Pore-forming moss protein bryoporin is structurally and mechanistically related to actinoporins from evolutionarily distant cnidarians.

Pore-forming proteins perforate lipid membranes and consequently affect their integrity and cell fitness. Therefore, it is not surprising that many of these proteins from bacteria, fungi, or certain animals act as toxins. While pore-forming proteins have also been found in plants, there is little information about their molecular structure and mode of action. Bryoporin is a protein from the moss Physcomitrium patens, and its corresponding gene was found to be upregulated by various abiotic stresses, especially dehydration, as well as upon fungal infection. Based on the amino acid sequence, it was suggested that bryoporin was related to the actinoporin family of pore-forming proteins, originally discovered in sea anemones. Here, we provide the first detailed structural and functional analysis of this plant cytolysin. The crystal structure of monomeric bryoporin is highly similar to those of actinoporins. Our cryo-EM analysis of its pores showed an actinoporin-like octameric structure, thereby revealing a close kinship of proteins from evolutionarily distant organisms. This was further confirmed by our observation of bryoporin's preferential binding to and formation of pores in membranes containing animal sphingolipids, such as sphingomyelin and ceramide phosphoethanolamine; however, its binding affinity was weaker than that of actinoporin equinatoxin II. We determined bryoporin did not bind to major sphingolipids found in fungi or plants, and its membrane-binding and pore-forming activity was enhanced by various sterols. Our results suggest that bryoporin could represent a part of the moss defense arsenal, acting as a pore-forming toxin against membranes of potential animal pathogens, parasites, or predators.

Fallopia japonica and Fallopia × bohemica extracts cause ultrastructural and biochemical changes in root tips of radish seedlings.

Japanese knotweed (Fallopia japonica) and Bohemian knotweed (Fallopia × bohemica) are invasive plants that use allelopathy as an additional mechanism for colonization of the new habitat. Allelochemicals affect the growth of roots of neighboring plants. In the present study, we analyze the early changes associated with the inhibited root growth of radish seedlings exposed to aqueous extracts of knotweed rhizomes for 3 days. Here, we show that cells in the root cap treated with the knotweed extracts exhibited reduced cell length and displayed several ultrastructural changes, including the increased abundance of dilated ER cisternae filled with electron-dense material (ER bodies) and the accumulation of dense inclusions. Moreover, mitochondrial damage was exhibited in the root cap and the meristem zone compared to the non-treated radish seedlings. Furthermore, malfunction of the intracellular redox balance system was detected as the increased total antioxidative capacity. We also detected increased metacaspase-like proteolytic activities and, in the case of 10% extract of F. japonica, increased caspase-like proteolytic activities. These ultrastructural and biochemical effects could be the reason for the more than 60% shorter root length of treated radish seedlings compared to controls.

Structure, function and development of the digestive system in malacostracan crustaceans and adaptation to different lifestyles.

The digestive system of the malacostracan crustaceans, namely the decapods, isopods, amphipods and mysids, is among the most complex organ systems of the animal kingdom serving multiple functions such as food processing, absorption and storage of nutrients, synthesis of digestive enzymes and blood proteins, detoxification of xenobiotics and osmoregulation. It is rather well investigated compared to other invertebrates because the Malacostraca include many ecological keystone species and food items for humans. The Decapoda and Peracarida share food processing with chewing and filtering structures of the stomach but differ with respect to morphology and ultrastructure of the digestive glands. In the Peracarida, the digestive glands are composed of few, relatively large lateral caeca, whereas in the Decapoda, hundreds to thousands of blindly ending tubules form a voluminous hepatopancreas. Morphogenesis and onset of functionality of the digestive system strongly depend on the mode of development. The digestive system is early developed in species with feeding planktonic larvae and appears late in species with direct lecithotrophic development. Some structures of the digestive system like the stomach ossicles are rather constant in higher taxa and are of taxonomic value, whereas others like the chewing structures are to some degree adapted to the feeding strategy. The nutrient absorbing and storing cells of the digestive glands show considerable ultrastructural variation during moult cycle, vitellogenesis and starvation. Some of the various functions of the digestive system are already assigned to specific sections of the digestive tract and cell types, but others still await precise localization.

Cholesterol Enriched Archaeosomes as a Molecular System for Studying Interactions of Cholesterol-Dependent Cytolysins with Membranes.

