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2673 Janelia Publications

Showing 211-220 of 2673 results
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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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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05/30/24 | Aging atlas reveals cell-type-specific effects of pro-longevity strategies.
Gao SM, Qi Y, Zhang Q, Guan Y, Lee Y, Ding L, Wang L, Mohammed AS, Li H, Fu Y, Wang MC
Nat Aging. 2024 May 30:. doi: 10.1038/s43587-024-00631-1

Organismal aging involves functional declines in both somatic and reproductive tissues. Multiple strategies have been discovered to extend lifespan across species. However, how age-related molecular changes differ among various tissues and how those lifespan-extending strategies slow tissue aging in distinct manners remain unclear. Here we generated the transcriptomic Cell Atlas of Worm Aging (CAWA, http://mengwanglab.org/atlas ) of wild-type and long-lived strains. We discovered cell-specific, age-related molecular and functional signatures across all somatic and germ cell types. We developed transcriptomic aging clocks for different tissues and quantitatively determined how three different pro-longevity strategies slow tissue aging distinctively. Furthermore, through genome-wide profiling of alternative polyadenylation (APA) events in different tissues, we discovered cell-type-specific APA changes during aging and revealed how these changes are differentially affected by the pro-longevity strategies. Together, this study offers fundamental molecular insights into both somatic and reproductive aging and provides a valuable resource for in-depth understanding of the diversity of pro-longevity mechanisms.

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06/04/24 | The physical and cellular mechanism of structural color change in zebrafish.
Gur D, Moore AS, Deis R, Song P, Wu X, Pinkas I, Deo C, Iyer N, Hess HF, Hammer JA, Lippincott-Schwartz J
Proc Natl Acad Sci U S A. 2024 Jun 04;121(23):e2308531121. doi: 10.1073/pnas.2308531121

Many animals exhibit remarkable colors that are produced by the constructive interference of light reflected from arrays of intracellular guanine crystals. These animals can fine-tune their crystal-based structural colors to communicate with each other, regulate body temperature, and create camouflage. While it is known that these changes in color are caused by changes in the angle of the crystal arrays relative to incident light, the cellular machinery that drives color change is not understood. Here, using a combination of 3D focused ion beam scanning electron microscopy (FIB-SEM), micro-focused X-ray diffraction, superresolution fluorescence light microscopy, and pharmacological perturbations, we characterized the dynamics and 3D cellular reorganization of crystal arrays within zebrafish iridophores during norepinephrine (NE)-induced color change. We found that color change results from a coordinated 20° tilting of the intracellular crystals, which alters both crystal packing and the angle at which impinging light hits the crystals. Importantly, addition of the dynein inhibitor dynapyrazole-a completely blocked this NE-induced red shift by hindering crystal dynamics upon NE addition. FIB-SEM and microtubule organizing center (MTOC) mapping showed that microtubules arise from two MTOCs located near the poles of the iridophore and run parallel to, and in between, individual crystals. This suggests that dynein drives crystal angle change in response to NE by binding to the limiting membrane surrounding individual crystals and walking toward microtubule minus ends. Finally, we found that intracellular cAMP regulates the color change process. Together, our results provide mechanistic insight into the cellular machinery that drives structural color change.

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05/23/24 | A neural circuit architecture for rapid learning in goal-directed navigation
Chuntao Dan , Brad K. Hulse , Ramya Kappagantula , Vivek Jayaraman , Ann M. Hermundstad
Neuron. 2024 May 23;112(15):2581-2599.e23. doi: https://doi.org/10.1016/j.neuron.2024.04.036

Anchoring goals to spatial representations enables flexible navigation but is challenging in novel environments when both representations must be acquired simultaneously. We propose a framework for how Drosophila uses internal representations of head direction (HD) to build goal representations upon selective thermal reinforcement. We show that flies use stochastically generated fixations and directed saccades to express heading preferences in an operant visual learning paradigm and that HD neurons are required to modify these preferences based on reinforcement. We used a symmetric visual setting to expose how flies' HD and goal representations co-evolve and how the reliability of these interacting representations impacts behavior. Finally, we describe how rapid learning of new goal headings may rest on a behavioral policy whose parameters are flexible but whose form is genetically encoded in circuit architecture. Such evolutionarily structured architectures, which enable rapidly adaptive behavior driven by internal representations, may be relevant across species.

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05/16/24 | Analysis of developmental gene expression using smFISH and in silico staging of C. elegans embryos
Breimann L, Bahry E, Zouinkhi M, Kolyvanov K, Street LA, Preibisch S, Ercan S
bioRxiv. 2024 May 16:. doi: 10.1101/2024.05.15.594414

Regulation of transcription during embryogenesis is key to development and differentiation. To study transcript expression throughout Caenorhabditis elegans embryogenesis at single-molecule resolution, we developed a high-throughput single-molecule fluorescence in situ hybridization (smFISH) method that relies on computational methods to developmentally stage embryos and quantify individual mRNA molecules in single embryos. We applied our system to sdc-2, a zygotically transcribed gene essential for hermaphrodite development and dosage compensation. We found that sdc-2 is rapidly activated during early embryogenesis by increasing both the number of mRNAs produced per transcription site and the frequency of sites engaged in transcription. Knockdown of sdc-2 and dpy-27, a subunit of the dosage compensation complex (DCC), increased the number of active transcription sites for the X chromosomal gene dpy-23 but not the autosomal gene mdh-1, suggesting that the DCC reduces the frequency of dpy-23 transcription. The temporal resolution from in silico staging of embryos showed that the deletion of a single DCC recruitment element near the dpy-23 gene causes higher dpy-23 mRNA expression after the start of dosage compensation, which could not be resolved using mRNAseq from mixed-stage embryos. In summary, we have established a computational approach to quantify temporal regulation of transcription throughout C. elegans embryogenesis and demonstrated its potential to provide new insights into developmental gene regulation.

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05/20/24 | Astrocyte Calcium Signaling
Ahrens MB, Khakh BS, Poskanzer KE
Cold Spring Harb Perspect Biol. 2024 May 20:. doi: 10.1101/cshperspect.a041353

Astrocytes are predominant glial cells that tile the central nervous system and participate in well-established functional and morphological interactions with neurons, blood vessels, and other glia. These ubiquitous cells display rich intracellular Ca signaling, which has now been studied for over 30 years. In this review, we provide a summary and perspective of recent progress concerning the study of astrocyte intracellular Ca signaling as well as discussion of its potential functions. Progress has occurred in the areas of imaging, silencing, activating, and analyzing astrocyte Ca signals. These insights have collectively permitted exploration of the relationships of astrocyte Ca signals to neural circuit function and behavior in a variety of species. We summarize these aspects along with a framework for mechanistically interpreting behavioral studies to identify directly causal effects. We finish by providing a perspective on new avenues of research concerning astrocyte Ca signaling.

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