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2508 Publications

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    06/25/24 | Hot-Distance: Combining One-Hot and Signed Distance Embeddings for Segmentation
    Marwan Zouinkhi , Jeff L. Rhoades , Aubrey V. Weigel
    arXiv. 2024 Jun 25:2406.17936. doi: 10.48550/arXiv.2406.17936

    Machine learning models are only as good as the data to which they are fit. As such, it is always preferable to use as much data as possible in training models. What data can be used for fitting a model depends a lot on the formulation of the task. We introduce Hot-Distance, a novel segmentation target that incorporates the strength of signed boundary distance prediction with the flexibility of one-hot encoding, to increase the amount of usable training data for segmentation of subcellular structures in focused ion beam scanning electron microscopy (FIB-SEM).

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    06/17/24 | Steering From the Rear: Coordination of Central Pattern Generators Underlying Navigation by Ascending Interneurons
    Jonaitis J, Hibbard KL, Layte KM, Hiramoto A, Cardona A, Truman JW, Nose A, Zwart MF, Pulver SR
    bioRxiv. 2024 Jun 17:. doi: 10.1101/2024.06.17.598162

    Understanding how animals coordinate movements to achieve goals is a fundamental pursuit in neuroscience. Here we explore how neurons that reside in posterior lower-order regions of a locomotor system and project to anterior higher-order regions influence steering and navigation. We characterized the anatomy and functional role of a population of ascending interneurons in the ventral nerve cord of Drosophila larvae. Through electron microscopy reconstructions and light microscopy, we determined that the cholinergic 19f cells receive input primarily from premotor interneurons and synapse upon a diverse array of postsynaptic targets within the anterior segments including other 19f cells. Calcium imaging of 19f activity in isolated CNS preparations in relation to motor neurons revealed that 19f neurons are recruited into most larval motor programmes. 19f activity lags behind motor neuron activity and as a population, the cells encode spatio-temporal patterns of locomotor activity in the larval CNS. Optogenetic manipulations of 19f cell activity in isolated CNS preparations revealed that they coordinate the activity of central pattern generators underlying exploratory headsweeps and forward locomotion in a context and location specific manner. In behaving animals, activating 19f cells suppressed exploratory headsweeps and slowed forward locomotion, while inhibition of 19f activity potentiated headsweeps, slowing forward movement. Inhibiting activity in 19f cells ultimately affected the ability of larvae to remain in the vicinity of an odor source during an olfactory navigation task. Overall, our findings provide insights into how ascending interneurons monitor motor activity and shape interactions amongst rhythm generators underlying complex navigational tasks.

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    05/31/24 | Salivary gland developmental mechanics
    Morales EA, Wang S
    Current Topics in Developmental Biology:. doi: 10.1016/bs.ctdb.2024.05.002

    The salivary gland undergoes branching morphogenesis to elaborate into a tree-like structure with numerous saliva-secreting acinar units, all joined by a hierarchical ductal system. The expansive epithelial surface generated by branching morphogenesis serves as the structural basis for the efficient production and delivery of saliva. Here, we elucidate the process of salivary gland morphogenesis, emphasizing the role of mechanics. Structurally, the developing salivary gland is characterized by a stratified epithelium tightly encased by the basement membrane, which is in turn surrounded by a mesenchyme consisting of a dense network of interstitial matrix and mesenchymal cells. Diverse cell types and extracellular matrices bestow this developing organ with organized, yet spatially varied mechanical properties. For instance, the surface epithelial sheet of the bud is highly fluidic due to its high cell motility and weak cell-cell adhesion, rendering it highly pliable. In contrast, the inner core of the bud is more rigid, characterized by reduced cell motility and strong cell-cell adhesion, which likely provide structural support for the tissue. The interactions between the surface epithelial sheet and the inner core give rise to budding morphogenesis. Furthermore, the basement membrane and the mesenchyme offer mechanical constraints that could play a pivotal role in determining the higher-order architecture of a fully mature salivary gland.

