Biology subjects

Vaughan, J. C.

Publications and source records attributed to Vaughan, J. C..

10 recordsLinked to original sources

bioRxiv2026

A Human Precision Cut Kidney Slice Platform for Studying Glomerular Biology and Therapeutic Responses

Introduction: Short term human precision cut kidney slice cultures (PCKS) represent a tractable experimental that contains mature kidney cell types within their native anatomic context and that can be manipulated in vitro. In this study, we employ genomic tools to analyze the PCKS response to culture conditions and their suitability for study of glomerular disease biology. Methods: The impact of culture medium and oxygenation on PCKS viability and gene expression was explored using spatial transcriptomics and traditional histologic approaches. The biological response of PCKS to IFN{gamma} stimulation and signaling suppression by the JAK1/2 inhibitor baricitinib were also explored. Results: A genome wide atlas of glomerular and tubulointerstitial gene expression patterns was generated from PCKS cultured in different media and oxygenation conditions and at various timepoints in culture. IFN{gamma} induced APOL1 gene and protein expression in glomeruli, which could be suppressed by baricitinib. Expression changes were reproducible in PCKS derived from multiple donors. Conclusions: PCKS are reproducibly responsive to IFN{gamma} stimulation, which will allow dissection of APOL1 mediated glomerular disease mechanisms in a human tissue model system. Spatial transcriptomic profiling of tissue responses helps to tailor culture medium/oxygenation to optimize the PCKS system to address specific biological questions.

cell biology↗

bioRxiv2026

High-throughput, organ-scale 3D tubule tracking using TubuleMAP

Advances in tissue clearing and lightsheet microscopy enable mesoscale imaging of intact and convoluted tubular networks, yet analytical tools to map tubule continuity and assess injury patterns within and across tubules are limited. Here, we introduce TubuleMAP, a semi-automated pipeline for 3D tubule tracking and reconstruction that adapts to various morphological and staining patterns, leverages parallel processing of terabyte-scale data for large-scale analysis of tubular networks, and uses a napari interface for human oversight. Using TubuleMAP, we reconstruct 1,000 intact mouse nephrons in [~]1-millimeter-thick kidney slab with [~]400-fold higher throughput and <1% human effort compared to prior approaches. These reconstructions enable analysis of mesoscale nephron organization, quantitative profiling of pathologic morphologies, whole-nephron cytometry, and identification of rare morphologies at unprecedented scales. We demonstrate generalizability by reconstructing all seminiferous tubules in a mouse testis within a day. TubuleMAP is released as an open-source Python package.

bioengineering↗

bioRxiv2024

Nanoscale Optical Imaging, Reconstruction, and Spatial Analysis of Whole Mouse Glomeruli

Renal glomeruli have traditionally been studied by micrometer-scale optical microscopy to interrogate overall physiology or molecular distributions and by nanoscale electron microscopy to interrogate the ultrastructure of thin sections. While these approaches are powerful, they have been limited in their ability to obtain detailed views of the glomeruli as holistic 3D functional units. To fill this knowledge gap, we have developed a novel pipeline for imaging, reconstructing, and analyzing whole mouse glomeruli at 100 nm resolution using super-resolution fluorescence microscopy. This pipeline integrates both manual and machine learning approaches to annotate and analyze glomerular structures. Using this method, we created 18 detailed glomerulus models, from a range of healthy, aged, and model diseased mice, that outline all major structures and cell types. These models have been made publicly accessible in an online repository, providing a valuable resource for further studies. Our results also uncovered a diverse set of novel phenotypes including nuclear enlargement in all glomerular cell types in aging and disease, as well as an aging-related pattern of regional thickening of the Bowmans capsule basement membrane near the tubular-glomerular junction.

physiology↗

bioRxiv2023

Highly Multiplexed Fluorescence Microscopy with Spectrally Tunable Semiconducting Polymer Dots

