Long-term imaging of the photosensitive, reef-building coral Acropora muricata using light-sheet illumination

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Laissue, P. P., Roberson, L., Gu, Y., Qian, C., & Smith, D. J. Long-term imaging of the photosensitive, reef-building coral Acropora muricata using light-sheet illumination. Scientific Reports, 10(1), (2020):10369, doi:10.1038/s41598-020-67144-w.Coral reefs are in alarming decline due to climate emergency, pollution and other man-made disturbances. The numerous ecosystem services derived from coral reefs are underpinned by the growth and physical complexity of reef-forming corals. Our knowledge of their fundamental biology is limited by available technology. We need a better understanding of larval settlement and development, skeletogenesis, interactions with pathogens and symbionts, and how this biology interacts with environmental factors such as light exposure, temperature, and ocean acidification. We here focus on a fast-growing key coloniser, Acropora muricata (Linnaeus, 1758). To enable dynamic imaging of this photosensitive organism at different scales, we developed light-sheet illumination for fluorescence microscopy of small coral colonies. Our approach reveals live polyps in previously unseen detail. An imaging range for Acropora muricata with no measurable photodamage is defined based upon polyp expansion, coral tissue reaction, and photobleaching. We quantify polyp retraction as a photosensitive behavioural response and show coral tissue rupture at higher irradiance with blue light. The simple and flexible technique enables non-invasive continuous dynamic imaging of highly photosensitive organisms with sizes between 1 mm3 and 5 cm3, for eight hours, at high temporal resolution, on a scale from multiple polyps down to cellular resolution. This live imaging tool opens a new window into the dynamics of reef-building corals.This work was made possible through a Royal Society Research Grant [RG120274], an innovation voucher from the University of Essex [DBF6000], a Royal Society Industry Fellowship [IF150018] and two Whitman Center Fellowships from the Marine Biological Laboratory (Woods Hole, USA) to PPL. PPL would like to thank Russell Smart for aquarium maintenance and Tony Jordan for production of customised parts. PPL also thanks the open-source communities OpenSPIM and µManager for support, as well as Cairn Research, 89North, Nikon Instruments UK, Alex Gardiol from Olympus Keymed UK, and Scott Young, Matt Preston and Daniel Croucher from Teledyne Photometrics for equipment loans. PPL is grateful to Amy Gladfelter, Hari Shroff, Abhishek Kumar, Louis Kerr, Philip M. Mullineaux, Marino Exposito-Rodriguez and Jean A. Laissue for support and critical discussions

Mineral formation in the primary polyps of pocilloporoid corals

In reef-building corals, larval settlement and its rapid calcification provides a unique opportunity to study the bio-calcium carbonate formation mechanism involving skeleton morphological changes. Here we investigate the mineral formation of primary polyps, just after settlement, in two species of the pocilloporoid corals: Stylophora pistillata (Esper, 1797) and Pocillopora acuta (Lamarck, 1816). We show that the initial mineral phase is nascent Mg-Calcite, with rod-like morphology in P. acuta, and dumbbell morphology in S. pistillata. These structures constitute the first layer of the basal plate which is comparable to Rapid Accretion Deposits (Centers of Calcification, CoC) in adult coral skeleton. We found also that the rod-like/dumbbell Mg-Calcite structures in subsequent growth step will merge into larger aggregates by deposition of aragonite needles. Our results suggest that a biologically controlled mineralization of initial skeletal deposits occurs in three steps: first, vesicles filled with divalent ions are formed intracellularly. These vesicles are then transferred to the calcification site, forming nascent Mg-Calcite rod/pristine dumbbell structures. During the third step, aragonite crystals develop between these structures forming spherulite-like aggregates

ROS-dependent signaling pathways in plants and algae exposed to high light: Comparisons with other eukaryotes

Abstract Like all aerobic organisms, plants and algae co-opt reactive oxygen species (ROS) as signaling molecules to drive cellular responses to changes in their environment. In this respect, there is considerable commonality between all eukaryotes imposed by the constraints of ROS chemistry, similar metabolism in many subcellular compartments, the requirement for a high degree of signal specificity and the deployment of thiol peroxidases as transducers of oxidizing equivalents to regulatory proteins. Nevertheless, plants and algae carry out specialised signaling arising from oxygenic photosynthesis in chloroplasts and photoautotropism, which often induce an imbalance between absorption of light energy and the capacity to use it productively. A key means of responding to this imbalance is through communication of chloroplasts with the nucleus to adjust cellular metabolism. Two ROS, singlet oxygen (1O2) and hydrogen peroxide (H2O2), initiate distinct signaling pathways when photosynthesis is perturbed. 1O2, because of its potent reactivity means that it initiates but does not transduce signaling. In contrast, the lower reactivity of H2O2 means that it can also be a mobile messenger in a spatially-defined signaling pathway. How plants translate a H2O2 message to bring about changes in gene expression is unknown and therefore, we draw on information from other eukaryotes to propose a working hypothesis. The role of these ROS generated in other subcellular compartments of plant cells in response to HL is critically considered alongside other eukaryotes. Finally, the responses of animal cells to oxidative stress upon high irradiance exposure is considered for new comparisons between plant and animal cells

