Structure-function properties of the gastrodigestive and hepatic systems of zebrafish (Danio rerio)

While they lack mammal-specific organs, zebrafish provide a high degree of resemblance in their genetic profile, molecular mechanisms and organ physiology to humans and have been established as an excellent complementary platform to rodents. However, their use in gastroenterology and hepatology is under-utilised, conceivably due to a lack of digestive system ultrastructural details as most anatomical studies were performed by light and fluorescence imaging. This thesis provides detailed insights into the structure and function of the zebrafish digestive system, particularly the liver. Multimodal bio-imaging approaches were developed in order to investigate the hepatic ultrastructure and function. Using a protocol that renders samples compatible with multiple imaging platforms, we produce a detailed map of the zebrafish gastrodigestive system from organ to subcellular levels. Findings were compared with the rodent/human counterparts and while some differences exist between the zebrafish and the rodent/human hepatic parenchymal cells and biliary system organisations, many similarities, at the sub/cellular levels, were also demonstrated. Using advances in genetics and a protocol that retains endogenous fluorescence within zebrafish at the same time as ultrastructure for electron microscopy, we further investigated key hepatic functional properties (e.g. macromolecular transport routes) by performing albumin injections and studying the liver macrophages. While we demonstrated similarities in the albumin uptake pathway and in the morphology of liver macrophages in zebrafish, we reveal that zebrafish liver macrophages lack of phagocytic function (a key aspect in rodents and human), which may limit their use in hepatic-immune diseases studies. Altogether, our studies provide new insights and novel protocols for the analysis of the zebrafish liver and lay a foundation to further evaluate uptake routes for gastro-digestive research and drug delivery in various diseases

A36-dependent Actin Filament Nucleation Promotes Release of Vaccinia Virus

Cell-to-cell transmission of vaccinia virus can be mediated by enveloped virions that remain attached to the outer surface of the cell or those released into the medium. During egress, the outer membrane of the double-enveloped virus fuses with the plasma membrane leaving extracellular virus attached to the cell surface via viral envelope proteins. Here we report that F-actin nucleation by the viral protein A36 promotes the disengagement of virus attachment and release of enveloped virus. Cells infected with the A36(YdF) virus, which has mutations at two critical tyrosine residues abrogating localised actin nucleation, displayed a 10-fold reduction in virus release. We examined A36(YdF) infected cells by transmission electron microscopy and observed that during release, virus appeared trapped in small invaginations at the plasma membrane. To further characterise the mechanism by which actin nucleation drives the dissociation of enveloped virus from the cell surface, we examined recombinant viruses by super-resolution microscopy. Fluorescently-tagged A36 was visualised at sub-viral resolution to image cell-virus attachment in mutant and parental backgrounds. We confirmed that A36(YdF) extracellular virus remained closely associated to the plasma membrane in small membrane pits. Virus-induced actin nucleation reduced the extent of association, thereby promoting the untethering of virus from the cell surface. Virus release can be enhanced via a point mutation in the luminal region of B5 (P189S), another virus envelope protein. We found that the B5(P189S) mutation led to reduced contact between extracellular virus and the host membrane during release, even in the absence of virus-induced actin nucleation. Our results posit that during release virus is tightly tethered to the host cell through interactions mediated by viral envelope proteins. Untethering of virus into the surrounding extracellular space requires these interactions be relieved, either through the force of actin nucleation or by mutations in luminal proteins that weaken these interactions.This work was outlined and supported by Project Grant #632785 of the National Health and Medical Research Council of Australia and The Australian Research Council Federation Discovery Project #1096623. CBW was supported by a National Health and Medical Research Council of Australia Senior Research Fellowship #571905. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Ultrastructural mapping of the zebrafish gastrointestinal system as a basis for experimental drug studies

Research in the field of gastroenterology is increasingly focused on the use of alternative nonrodent model organisms to provide new experimental tools to study chronic diseases. The zebrafish is a particularly valuable experimental platform to explore organ and cell structure-function relationships under relevant biological and pathobiological settings. This is due to its optical transparency and its close-to-human genetic makeup. To-date, the structure-function properties of the GIS of the zebrafish are relatively unexplored and limited to histology and fluorescent microscopy. Occasionally those studies include EM of a given subcellular process but lack the required full histological picture. In this work, we employed a novel combined biomolecular imaging approach in order to cross-correlate 3D ultrastructure over different length scales (optical-, X-ray micro-CT, and high-resolution EM). Our correlated imaging studies and subsequent data modelling provide to our knowledge the first detailed 3D picture of the zebrafish larvae GIS. Our results provide unequivocally a limit of confidence for studying various digestive disorders and drug delivery pathways in the zebrafish.13 page(s

3-D EM exploration of the hepatic microarchitecture - lessons learned from large-volume in situ serial sectioning

