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2508 Publications

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    05/07/24 | YAP condensates are highly organized hubs
    Siyuan Hao , Ye Jin Lee , Nadav Benhamou Goldfajn , Eduardo Flores , Jindayi Liang , Hannah Fuehrer , Justin Demmerle , Jennifer Lippincott-Schwartz , Zhe Liu , Shahar Sukenik , Danfeng Cai
    iScience. 2024 May 07:109927. doi: https://doi.org/10.1016/j.isci.2024.109927

    YAP/TEAD signaling is essential for organismal development, cell proliferation, and cancer progression. As a transcriptional coactivator, how YAP activates its downstream target genes is incompletely understood. YAP forms biomolecular condensates in response to hyperosmotic stress, concentrating transcription-related factors to activate downstream target genes. However, whether YAP forms condensates under other signals, how YAP condensates organize and function, and how YAP condensates activate transcription in general are unknown. Here, we report that endogenous YAP forms sub-micron scale condensates in response to Hippo pathway regulation and actin cytoskeletal tension. YAP condensates are stabilized by the transcription factor TEAD1, and recruit BRD4, a coactivator that is enriched at active enhancers. Using single-particle tracking, we found that YAP condensates slowed YAP diffusion within condensate boundaries, a possible mechanism for promoting YAP target search. These results reveal that YAP condensate formation is a highly regulated process that is critical for YAP/TEAD target gene expression.

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    05/01/24 | A survival-critical role for Drosophila giant interneurons during predation
    Cynthia M. Chai , Carmen Morrow , Dhyey D. Parikh , Catherine R. von Reyn , Anthony Leonardo , Gwyneth M Card
    bioRxiv. 2024 May 1:. doi: 10.1101/2024.04.30.591368

    Large axon-diameter descending neurons are metabolically costly but transmit information rapidly from sensory neurons in the brain to motor neurons in the nerve cord. They have thus endured as a common feature of escape circuits in many animal species where speed is paramount. Though often considered isolated command neurons triggering fast-reaction-time, all-or-none escape responses, giant neurons are just one of multiple parallel pathways enabling selection between behavioral alternatives. Such degeneracy among escape circuits makes it unclear if and how giant neurons benefit prey fitness. Here we competed Drosophila melanogaster flies with genetically-silenced Giant Fibers (GFs) against flies with functional GFs in an arena with wild-caught damselfly predators and find that GF silencing decreases prey survival. Kinematic analysis of damselfly attack trajectories shows that decreased prey survival fitness results from GF-silenced flies failing to escape during predator attack speeds and approach distances that would normally elicit successful escapes. When challenged with a virtual looming predator, fly GFs promote survival by enforcing selection of a short-duration takeoff sequence as opposed to reducing reaction time. Our findings support a role for the GFs in promoting prey survival by influencing action selection as a means to enhance escape performance during realistically complex predation scenarios.

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    04/25/24 | Connectomic Analysis of Mitochondria in the Central Brain of Drosophila
    Patricia K Rivlin , Michal Januszewski , Kit D Longden , Erika Neace , Louis K Scheffer , Christopher Ordish , Jody Clements , Elliott Phillips , Natalie Smith , Satoko Takemura , Lowell Umayam , Claire Walsh , Emily A Yakal , Stephen M Plaza , Stuart Berg
    bioRxiv. 2024 Apr 25:. doi: 10.1101/2024.04.21.590464

    Mitochondria are an integral part of the metabolism of a neuron. EM images of fly brain volumes, taken for connectomics, contain mitochondria as well as the cells and synapses that have already been reported. Here, from the Drosophila hemibrain dataset, we extract, classify, and measure approximately 6 million mitochondria among roughly 21 thousand neurons of more than 5500 cell types. Each mitochondrion is classified by its appearance - dark and dense, light and sparse, or intermediate - and the location, orientation, and size (in voxels) are annotated. These mitochondria are added to our publicly available data portal, and each synapse is linked to its closest mitochondrion. Using this data, we show quantitative evidence that mitochodrial trafficing extends to the smallest dimensions in neurons. The most basic characteristics of mitochondria - volume, distance from synapses, and color - vary considerably between cell types, and between neurons with different neurotransmitters. We find that polyadic synapses with more post-synaptic densities (PSDs) have closer and larger mitochondria on the pre-synaptic side, but smaller and more distant mitochondria on the PSD side. We note that this relationship breaks down for synapses with only one PSD, suggesting a different role for such synapses.Competing Interest StatementThe authors have declared no competing interest.