Current studies of biological tissues require visualizing diverse cell types and molecular interactions, creating a growing need for versatile techniques to simultaneously probe numerous targets. Traditional multiplexed imaging is limited to around five targets at once. Emerging methods utilizing sequential rounds of staining, imaging, and signal removal can probe tens of targets but require specialized hardware, time-consuming workflows, and face some challenges with sample distortion and artifacts. Here we present a new method for highly-multiplexed fluorescence microscopy using semiconducting polymer dots (Pdots) in a single round of staining and imaging. Pdots are small, bright, and photostable fluorescent probes with a wide range of tunable Stokes shifts (20-450 nm). Multiple series of Pdots with varying excitation wavelengths allow for fast (<1 minute) and single-round imaging of up to 21 targets in brain and kidney. This method is based on a simple immunofluorescence workflow, efficient use of spectral space, standard hardware, and straightforward analysis, making it widely applicable for bioimaging laboratories.

biochemistry↗

bioRxiv2023

An end-to-end workflow for non-destructive 3D pathology

Recent advances in 3D pathology offer the ability to image orders-of-magnitude more tissue than conventional pathology while providing a volumetric context that is lacking with 2D tissue sections, all without requiring destructive tissue sectioning. Generating high-quality 3D pathology datasets on a consistent basis is non-trivial, requiring careful attention to many details regarding tissue preparation, imaging, and data/image processing in an iterative process. Here we provide an end-to-end protocol covering all aspects of a 3D pathology workflow (using light-sheet microscopy as an illustrative imaging platform) with sufficient detail to perform well-controlled preclinical and clinical studies. While 3D pathology is compatible with diverse staining protocols and computationally generated color palettes for visual analysis, this protocol will focus on a fluorescent analog of hematoxylin and eosin (H&E), which remains the most common stain for gold-standard diagnostic determinations. We present our guidelines for a broad range of end-users (e.g., biologists, clinical researchers, and engineers) in a simple tutorial format.

pathology↗

bioRxiv2021

Versatile, do-it-yourself, low-cost spinning disk confocal microscope

Confocal microscopy is an invaluable tool for 3D imaging of biological specimens, however, accessibility is often limited to core facilities due to the high cost of the hardware. We describe an inexpensive do-it-yourself (DIY) spinning disk confocal microscope (SDCM) module based on a commercially fabricated chromium photomask that can be added on to a laser-illuminated epifluorescence microscope. The SDCM achieves strong performance across a wide wavelength range (~400-800 nm) as demonstrated through a series of biological imaging applications that include conventional microscopy (immunofluorescence, small-molecule stains, and fluorescence in situ hybridization) and super-resolution microscopy (single-molecule localization microscopy and expansion microscopy). This low-cost and simple DIY SDCM is well-documented and should help increase accessibility to confocal microscopy for researchers.

biophysics↗

bioRxiv2021

The Giardia lamellipodium-like ventrolateral flange supports attachment and rapid cytokinesis

Attachment to the intestinal epithelium is critical to the lifestyle of the ubiquitous parasite Giardia lamblia. The microtubule cytoskeleton plays a well characterized role in attachment via the ventral adhesive disc, whereas the role of the unconventional actin cytoskeleton is controversial. We identified a novel actin associated protein with putative WH2-like actin binding domains we named Flangin. Flangin complexes with Giardia actin and is enriched in the ventrolateral flange (VLF), a lamellipodium-like membrane protrusion at the interface between parasites and attached surfaces. Live imaging revealed that the VLF grows to ~1 m in width after cytokinesis, then remains size-uniform in interphase, grows during mitosis, and is resorbed during cytokinesis. A Flangin truncation mutant stabilizes the VLF and blocks cytokinesis, indicating that the VLF is a membrane reservoir supporting rapid myosin-independent cytokinesis in Giardia. Rho family GTPases are important regulators of membrane protrusions, GlRac, the sole Rho family GTPase in Giardia, was localized to the VLF. Knockdown of Flangin, actin, and GlRac result in VLF formation defects indicating a conserved role for GlRac and actin in forming membrane protrusions, despite the absence of canonical actin binding proteins that link Rho GTPase signaling to lamellipodia formation. Flangin-depleted parasites challenged with fluid shear force in flow chambers had a reduced ability to remain attached, indicating a role for the VLF in attachment. This secondary attachment mechanism complements the microtubule based adhesive ventral disc, a feature that is particularly important during mitosis when the parental ventral disc begins disassembly in preparation for cytokinesis. ImportanceThe ventrolateral flange (VLF) is a lamellipodium-like structure found at the host-parasite interface that has long been thought to be involved in parasite attachment. The proteins responsible for building the VLF have remained unidentified precluding manipulation of the VLF to determine its role in Giardia biology. We identified Flangin, a novel actin associated protein that localizes to the VLF, implicating Giardia actin in VLF formation. We demonstrate that: 1.) Flangin, actin, and GlRac are required for VLF formation, 2.) the VLF serves as a membrane reservoir to support Giardias incredibly fast cytokinesis, and 3) the VLF augments attachment, which is critical to parasitism. The microtubule-based adhesive ventral disc and the actin-based ventrolateral flange represent redundant means of maintaining attachment, the presence of redundant systems illustrate the importance of attachment to the lifestyle of this ubiquitous parasite.

