Bacteria stretch and bend oil to feed their appetite.

Microbes reshape oil droplets to speed biodegradation.

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.

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

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.

Spatial chloroplast-to-nucleus signalling involving plastid-nuclear complexes and stromules.

Communication between chloroplasts and the nucleus in response to various environmental cues may be mediated by various small molecules. Signalling specificity could be enhanced if the physical contact between these organelles facilitates direct transfer and prevents interference from other subcellular sources of the same molecules. Plant cells have plastid-nuclear complexes, which provide close physical contact between these organelles. Plastid-nuclear complexes have been proposed to facilitate transfer of photosynthesis-derived H2O2 to the nucleus in high light. Stromules (stroma filled tubular plastid extensions) may provide an additional conduit for transfer of a wider range of signalling molecules, including proteins. However, plastid-nuclear complexes and stromules have been hitherto treated as distinct phenomena. We suggest that plastid-nuclear complexes and stromules work in a coordinated manner so that, according to environmental conditions or developmental state, the two modes of connection contribute to varying extents. We hypothesize that this association is dynamic and that there may be a link between plastid-nuclear complexes and the development of stromules. Furthermore, the changes in contact could alter signalling specificity by allowing an extended or different range of signalling molecules to be delivered to the nucleus. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.

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. STATEMENT OF SIGNIFICANCE: Coral settlement and recruitment periods are highly sensitive to environmental conditions. Successful mineralization during these periods is vital and influences the coral's chances of survival. Therefore, understanding the exact mechanism underlying carbonate precipitation is highly important. Here, we used in vivo microscopy, spectroscopy and molecular methods to provide new insights into mineral development. We show that the primary polyp's mineral arsenal consists of two types of minerals: Mg-Calcite and aragonite. In addition, we provide new insights into the ion pathway by showing that divalent ions are concentrated in intracellular vesicles and are eventually deposited at the calcification site.

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

Like all aerobic organisms, plants and algae co-opt reactive oxygen species (ROS) as signalling 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 oxidising equivalents to regulatory proteins. Nevertheless, plants and algae carry out specialised signalling 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 signalling pathways when photosynthesis is perturbed. 1O2, because of its potent reactivity means that it initiates but does not transduce signalling. In contrast, the lower reactivity of H2O2 means that it can also be a mobile messenger in a spatially-defined signalling 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.

Photosynthesis-dependent H2O2 transfer from chloroplasts to nuclei provides a high-light signalling mechanism.

Chloroplasts communicate information by signalling to nuclei during acclimation to fluctuating light. Several potential operating signals originating from chloroplasts have been proposed, but none have been shown to move to nuclei to modulate gene expression. One proposed signal is hydrogen peroxide (H2O2) produced by chloroplasts in a light-dependent manner. Using HyPer2, a genetically encoded fluorescent H2O2 sensor, we show that in photosynthetic Nicotiana benthamiana epidermal cells, exposure to high light increases H2O2 production in chloroplast stroma, cytosol and nuclei. Critically, over-expression of stromal ascorbate peroxidase (H2O2 scavenger) or treatment with DCMU (photosynthesis inhibitor) attenuates nuclear H2O2 accumulation and high light-responsive gene expression. Cytosolic ascorbate peroxidase over-expression has little effect on nuclear H2O2 accumulation and high light-responsive gene expression. This is because the H2O2 derives from a sub-population of chloroplasts closely associated with nuclei. Therefore, direct H2O2 transfer from chloroplasts to nuclei, avoiding the cytosol, enables photosynthetic control over gene expression.Multiple plastid-derived signals have been proposed but not shown to move to the nucleus to promote plant acclimation to fluctuating light. Here the authors use a fluorescent hydrogen peroxide sensor to provide evidence that H2O2 is transferred directly from chloroplasts to nuclei to control nuclear gene expression.

Quantified colocalization reveals heterotypic histocompatibility class I antigen associations on trophoblast cell membranes: relevance for human pregnancy.

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 associations.

The use of HyPer to examine spatial and temporal changes in H2O2 in high light-exposed plants.

Exposure of photosynthetic cells of leaf tissues of Arabidopsis thaliana (Arabidopsis) to high light intensities (HL) may provoke a rapid rise in hydrogen peroxide (H2O2) levels in chloroplasts and subcellular compartments, such as peroxisomes, associated with photosynthetic metabolism. It has been hypothesized that when H2O2 is contained at or near its site of production then it plays an important role in signaling to induce acclimation to HL. However, should this discrete containment fail and H2O2 levels exceed the capacity of antioxidant systems to scavenge them, then oxidative stress ensues which triggers cell death. To test this hypothesis, the spatiotemporal accumulation of H2O2 needs to be quantified in different subcellular compartments. In this chapter, preliminary experiments are presented on the use of Arabidopsis seedlings transformed with a nuclear-encoded cytosol-located yellow fluorescent protein-based sensor for H2O2, called HyPer. HyPer allows ratiometric determination of its fluorescence at two excitation wavelengths, which frees quantification of H2O2 from the variable levels of HyPer in vivo. HyPer fluorescence was shown to have the potential to provide the necessary spatial, temporal, and quantitative resolution to study HL responses of seedlings using confocal microscopy. Chlorophyll fluorescence imaging was used to quantify photoinhibition of photosynthesis induced by HL treatment of seedlings on the microscope staging. However, several technical issues remain, the most challenging of which is the silencing of HyPer expression beyond the seedling stage. This limited our pilot studies to cotyledon epidermal cells, which while not photosynthetic, nevertheless responded to HL with 45% increase in cytosolic H2O2.

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.

Response of the rat spinal cord to X-ray microbeams.

BACKGROUND AND PURPOSE: To quantify the late dose-related responses of the rat cervical spinal cord to X-ray irradiations by an array of microbeams or by a single millimeter beam. MATERIALS AND METHODS: Necks of anesthetized rats were irradiated transversely by an 11 mm wide array of 52 parallel, 35 µm wide, vertical X-ray microbeams, separated by 210 µm intervals between centers. Comparison was made with rats irradiated with a 1.35 mm wide single beam of similar X-rays. Rats were killed when paresis developed, or up to 383 days post irradiation (dpi). RESULTS: Microbeam peak/valley doses of ≈357/12.7 Gy to 715/25.4 Gy to an 11 mm long segment of the spinal cord, or single beam doses of ≈146-454 Gy to a 1.35 mm long segment caused foreleg paresis and histopathologically verified spinal cord damage; rats exposed to peak/valley doses up to 253/9 Gy were paresis-free at 383 dpi. CONCLUSIONS: Whereas microbeam radiation therapy [MRT] for malignant gliomas implanted in rat brains can be safe, palliative or curative, the high tolerance of normal rat spinal cords to similar microbeam exposures justifies testing MRT for autochthonous malignancies in the central nervous system of larger animals with a view to subsequent clinical applications.

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.