Morphogenesis of a filamentous fungus : dynamics of the actin cytoskeleton and control of hyphal integrity in "Ashbya gossypii"

PART I - THE DYNAMIC ACTIN CYTOSKELETON OF ASHBYA GOSSYPII: Polarized growth is an intriguing aspect in a continuously elongating organism like A. gossypii. We therefore attempted a detailed study of the live actin cytoskeleton in this model filamentous fungus. We analyse the different components of the actin cytoskeleton tagged with Green Fluorescent Protein (GFP) by means of rapid, multi-dimensional video microscopy, studying their structural and dynamic properties. Cap1p and Cap2p are the subunits making up capping protein, a heterodimer which binds the barbed end of actin filaments. GFP-labelled variants of each were studied. Cap1-GFP and Cap2-GFP colocalize with actin patches in rhodamine-phalloidin stainings. They are highly enriched in the first six micrometers from the tip, mostly cortical, and at sites of septation and branch formation. Cap1p-GFP and Cap2p-GFP patches moved at 224 (+-98) nm/s over distances of 0.8 μm (+/-0.7μm) and generally had a lifetime of 14 seconds ((+/-6.5). Sequential recordings of the entire hypha were analysed, suggesting that these particles undergo a pattern of movement consistent with their role in endocytosis. That is, following an initial non-motile stage, actin patches undergo random movement near their site of formation, often followed by a secondary, linear retrograde movement away from the tip. Co-stainings with the endocytosis marker FM4-64 show partial colocalization, further supporting the notion that actin patches are involved in endocytosis. A second movement type is that of retrograde patches returning to the tip, resulting in a cycling pattern. This suggests maintenance of polarization by endocytic recycling, a mechanism which was corroborated by experiments concerning lateral diffusion in the apical membrane. Application of Latrunculin A results in depolarized, spherical tips. The combination of these results suggests that apart from their role in endocytosis, Cap-GFP patches are charged with the task of maintaining polarization by endocytic recycling. Actin cables and actin rings were made visible by using a GFP tagged variant of Abp140p, an F-actin binding and crosslinking protein. Abp140p-GFP colocalizes fully with actin cables, actin patches and actin rings in rhodamine phalloidin stainings. Abp140p-GFP cables are mostly cortical, often helical, can be as long as 40μm and are highly motile. The different fluorescent intensities indicate existence of actin bundles with different numbers of cables. Elongation of the tip of a cable is 184 (+/-62) nm/s. Fine cables in the apical zone often feature Abp140p-GFP patches moving to the tip, where they desintegrate. This is strongly reminiscent of the short, straight actin cables in S. cerevisiae, which have been shown to transport exocytic vesicles to the site where a new cell wall is formed. We conclude with a model of the hyphal organisation of the actin cytoskeleton in A. gossypii. PART II - FAR11P IS REQUIRED TO PREVENT PREMATURE HYPHAL ABSCISSION IN THE FILAMENTOUS FUNGUS ASHBYA GOSSYPII: AgFar11p belongs to the Far proteins which have diverse functions. In the budding yeast Saccharomyces cerevisiae, the syntenic homolog ScFar11p links pheromone response to the cell cycle. In the filamentous fungus Neurospora crassa, the Far11p homolog (NcHAM-2) is required for hyphal fusion. While this process is important for communication and homeostasis in filamentous fungi, it has not been observed in A.gossypii. We investigated the structure and role of AgFar11p. It is a putative transmembrane protein and bears conserved domains found in the homologs of S.cerevisiae and N.crassa. Deletion of the FAR11 gene in Ashbya gossypii leads to premature hyphal abscission at septa and lysis of hyphal compartments. This chain of events occurs in wild type only at the end of the life cycle, when spores are released from hyphal compartments. We conclude that hyphal abscission in far11Δ strains is premature and suggest that in A.gossypii, Far11p is involved in the timing of sporangium formation

Three-dimensional visualization and quantification of lipids in microalgae using confocal laser scanning microscopy

Fluorescence microscopy and digital imaging allow the selective visualisation and quantification of cellular components and can convey research findings in an appealing and intuitive way. These techniques are regularly used in biomedical research laboratories, but have less widespread application in marine sciences. We present here an approach to label and volumetrically quantify neutral lipids, chloroplasts, DNA and cell volumes in microalgae. Using fluorescence microscopy techniques on “turn-key” systems commonly available to environmental research labs, imaging facilities or accessible groups in other disciplines ensure that this approach can be widely reproduced

From function to shape - a novel role of a formin in morphogenesis of the fungus Ashbya gossypii

