomain TF (Ap) to activate DIP-β, thereby generating precise synaptic connectivity. We speculate that hierarchical homeodomain TF function may represent a general principle for coordinating neuronal fate specification and circuit assembly.

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    08/13/22 | A vast space of compact strategies for highly efficient decisions
    Tzuhsuan Ma , Ann M Hermundstad
    bioRxiv. 2022 Aug 13:. doi: 10.1101/2022.08.10.503471

    When foraging in dynamic and uncertain environments, animals can benefit from basing their decisions on smart inferences about hidden properties of the world. Typical theoretical approaches to understand the strategies that animals use in such settings combine Bayesian inference and value iteration to derive optimal behavioral policies that maximize total reward given changing beliefs about the environment. However, specifying these beliefs requires infinite numerical precision; with limited resources, this problem can no longer be separated into optimizing inference and optimizing action selections. To understand the space of behavioral policies in this constrained setting, we enumerate and evaluate all possible behavioral programs that can be constructed from just a handful of states. We show that only a small fraction of the top-performing programs can be constructed by approximating Bayesian inference; the remaining programs are structurally or even functionally distinct from Bayesian. To assess structural and functional relationships among all programs, we developed novel tree embedding algorithms; these embeddings, which are capable of extracting different relational structures within the program space, reveal that nearly all good programs are closely connected through single algorithmic “mutations”. We demonstrate how one can use such relational structures to efficiently search for good solutions via an evolutionary algorithm. Moreover, these embeddings reveal that the diversity of non-Bayesian behaviors originates from a handful of key mutations that broaden the functional repertoire within the space of good programs. The fact that this diversity of behaviors does not significantly compromise performance suggests a novel approach for studying how these strategies generalize across tasks.

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    08/12/22 | Isolation, cryo-laser scanning confocal microscope imaging and cryo-FIB milling of mouse glutamatergic synaptosomes.
    Gogoi P, Shiozaki M, Gouaux E
    PLoS One. 2022 Aug 12;17(8):e0271799. doi: 10.1371/journal.pone.0271799

    Ionotropic glutamate receptors (iGluRs) at postsynaptic terminals mediate the majority of fast excitatory neurotransmission in response to release of glutamate from the presynaptic terminal. Obtaining structural information on the molecular organization of iGluRs in their native environment, along with other signaling and scaffolding proteins in the postsynaptic density (PSD), and associated proteins on the presynaptic terminal, would enhance understanding of the molecular basis for excitatory synaptic transmission in normal and in disease states. Cryo-electron tomography (ET) studies of synaptosomes is one attractive vehicle by which to study iGluR-containing excitatory synapses. Here we describe a workflow for the preparation of glutamatergic synaptosomes for cryo-ET studies. We describe the utilization of fluorescent markers for the facile detection of the pre and postsynaptic terminals of glutamatergic synaptosomes using cryo-laser scanning confocal microscope (cryo-LSM). We further provide the details for preparation of lamellae, between ~100 to 200 nm thick, of glutamatergic synaptosomes using cryo-focused ion-beam (FIB) milling. We monitor the lamella preparation using a scanning electron microscope (SEM) and following lamella production, we identify regions for subsequent cryo-ET studies by confocal fluorescent imaging, exploiting the pre and postsynaptic fluorophores.

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    Looger Lab
    08/12/22 | Selective Serotonin Reuptake Inhibitors Within Cells: Temporal Resolution in Cytoplasm, Endoplasmic Reticulum, and Membrane
    Aaron L. Nichols , Zack Blumenfeld , Laura Luebbert , Hailey J. Knox , Anand K. Muthusamy , Jonathan S. Marvin , Charlene H. Kim , Stephen N. Grant , David P. Walton , Bruce N. Cohen , Rebekkah Hammar , Loren L. Looger , Per Artursson , Dennis A. Dougherty , Henry A. Lester
    bioRxiv. 2022 Aug 12:. doi: 10.1101/2022.08.09.502705

    Selective serotonin reuptake inhibitors (SSRIs) are the most prescribed treatment for individuals experiencing major depressive disorder (MDD). The therapeutic mechanisms that take place before, during, or after SSRIs bind the serotonin transporter (SERT) are poorly understood, partially because no studies exist of the cellular and subcellular pharmacokinetic properties of SSRIs in living cells. We studied escitalopram and fluoxetine using new intensity- based drug-sensing fluorescent reporters (“iDrugSnFRs”) targeted to the plasma membrane (PM), cytoplasm, or endoplasmic reticulum (ER) of cultured neurons and mammalian cell lines. We also employed chemical detection of drug within cells and phospholipid membranes. The drugs attain equilibrium in neuronal cytoplasm and ER, at approximately the same concentration as the externally applied solution, with time constants of a few s (escitalopram) or 200-300 s (fluoxetine). Simultaneously, the drugs accumulate within lipid membranes by ≥ 18-fold (escitalopram) or 180-fold (fluoxetine), and possibly by much larger factors. Both drugs leave cytoplasm, lumen, and membranes just as quickly during washout. We synthesized membrane-impermeant quaternary amine derivatives of the two SSRIs. The quaternary derivatives are substantially excluded from membrane, cytoplasm, and ER for > 2.4 h. They inhibit SERT transport-associated currents 6- or 11-fold less potently than the SSRIs (escitalopram or fluoxetine derivative, respectively), providing useful probes for distinguishing compartmentalized SSRI effects. Although our measurements are orders of magnitude faster than the “therapeutic lag” of SSRIs, these data suggest that SSRI-SERT interactions within organelles or membranes may play roles during either the therapeutic effects or the “antidepressant discontinuation syndrome”.

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    08/09/22 | Biomechanical origins of proprioceptive maps in the Drosophila leg
    Akira Mamiya , Pralaksha Gurung , Igor Siwanowicz , Anne Sustar , Chenghao Chen , Jasper S. Phelps , Aaron T. Kuan , Alexandra Pacureanu , Wei-Chung Allen Lee , Natasha Mhatre , John C. Tuthill
    bioRxiv. 2022 Aug 09:. doi: 10.1101/2022.08.08.503192

    Proprioception, the sense of body position and movement, is essential for effective motor control. Because proprioceptive sensory neurons are embedded in complex and dynamic tissues, it has been challenging to understand how they sense and encode mechanical stimuli. Here, we find that proprioceptor neurons in the Drosophila femur are organized into functional groups that are biomechanically specialized to detect features of tibia joint kinematics. The dendrites of position and vibration-tuned proprioceptors receive distinct mechanical signals via the arculum, an elegant mechanical structure that decomposes movement of the tibia joint into orthogonal components. The cell bodies of position-tuned proprioceptors form a goniotopic map of joint angle, whereas the dendrites of vibration-tuned proprioceptors form a tonotopic map of tibia vibration frequency. Our findings reveal biomechanical mechanisms that underlie proprioceptor feature selectivity and identify common organizational principles between proprioception and other topographically organized sensory systems.

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