To-date serial block-face scanning electron microscopy (SBF-SEM) dominates as the premier technique for generating three-dimensional (3-D) data of resin-embedded biological samples at an unprecedented depth volume. Given the infancy of the technique, limited literature is currently available regarding the applicability of SBF-SEM for the ultrastructural investigation of tissues. Herein, we provide a comprehensive and rigorous appraisal of five different SBF-SEM sample preparation protocols for the large-volume exploration of the hepatic microarchitecture at an unparalleled X, Y and Z resolution. In so doing, we qualitatively and quantitatively validate the use of a comprehensive SBF-SEM sample preparation protocol, based on the application of heavy metal fixatives, stains and mordanting agents. Employing the best-tested SBF-SEM approach, enabled us to assess large-volume morphometric data on murine parenchymal cells, sinusoids and bile canaliculi. Finally, we integrated the validated SBF-SEM protocol with a correlative light and electron microscopy (CLEM) approach. The combination of confocal scanning laser microscopy and SBF-SEM provided a novel way to picture subcellular detail. We appreciate that this multidimensional approach will aid the subsequent research of liver tissue under relevant experimental and disease conditions

Redistribution of A36 during virus exit.

<p>(<b>A</b>) 3D-SIM fluorescent micrograph of A36-YFP/B5-RFP infected BSC-1 cell. Virus particles associated with F-actin (red, left panel; visualised with Lifeact-cerulean) show polarisation of A36 (green) whereas B5 (red, right panel) localises to the CEV circumference (close-ups). 3D-SIM fluorescent micrographs of (<b>B</b>) A36-YFP, (<b>C</b>) A36^YdF-YFP in non-permeabilised BSC-1 cells probed with rat anti-B5 primary antibody and Alexafluor 568 anti-rat secondary antibody. Close-ups show A36 (green) localisation in two representative intracellular (no B5 visible) and extracellular virus particles. (<b>D</b>) 3D-SIM fluorescent micrographs of representative intracellular and extracellular B5^P189S/A36-YFP, B5^P189S/A36^YdF-YFP, A34^K151E/A36-YFP or A34^K151E/A36^YdF-YFP viruses in non-permeabilised BSC-1 cells probed with rat anti-B5 primary antibody and Alexafluor 568 anti-rat secondary antibody. All close-ups from larger panels are arranged in order from top to bottom. Scale bar = 5 µm.</p

A second site mutation in B5 restores EEV release in A36^YdF.

<p>(<b>A</b>) Plaque (semi-solid overlay) and comet (liquid overlay) assays of A36^YdF, B5^P189S, B5^P189S/A36^YdF, A34^K151E or A34^K151E/A36^YdF stained at 72 hpi. (<b>B</b>) Levels of infectious EEV in supernatants collected at 16 hpi from BSC-1 cells infected at an MOI of 0.1. Error bars represent s.e.m. from 3 replicate wells in 3 independent experiments. P values of <0.05 and <0.01 are represented by * and **, respectively.</p

Modified N-linked glycosylation status predicts trafficking defective human Piezo1 channel mutations

10.1038/s42003-021-02528-wCommunications Biology41103

Plaque size and release of VACV is inhibited by mutation of Y112 and Y132 residues in A36.

<p>(<b>A</b>) BSC-1 cells were infected with the indicated viruses, incubated with a semi-solid (plaque assay, 1.5% CMC) or liquid (comet assay, DMEM) overlay, with either imatinib or carrier (DMSO), stained at 72 hpi. (<b>B</b>) Average plaque size of WR, A36^Y112F, A36^Y132F and A36^YdF in BSC-1 or (<b>C</b>) NIH 3T3 cells. Error bars represent s.e.m. from 50 plaques. (<b>D</b>) Single-step growth analysis of VACV strains. Monolayers of BSC-1 cells were infected with the indicated viruses at a MOI of 5. At 4, 8, 12 and 24 hpi cells and supernatants were harvested and virus titres were determined by titration with BSC-1s. The average titres of two independent experiments are plotted at each time point. (<b>E</b>) Levels of infectious EEV in supernatants collected 16 hpi from BSC-1 cells or (<b>F</b>) NIH3T3 cells infected at an MOI of 0.1 and overlayed with DMEM containing 0.01% DMSO (unfilled) or 10 µM imatinib (filled). Error bars represent s.e.m. from 3 replicate wells in 3 independent experiments. P values of <0.05, <0.01 and <0.001 are represented by *, ** and ***, respectively.</p

A36^YdF is invaginated at the cell membrane during egress.

<p>Representative transmission electron micrographs of BSC-1 cells infected with (<b>A</b>) WR and (<b>B</b>) A36^YdF at an MOI of 5 and fixed at 9 hpi. In contrast to WR, distinct membrane pits containing virus are apparent during A36^YdF virus egress. Scale bar = 0.2 µm. (<b>C</b>) Average percentage of viral membrane circumference in contact with cell membrane calculated from 14 (WR) or 16 (YdF) viruses randomly selected from the transmission electron micrographs. *** = P value of <0.001.</p