Archaeosomes are vesicles made of lipids from archaea. They possess many unique features in comparison to other lipid systems, with their high stability being the most prominent one, making them a promising system for biotechnological applications. Here, we report a preparation protocol of large unilamellar vesicles, giant unilamellar vesicles (GUVs), and nanodiscs from archaeal lipids with incorporated cholesterol. Incorporation of cholesterol led to additional increase in thermal stability of vesicles. Surface plasmon resonance, sedimentation assays, intrinsic tryptophan fluorescence measurements, calcein release experiments, and GUVs experiments showed that members of cholesterol-dependent cytolysins, listeriolysin O (LLO), and perfringolysin O (PFO), bind to cholesterol-rich archaeosomes and thereby retain their pore-forming activity. Interestingly, we observed specific binding of LLO, but not PFO, to archaeosomes even in the absence of cholesterol. This suggests a new capacity of LLO to bind to carbohydrate headgroups of archaeal lipids. Furthermore, we were able to express LLO inside GUVs by cell-free expression. GUVs made from archaeal lipids were highly stable, which could be beneficial for synthetic biology applications. In summary, our results describe novel model membrane systems for studying membrane interactions of proteins and their potential use in biotechnology.

Diabrotica v. virgifera Seems Not Affected by Entomotoxic Protease Inhibitors from Higher Fungi.

Certain soil insects, such as the root-damaging larvae of the maize pest Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), are increasingly difficult to control because of recent bans of some insecticides. An alternative and safer approach may be the development of biopesticides based on entomotoxic defense proteins of higher fungi. Many of these potentially interesting proteins are protease inhibitors, and some have been shown to adversely affect insects. We examined the effects of the cysteine protease inhibitors macrocypin 1, 3, and 4 from Macrolepiota procera, clitocypin from Clitocybe nebularis, and cocaprin 1 and the serine protease inhibitor cospin 1 from Coprinopsis cinerea on D. v. virgifera. We confirmed the inhibition by mycocypins of the cysteine catalytic-type proteolytic activities in gut extracts of larvae and adults. The inhibition of pGlu-Phe-Leu-hydrolyzing activity was stronger than that of Z-Phe-Arg-hydrolyzing activity. Mycocypins and cospin resisted long-term proteolytic digestion, whereas cocaprin 1 was digested. Bioassays with overlaid artificial diet revealed no effects of proteins on neonatal mortality or stunting, and no effects on adult mortality. Immersion of eggs in protein solutions had little effect on egg hatching or mortality of hatching neonates. Microscopic analysis of the peritrophic matrix and apical surface of the midguts revealed the similarity between larvae of D. v. virgifera and the chrysomelid Leptinotarsa decemlineata, which are sensitive to these inhibitors. The resistance of D. v. virgifera to fungal protease inhibitors is likely due to effective adaptation of digestive enzyme expression to dietary protease inhibitors. We continue to study unique protein complexes of higher fungi for the development of new approaches to pest control.

Leafhopper males compensate for unclear directional cues in vibration-mediated mate localization.

Ambient noise and transmission properties of the substrate pose challenges in vibrational signal-mediated mating behavior of arthropods, because vibrational signal production is energetically demanding. We explored implications of these challenges in the leafhopper Aphrodes makarovi (Insecta: Hemiptera: Cicadellidae) by exposing males to various kinds of vibrational noise on a natural substrate and challenging them to find the source of the female playback. Contrary to expectations, males exposed to noise were at least as efficient as control males on account of similar searching success with less signaling effort, while playing back male-female duets allowed the males to switch to satellite behavior and locate the target without signaling, as expected. We found altered mitochondrial structure in males with high signaling effort that likely indicate early damaging processes at the cellular level in tymbal muscle, but no relation between biochemical markers of oxidative stress and signaling effort. Analysis of signal transmission revealed ambiguous amplitude gradients, which might explain relatively low searching success, but it also indicates the existence of behavioral adaptations to complex vibrational environments. We conclude that the observed searching tactic, emphasizing speed rather than thorough evaluation of directional cues, may compensate for unclear stimuli when the target is near.

Calcium bodies of Titanethes albus (Crustacea: Isopoda): molt-related structural dynamics and calcified matrix-associated bacteria.