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    04/25/24 | Expansion of in vitro Toxoplasma gondii cysts using enzymatically enhanced ultrastructure expansion microscopy
    Kseniia Bondarenko , Floriane Limoge , Kayvon Pedram , Mathieu Gissot , Joanna C. Young
    bioRxiv. 2024 Apr 25:. doi: 10.1101/2024.04.24.590991

    Expansion microscopy (ExM) is an innovative approach to achieve super-resolution images without using super-resolution microscopes, based on the physical expansion of the sample. The advent of ExM has unlocked super-resolution imaging for a broader scientific circle, lowering the cost and entry skill requirements to the field. One of its branches, ultrastructure ExM (U-ExM), has become popular among research groups studying Apicomplexan parasites, including the acute stage of Toxoplasma gondii infection. The chronic cyst-forming stage of Toxoplasma, however, resists U-ExM expansion, impeding precise protein localisation. Here, we solve the in vitro cyst’s resistance to denaturation required for successful U-ExM of the encapsulated parasites. As the cyst’s main structural protein CST1 contains a mucin domain, we added an enzymatic digestion step using the pan-mucinase StcE prior to the expansion protocol. This allowed full expansion of the cysts in fibroblasts and primary neuronal cell culture without interference with the epitopes of the cyst-wall associated proteins. Using StcE-enhanced U-ExM, we clarified the shape and location of the GRA2 protein important for establishing a normal cyst. Expanded cysts revealed GRA2 granules spanning across the cyst wall, with a notable presence observed outside on both sides of the CST1-positive layer.

    Importance Toxoplasma gondii is an intracellular parasite capable of establishing long-term chronic infection in nearly all warm-blooded animals. During the chronic stage, parasites encapsulate into cysts in a wide range of tissues but particularly in neurons of the central nervous system and in skeletal muscle. Current anti-Toxoplasma drugs do not eradicate chronic parasites and leave behind a reservoir of infection. As the cyst is critical for both transmission and pathology of the disease, we need to understand more fully the biology of the cyst and its vulnerabilities.

    The advent of a new super-resolution approach called ultrastructure expansion microscopy allowed in-depth studies of the acute stage of Toxoplasma infection but not the cyst-forming stage, which resists protocol-specific denaturation. Here, we show that an additional step of enzymatic digestion using mucinase StcE allows full expansion of the Toxoplasma cysts, offering a new avenue for a comprehensive examination of the chronic stage of infection using an accessible super-resolution technique.

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    04/17/24 | Machine learning reveals the control mechanics of an insect wing hinge
    Melis JM, Siwanowicz I, Dickinson MH
    Nature. 2024 Apr 17;628(8009):795-803. doi: 10.1038/s41586-024-07293-4

    Insects constitute the most species-rich radiation of metazoa, a success that is due to the evolution of active flight. Unlike pterosaurs, birds and bats, the wings of insects did not evolve from legs, but are novel structures that are attached to the body via a biomechanically complex hinge that transforms tiny, high-frequency oscillations of specialized power muscles into the sweeping back-and-forth motion of the wings. The hinge consists of a system of tiny, hardened structures called sclerites that are interconnected to one another via flexible joints and regulated by the activity of specialized control muscles. Here we imaged the activity of these muscles in a fly using a genetically encoded calcium indicator, while simultaneously tracking the three-dimensional motion of the wings with high-speed cameras. Using machine learning, we created a convolutional neural network that accurately predicts wing motion from the activity of the steering muscles, and an encoder-decoder that predicts the role of the individual sclerites on wing motion. By replaying patterns of wing motion on a dynamically scaled robotic fly, we quantified the effects of steering muscle activity on aerodynamic forces. A physics-based simulation incorporating our hinge model generates flight manoeuvres that are remarkably similar to those of free-flying flies. This integrative, multi-disciplinary approach reveals the mechanical control logic of the insect wing hinge, arguably among the most sophisticated and evolutionarily important skeletal structures in the natural world.

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    04/30/24 | Mitochondrially-associated actin waves maintain organelle homeostasis and equitable inheritance.
    Coscia SM, Moore AS, Wong YC, Holzbaur EL
    Curr Opin Cell Biol. 2024 Apr 30;88:102364. doi: 10.1016/j.ceb.2024.102364

    First identified in dividing cells as revolving clusters of actin filaments, these are now understood as mitochondrially-associated actin waves that are active throughout the cell cycle. These waves are formed from the polymerization of actin onto a subset of mitochondria. Within minutes, this F-actin depolymerizes while newly formed actin filaments assemble onto neighboring mitochondria. In interphase, actin waves locally fragment the mitochondrial network, enhancing mitochondrial content mixing to maintain organelle homeostasis. In dividing cells actin waves spatially mix mitochondria in the mother cell to ensure equitable partitioning of these organelles between daughter cells. Progress has been made in understanding the consequences of actin cycling as well as the underlying molecular mechanisms, but many questions remain, and here we review these elements. Also, we draw parallels between mitochondrially-associated actin cycling and cortical actin waves. These dynamic systems highlight the remarkable plasticity of the actin cytoskeleton.