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    06/10/24 | Transport and Organization of Individual Vimentin Filaments Within Dense Networks Revealed by Single Particle Tracking and 3D FIB-SEM
    Renganathan B, Moore AS, Yeo W, Petruncio A, Ackerman D, Wiegel A, CellMap Team , Pasolli HA, Xu CS, Hess HF, Serpinskaya AS, Zhang HF, Lippincott-Schwartz J, Gelfand VI
    bioRxiv. 2024 Jun 10:. doi: 10.1101/2024.06.10.598346

    Vimentin intermediate filaments (VIFs) form complex, tight-packed networks; due to this density, traditional ensemble labeling and imaging approaches cannot accurately discern single filament behavior. To address this, we introduce a sparse vimentin-SunTag labeling strategy to unambiguously visualize individual filament dynamics. This technique confirmed known long-range dynein and kinesin transport of peripheral VIFs and uncovered extensive bidirectional VIF motion within the perinuclear vimentin network, a region we had thought too densely bundled to permit such motility. To examine the nanoscale organization of perinuclear vimentin, we acquired high-resolution electron microscopy volumes of a vitreously frozen cell and reconstructed VIFs and microtubules within a 50 um3 window. Of 583 VIFs identified, most were integrated into long, semi-coherent bundles that fluctuated in width and filament packing density. Unexpectedly, VIFs displayed minimal local co-alignment with microtubules, save for sporadic cross-over sites that we predict facilitate cytoskeletal crosstalk. Overall, this work demonstrates single VIF dynamics and organization in the cellular milieu for the first time.

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    06/06/24 | Comparative connectomics of the descending and ascending neurons of the Drosophila nervous system: stereotypy and sexual dimorphism
    Stürner T, Brooks P, Serratosa Capdevila L, Morris BJ, Javier A, Fang S, Gkantia M, Cachero S, Beckett IR, Champion AS, Moitra I, Richards A, Klemm F, Kugel L, Namiki S, Cheong HS, Kovalyak J, Tenshaw E, Parekh R, Schlegel P, Phelps JS, Mark B, Dorkenwald S, Bates AS, Matsliah A, Yu S, McKellar CE, Sterling A, Seung S, Murthy M, Tuthill J, Lee WA, Card GM, Costa M, Jefferis GS, Eichler K
    bioRxiv. 2024 Jun 06:. doi: 10.1101/2024.06.04.596633

    In most complex nervous systems there is a clear anatomical separation between the nerve cord, which contains most of the final motor outputs necessary for behaviour, and the brain. In insects, the neck connective is both a physical and information bottleneck connecting the brain and the ventral nerve cord (VNC, spinal cord analogue) and comprises diverse populations of descending (DN), ascending (AN) and sensory ascending neurons, which are crucial for sensorimotor signalling and control.Integrating three separate EM datasets, we now provide a complete connectomic description of the ascending and descending neurons of the female nervous system of Drosophila and compare them with neurons of the male nerve cord. Proofread neuronal reconstructions have been matched across hemispheres, datasets and sexes. Crucially, we have also matched 51% of DN cell types to light level data defining specific driver lines as well as classifying all ascending populations.We use these results to reveal the general architecture, tracts, neuropil innervation and connectivity of neck connective neurons. We observe connected chains of descending and ascending neurons spanning the neck, which may subserve motor sequences. We provide a complete description of sexually dimorphic DN and AN populations, with detailed analysis of circuits implicated in sex-related behaviours, including female ovipositor extrusion (DNp13), male courtship (DNa12/aSP22) and song production (AN hemilineage 08B). Our work represents the first EM-level circuit analyses spanning the entire central nervous system of an adult animal.