Morphogenesis of filamentous ascomycetes includes continuously elongating hyphae, frequently emerging lateral branches, and, under certain circumstances, symmetrically dividing hyphal tips. We identified the formin AgBni1p of the model fungus Ashbya gossypii as an essential factor in these processes. AgBni1p is an essential protein apparently lacking functional overlaps with the two additional A. gossypii formins that are nonessential. Agbni1 null mutants fail to develop hyphae and instead expand to potato-shaped giant cells, which lack actin cables and thus tip-directed transport of secretory vesicles. Consistent with the essential role in hyphal development, AgBni1p locates to tips, but not to septa. The presence of a diaphanous autoregulatory domain (DAD) indicates that the activation of AgBni1p depends on Rho-type GTPases. Deletion of this domain, which should render AgBni1p constitutively active, completely changes the branching pattern of young hyphae. New axes of polarity are no longer established subapically (lateral branching) but by symmetric divisions of hyphal tips (tip splitting). In wild-type hyphae, tip splitting is induced much later and only at much higher elongation speed. When GTP-locked Rho-type GTPases were tested, only the young hyphae with mutated AgCdc42p split at their tips, similar to the DAD deletion mutant. Two-hybrid experiments confirmed that AgBni1p interacts with GTP-bound AgCdc42p. These data suggest a pathway for transforming one axis into two new axes of polar growth, in which an increased activation of AgBni1p by a pulse of activated AgCdc42p stimulates additional actin cable formation and tip-directed vesicle transport, thus enlarging and ultimately splitting the polarity site

Tolerance of Normal Rabbit Facial Bones and Teeth to Synchrotron X-Ray Microbeam Irradiation

Microbeam radiation therapy, an alternative radiosurgical treatment under preclinical investigation, aims to safely treat muzzle tumors in pet animals. This will require data on the largely unknown radiation toxicity of microbeam arrays for bones and teeth. To this end, the muzzle of six young adult New Zealand rabbits was irradiated by a lateral array of microplanar beamlets with peak entrance doses of 200, 330 or 500 Gy. The muzzles were examined 431 days postirradiation by computed microtomographic imaging (micro-CT) ex vivo, and extensive histopathology. The boundaries of the radiation field were identified histologically by microbeam tracks in cartilage and other tissues. There was no radionecrosis of facial bones in any rabbit. Conversely, normal incisor teeth exposed to peak entrance doses of 330 Gy or 500 Gy developed marked caries-like damage, whereas the incisors of the two rabbits exposed to 200 Gy remained unscathed. A single, unidirectional array of microbeams with a peak entrance dose ≤200 Gy (valley dose14 Gy) did not damage normal bone, teeth and soft tissues of the muzzle of normal rabbits longer than one year after irradiation. Because of that, Microbeam radiation therapy of muzzle tumors in pet animals is unlikely to cause sizeable damage to normal teeth, bone and soft tissues, if a single array as used here delivers a limited entrance dose of 200 Gy and a valley dose of ≤14 Gy

Quantified Colocalization Reveals Heterotypic Histocompatibility Class I Antigen Associations on Trophoblast Cell Membranes: Relevance for Human Pregnancy1

Human placental syncytiotrophoblasts lack expression of most types of human leukocyte antigen (HLA) class I and class II molecules; this is thought to contribute to a successful pregnancy. However, the HLA class Ib antigens HLA-G, -E, and -F and the HLA class Ia antigen HLA-C are selectively expressed on extravillous trophoblast cells, and they are thought to play a major role in controlling feto-maternal tolerance. We have hypothesized that selective expression, coupled with the preferential physical association of pairs of HLA molecules, contribute to the function of HLA at the feto-maternal interface and the maternal recognition of the fetus. We have developed a unique analytical model that allows detection and quantification of the heterotypic physical associations of HLA class I molecules expressed on the membrane of human trophoblast choriocarcinoma cells, ACH-3P and JEG-3. Automated image analysis was used to estimate the degree of overlap of HLA molecules labeled with different fluorochromes. This approach yields an accurate measurement of the degree of colocalization. In both JEG-3 and ACH-3P cells, HLA-C, -E, and -G were detected on the cell membrane, while the expression of HLA-F was restricted to the cytoplasm. Progesterone treatment alone induced a significant increase in the expression level of the HLA-G/HLA-E association, suggesting that this heterotypic association is modulated by this hormone. Our data shows that the cell-surface HLA class I molecules HLA-G, -E, and -C colocalize with each other and have the potential to form preferential heterotypic association

A novel method for quantified, superresolved, three-dimensional colocalisation of isotropic, fluorescent particles

Colocalisation, the overlap of subcellular structures labelled with different colours, is a key step to characterise cellular phenotypes. We have developed a novel bioimage informatics approach for quantifying colocalisation of round, blob-like structures in two-colour, highly resolved, three-dimensional fluorescence microscopy datasets. First, the algorithm identifies isotropic fluorescent particles, of relative brightness compared to their immediate neighbourhood, in three dimensions and for each colour. The centroids of these spots are then determined, and each object in one location of a colour image is checked for a corresponding object in the other colour image. Three-dimensional distance maps between the centroids of differently coloured spots then display where and how closely they colocalise, while histograms allow to analyse all colocalisation distances. We use the method to reveal sparse colocalisation of different human leukocyte antigen receptors in choriocarcinoma cells. It can also be applied to other isotropic subcellular structures such as vesicles, aggresomes and chloroplasts. The simple, robust and fast approach yields superresolved, object-based colocalisation maps and provides a first indication of protein–protein interactions of fluorescent, isotropic particles