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    04/06/24 | NMDAR-mediated activation of pannexin1 channels contributes to the detonator properties of hippocampal mossy fiber synapses.
    Rangel-Sandoval C, Soula M, Li W, Castillo PE, Hunt DL
    iScience. 2024 Apr 06;27(5):109681. doi: 10.1016/j.isci.2024.109681

    Pannexins are large-pore ion channels expressed throughout the mammalian brain that participate in various neuropathologies; however, their physiological roles remain obscure. Here, we report that pannexin1 channels (Panx1) can be synaptically activated under physiological recording conditions in rodent acute hippocampal slices. Specifically, NMDA receptor (NMDAR)-mediated responses at the mossy fiber to CA3 pyramidal cell synapse were followed by a slow postsynaptic inward current that could activate CA3 pyramidal cells but was absent in Panx1 knockout mice. Immunoelectron microscopy revealed that Panx1 was localized near the postsynaptic density. Further, Panx1-mediated currents were potentiated by metabotropic receptors and bidirectionally modulated by burst-timing-dependent plasticity of NMDAR-mediated transmission. Lastly, Panx1 channels were preferentially recruited when NMDAR activation enters a supralinear regime, resulting in temporally delayed burst-firing. Thus, Panx1 can contribute to synaptic amplification and broadening the temporal associativity window for co-activated pyramidal cells, thereby supporting the auto-associative functions of the CA3 region.

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    Fitzgerald Lab
    04/25/24 | Optimization in Visual Motion Estimation.
    Clark DA, Fitzgerald JE
    Annu Rev Vis Sci. 2024 Apr 25:. doi: 10.1146/annurev-vision-101623-025432

    Sighted animals use visual signals to discern directional motion in their environment. Motion is not directly detected by visual neurons, and it must instead be computed from light signals that vary over space and time. This makes visual motion estimation a near universal neural computation, and decades of research have revealed much about the algorithms and mechanisms that generate directional signals. The idea that sensory systems are optimized for performance in natural environments has deeply impacted this research. In this article, we review the many ways that optimization has been used to quantitatively model visual motion estimation and reveal its underlying principles. We emphasize that no single optimization theory has dominated the literature. Instead, researchers have adeptly incorporated different computational demands and biological constraints that are pertinent to the specific brain system and animal model under study. The successes and failures of the resulting optimization models have thereby provided insights into how computational demands and biological constraints together shape neural computation.

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    05/01/24 | Recognising the importance and impact of Imaging Scientists: Global guidelines for establishing career paths within core facilities
    Wright GD, Thompson KA, Reis Y, Bischof J, Hockberger PE, Itano MS, Yen L, Adelodun ST, Bialy N, Brown CM, Chaabane L, Chew T, Chitty AI, Cordelières FP, De Niz M, Ellenberg J, Engelbrecht L, Fabian-Morales E, Fazeli E, Fernandez-Rodriguez J, Ferrando-May E, Fletcher G, Galloway GJ, Guerrero A, Guimarães JM, Jacobs CA, Jayasinghe S, Kable E, Kitten GT, Komoto S, Ma X, Marques JA, Millis BA, Miranda K, JohnO'Toole P, Olatunji SY, Paina F, Pollak CN, Prats C, Pylvänäinen JW, Rahmoon MA, Reiche MA, Riches JD, Rossi AH, Salamero J, Thiriet C, Terjung S, Vasconcelos AD, Keppler A
    J Microsc. 2024 May 01:. doi: 10.1111/jmi.13307

    In the dynamic landscape of scientific research, imaging core facilities are vital hubs propelling collaboration and innovation at the technology development and dissemination frontier. Here, we present a collaborative effort led by Global BioImaging (GBI), introducing international recommendations geared towards elevating the careers of Imaging Scientists in core facilities. Despite the critical role of Imaging Scientists in modern research ecosystems, challenges persist in recognising their value, aligning performance metrics and providing avenues for career progression and job security. The challenges encompass a mismatch between classic academic career paths and service-oriented roles, resulting in a lack of understanding regarding the value and impact of Imaging Scientists and core facilities and how to evaluate them properly. They further include challenges around sustainability, dedicated training opportunities and the recruitment and retention of talent. Structured across these interrelated sections, the recommendations within this publication aim to propose globally applicable solutions to navigate these challenges. These recommendations apply equally to colleagues working in other core facilities and research institutions through which access to technologies is facilitated and supported. This publication emphasises the pivotal role of Imaging Scientists in advancing research programs and presents a blueprint for fostering their career progression within institutions all around the world.

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    04/22/24 | A Bayesian Solution to Count the Number of Molecules within a Diffraction Limited Spot
    Alexander Hillsley , Johannes Stein , Paul W. Tillberg , David L. Stern , Jan Funke
    bioRxiv. 2024 Apr 22:. doi: 10.1101/2024.04.18.590066

    We address the problem of inferring the number of independently blinking fluorescent light emitters, when only their combined intensity contributions can be observed at each timepoint. This problem occurs regularly in light microscopy of objects that are smaller than the diffraction limit, where one wishes to count the number of fluorescently labelled subunits. Our proposed solution directly models the photo-physics of the system, as well as the blinking kinetics of the fluorescent emitters as a fully differentiable hidden Markov model. Given a trace of intensity over time, our model jointly estimates the parameters of the intensity distribution per emitter, their blinking rates, as well as a posterior distribution of the total number of fluorescent emitters. We show that our model is consistently more accurate and increases the range of countable subunits by a factor of two compared to current state-of-the-art methods, which count based on autocorrelation and blinking frequency, Further-more, we demonstrate that our model can be used to investigate the effect of blinking kinetics on counting ability, and therefore can inform experimental conditions that will maximize counting accuracy.

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