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    05/31/24 | Periodic ER-plasma membrane junctions support long-range Ca2+ signal integration in dendrites
    Benedetti L, Fan R, Weigel AV, Moore AS, Houlihan PR, Kittisopikul M, Park G, Petruncio A, Hubbard PM, Pang S, Xu CS, Hess HF, Saalfeld S, Rangaraju V, Clapham DE, De Camilli P, Ryan TA, Lippincott-Schwartz J
    bioRxiv. 2024 May 31:. doi: 10.1101/2024.05.27.596121

    Neuronal dendrites must relay synaptic inputs over long distances, but the mechanisms by which activity-evoked intracellular signals propagate over macroscopic distances remain unclear. Here, we discovered a system of periodically arranged endoplasmic reticulum-plasma membrane (ER-PM) junctions tiling the plasma membrane of dendrites at \~1 μm intervals, interlinked by a meshwork of ER tubules patterned in a ladder-like array. Populated with Junctophilin-linked plasma membrane voltage-gated Ca2+ channels and ER Ca2+-release channels (ryanodine receptors), ER-PM junctions are hubs for ER-PM crosstalk, fine-tuning of Ca2+ homeostasis, and local activation of the Ca2+/calmodulin-dependent protein kinase II. Local spine stimulation activates the Ca2+ modulatory machinery facilitating voltage-independent signal transmission and ryanodine receptor-dependent Ca2+ release at ER-PM junctions over 20 μm away. Thus, interconnected ER-PM junctions support signal propagation and Ca2+ release from the spine-adjacent ER. The capacity of this subcellular architecture to modify both local and distant membrane-proximal biochemistry potentially contributes to dendritic computations.HighlightsPeriodic ER-PM junctions tile neuronal dendritic plasma membrane in rodent and fly.ER-PM junctions are populated by ER tethering and Ca2+ release and influx machinery.ER-PM junctions act as sites for local activation of CaMKII.Local spine activation drives Ca2+ release from RyRs at ER-PM junctions over 20 μm.

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    06/05/24 | Spatial Single-cell Analysis Decodes Cortical Layer and Area Specification
    Qian X, Coleman K, Jiang S, Kriz AJ, Marciano JH, Luo C, Cai C, Manam MD, Caglayan E, Otani A, Ghosh U, Shao DD, Andersen RE, Neil JE, Johnson R, LeFevre A, Hecht JL, Miller MB, Sun L, Stringer C, Li M, Walsh CA
    bioRxiv. 2024 Jun 05:. doi: 10.1101/2024.06.05.597673

    The human cerebral cortex, pivotal for advanced cognitive functions, is composed of six distinct layers and dozens of functionally specialized areas. The layers and areas are distinguished both molecularly, by diverse neuronal and glial cell subtypes, and structurally, through intricate spatial organization3,4. While single-cell transcriptomics studies have advanced molecular characterization of human cortical development, a critical gap exists due to the loss of spatial context during cell dissociation. Here, we utilized multiplexed error-robust fluorescence in situ hybridization (MERFISH)9, augmented with deep-learning-based cell segmentation, to examine the molecular, cellular, and cytoarchitectural development of human fetal cortex with spatially resolved single-cell resolution. Our extensive spatial atlas, encompassing 16 million single cells, spans eight cortical areas across four time points in the second and third trimesters. We uncovered an early establishment of the six-layer structure, identifiable in the laminar distribution of excitatory neuronal subtypes by mid-gestation, long before the emergence of cytoarchitectural layers. Notably, while anterior-posterior gradients of neuronal subtypes were generally observed in most cortical areas, a striking exception was the sharp molecular border between primary (V1) and secondary visual cortices (V2) at gestational week 20. Here we discovered an abrupt binary shift in neuronal subtype specification at the earliest stages, challenging the notion that continuous morphogen gradients dictate mid-gestation cortical arealization. Moreover, integrating single-nuclei RNA-sequencing and in situ whole transcriptomics revealed an early upregulation of synaptogenesis in V1-specific Layer 4 neurons, suggesting a role of synaptogenesis in this discrete border formation. Collectively, our findings underscore the crucial role of spatial relationships in determining the molecular specification of cortical layers and areas. This work not only provides a valuable resource for the field, but also establishes a spatially resolved single-cell analysis paradigm that paves the way for a comprehensive developmental atlas of the human brain.