cell biology↗

bioRxiv2020

Multiplexed single-cell profiling of chromatin states at genomic loci by expansion microscopy

Proper regulation of genome architecture and activity is essential for the development and function of multicellular organisms. Histone modifications, acting in combination, specify these activity states at individual genomic loci. However, the methods used to study these modifications often require either a large number of cells or are limited to targeting one histone mark at a time. Here, we developed a new method called Single Cell Evaluation of Post-TRanslational Epigenetic Encoding (SCEPTRE) that uses Expansion Microscopy (ExM) to visualize and quantify multiple histone modifications at non-repetitive genomic regions in single cells at a spatial resolution of ~75 nm. Using SCEPTRE, we distinguished multiple histone modifications at a single housekeeping gene, quantified histone modification levels at multiple developmentally-regulated genes in individual cells, and identified a relationship between histone H3K4 trimethylation and the loading of paused RNA polymerase II at individual loci. Thus, SCEPTRE enables multiplexed detection of combinatorial chromatin states at single genomic loci in single cells.

genomics↗

bioRxiv2019

Multi-immersion open-top light-sheet microscope for high-throughput imaging of cleared tissues

Recent advances in optical clearing and light-sheet microscopy have provided unprecedented access to structural and molecular information from intact tissues. However, current light-sheet microscopes have imposed constraints on the size, shape, number of specimens, and compatibility with various clearing protocols. Here we present a multi-immersion open-top light-sheet microscope that enables simple mounting of multiple specimens processed with a variety of protocols, which will facilitate wider adoption by preclinical researchers and clinical laboratories. One Sentence SummaryGlaser et al. describe a multi-immersion open-top light-sheet microscope that enables simple and high-throughput imaging of large numbers of preclinical and clinical specimens prepared with a variety of clearing protocols.

bioengineering↗

bioRxiv2018

A conserved morphogenetic mechanism for epidermal ensheathment of nociceptive sensory neurites

Interactions between epithelial cells and neurons influence a range of sensory modalities including taste, touch, and smell. Vertebrate and invertebrate keratinocytes/keratinocyte-like epidermal cells ensheath peripheral arbors of somatosensory neurons, including nociceptors, yet the developmental origins and functional roles of this ensheathment are largely unknown. Here, we describe an evolutionarily conserved morphogenetic mechanism for epidermal ensheathment of somatosensory neurites. We found that somatosensory neurons in Drosophila and zebrafish induce formation of epidermal sheaths, which wrap neurites of different types of neurons to different extents. Neurites induce formation of plasma membrane phosphatidylinositol 4,5-bisphosphate microdomains at nascent sheaths, followed by a filamentous actin network, and recruitment of junctional proteins that likely form autotypic junctions to seal sheaths. Finally, blocking epidermal sheath formation destabilized dendrite branches and reduced nociceptive sensitivity in Drosophila. Epidermal somatosensory neurite ensheathment is thus a deeply conserved cellular process that contributes to the morphogenesis and function of nociceptive sensory neurons.

neuroscience↗