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    06/02/24 | Mechanosensory representation of wing deformations
    Yarger AM, Maeda M, Siwanowicz I, Kajiyama H, Walker SM, Bomphrey RJ, Lin H
    bioRxiv. 2024 Jun 02:. doi: 10.1101/2024.06.02.596338

    Efficient representation of structural deformations is crucial for monitoring the instantaneous state of biological structures. Insects’ ability to encode wing deformations during flight demonstrates a general morphological computing principle applicable across sensory systems in nature as well as engineered systems. To characterize how relevant features are encoded, we measured and modelled displacement and strain across dragonfly wing surfaces in tethered and free flight. Functional interpretations were supported by neuroanatomical maps, and ablation and perturbation experiments. We find that signal redundancy is reduced by non-random sensor distributions and that morphology limits the stimulus space such that sensory systems can monitor natural states with few sensors. Deviations from the natural states are detected by a flexible population of additional sensors with many distinguishable activation patterns.

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    06/01/24 | A fluorogenic complementation tool kit for interrogating lipid droplet-organelle interaction
    Xiao Li , Rico Gamuyao , Ming-Lun Wu , Woo Jung Cho , Nathan B. Kurtz , Sharon V. King , R.A. Petersen , Daniel R. Stabley , Caleb Lindow , Leslie Climer , Abbas Shirinifard , Francesca Ferrara , Robert E. Throm , Camenzind G. Robinson , Alex Carisey , Alison G. Tebo , Chi-Lun Chang
    J. Cell Biol.. 2024 Jul 01;223(9):e202311126. doi: 10.1083/jcb.202311126

    Contact sites between lipid droplets and other organelles are essential for cellular lipid and energy homeostasis upon metabolic demands. Detection of these contact sites at the nanometer scale over time in living cells is challenging. We developed a tool kit for detecting contact sites based on fluorogen-activated bimolecular complementation at CONtact sites, FABCON, using a reversible, low-affinity split fluorescent protein, splitFAST. FABCON labels contact sites with minimal perturbation to organelle interaction. Via FABCON, we quantitatively demonstrated that endoplasmic reticulum (ER)- and mitochondria (mito)-lipid droplet contact sites are dynamic foci in distinct metabolic conditions, such as during lipid droplet biogenesis and consumption. An automated analysis pipeline further classified individual contact sites into distinct subgroups based on size, likely reflecting differential regulation and function. Moreover, FABCON is generalizable to visualize a repertoire of organelle contact sites including ER-mito. Altogether, FABCON reveals insights into the dynamic regulation of lipid droplet-organelle contact sites and generates new hypotheses for further mechanistical interrogation during metabolic regulation.

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    06/01/24 | It only takes seconds for a human monoclonal autoantibody to inhibit N-methyl-D-aspartate receptors
    Yang S, Heckmann J, Taha A, Gao S, Steinke S, Hust M, Prüß H, Furukawa H, Geis C, Heckmann M, Yu-Strzelczyk J
    bioRxiv. 2024 Jun 01:. doi: 10.1101/2024.05.28.595700

    Transfer of autoantibodies targeting ionotropic N-methyl-D-aspartate receptors in autoimmune encephalitis patients into mice leads to typical disease signs. Long-term effects of the pathogenic antibodies consist of immunoglobulin G-induced crosslinking and receptor internalization. We focused on the direct and immediate impact of a specific pathogenic patient-derived monoclonal autoantibody (immunoglobulin G #003-102) on receptor function.We performed cell-attached recordings in cells transfected with the GluN1 and GluN2A subunit of the N-methyl-D-aspartate receptor. Immunoglobulin G #003-102 binds to the amino-terminal domain of the glycine-binding GluN1 subunit. It reduced simultaneous receptor openings significantly compared to controls at both low and high glutamate and glycine concentrations. Closer examination of our data in 50-second to 2-second intervals revealed, that Immunoglobulin G #003-102 rapidly decreases the number of open receptors. However, antigen-binding fragments of immunoglobulin G #003-102 did not reduce the receptor openings.In conclusion, patient-derived immunoglobulin G #003-102 inhibits N-methyl-D-aspartate receptors rapidly and directly before receptor internalization occurs and the entire immunoglobulin G is necessary for this acute inhibitory effect. This suggests an application of the antigen-binding fragment-like constructs of #003-102 as a potential new treatment strategy for shielding the pathogenic epitopes on the N-methyl-D-aspartate receptors.

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