Crustaceans form a variety of calcium deposits in which they store calcium necessary for the mineralization of their exoskeletons. Calcium bodies, organs containing large amounts of calcium, have been reported in some terrestrial isopod crustaceans, but have not yet been extensively studied. We analyzed the architecture of these organs during the molt cycle in the isopod Titanethes albus. Two pairs of calcium bodies are positioned ventrolaterally in posterior pereonites of T. albus. Individual organs are epithelial sacs that contain material arranged in concentric layers delimited by thin laminae. As demonstrated by electron microscopy and fluorescence in situ hybridization, abundant bacteria are present within the calcium bodies. Regardless of the molt cycle stage, crystalline concretions are present in the central areas of the calcium bodies. Energy dispersive X-ray spectrometry of the concretions demonstrated that they are composed predominantly of calcium and phosphorus and selected area electron diffraction indicated the presence of hydroxyapatite. In molting animals, a glassy layer of mineralized matrix is formed between the envelope and the outermost lamina of the calcium body. This layer consists of an amorphous calcium mineral which contains less phosphorus than the central concretions and is resorbed after molt. Since changes in the mineralized matrix are synchronized with the molt cycle, the calcium bodies likely function as a storage compartment that complements sternal deposits as a source of calcium for the mineralization of the exoskeleton. Bacteria associated with the mineralized matrix of calcium bodies are evidently involved in calcium dynamics.

Root growth inhibition and ultrastructural changes in radish root tips after treatment with aqueous extracts of Fallopia japonica and F. ×bohemica rhizomes.

Allelopathic compounds released by invasive alien plants can suppress the growth of plants in their vicinity. The aim of this study was to investigate changes in tissue and cell structure in roots of radish seedlings treated with 10% aqueous extracts of rhizomes from the invasive knotweeds Fallopia japonica and F. ×bohemica. After 7 days of growth without and with aqueous extracts from these rhizomes, the anatomical and ultrastructural changes in the radish seedling roots were analyzed with light and transmission electron microscopy, and hydrogen peroxide was localized with diaminobenzidine, to define oxidative stress. The roots of radish seedlings treated with the knotweed extracts were shorter and thicker, due to the shorter and wider shapes of their cortex cells, which were organized in more columns than the control roots. There were signs of cell damage and oxidative stress in the root cap cells, and to a lesser extent in the meristematic zone. As well as the irregularly shaped nuclei and plasma membrane detached from the cell wall, the most prominent ultrastructural effects in the root cap cells of these aqueous rhizome extracts were the ring-shaped form of the mitochondria and large endoplasmic reticulum bodies. Excessive vacuolization was seen for the cells of the root apical meristem.

Formation and remodelling of septate junctions in the epidermis of isopod Porcellioscaber during development.

Septate junctions (SJs) perform an occluding function in invertebrate epithelia and consist of parallel septa extending across the intercellular space between neighbouring cells. In addition, they are required for several morphogenetic processes in arthropods. The biogenesis of SJs during development is inadequately studied and it was characterised in detail only for various epithelia of Drosophilamelanogaster. This paper provides a detailed analysis of the ultrastructural differentiation of SJs in the epidermis of the terrestrial isopod Porcellioscaber during embryonic and postembryonic development. In this study, mid-stage embryo S13 was the earliest stage in which single septa were observed basally to the adherens junction (AJ). Differentiation of SJs during further development includes gradual elongation of septa arrays and formation of continuous arrays of septa. The enlargement of SJs in the epidermis is most pronounced at the transition from embryonic to postembryonic development and after the release of mancae from the marsupium. SJs of postmarsupial mancae are similar to those of adults, but are not yet as extensive. Comparison of the differentiation of SJs in the epidermis and hindgut of P.scaber, reveals a similar sequence of events. In addition, remodelling of SJs was observed in the epidermis of late marsupial mancae, the stage of cuticle renewal. Common features of SJs' biogenesis in P.scaber and D.melanogaster ectodermal epithelia are indicated.

Nanoscale transformations of amphiboles within human alveolar epithelial cells.

Amphibole asbestos is related to lung fibrosis and several types of lung tumors. The disease-triggering mechanisms still challenge our diagnostic capabilities and are still far from being fully understood. The literature focuses primarily on the role and formation of asbestos bodies in lung tissues, but there is a distinct lack of studies on amphibole particles that have been internalized by alveolar epithelial cells (AECs). These internalized particles may directly interact with the cell nucleus and the organelles, exerting a synergistic action with asbestos bodies (AB) from a different location. Here we document the near-atomic- to nano-scale transformations induced by, and taking place within, AECs of three distinct amphiboles (anthophyllite, grunerite, "amosite") with different Fe-content and morphologic features. We show that: (i) an Fe-rich layer is formed on the internalized particles, (ii) particle grain boundaries are transformed abiotically by the internal chemical environment of AECs and/or by a biologically induced mineralization mechanism, (iii) the Fe-rich material produced on the particle surface does not contain large amounts of P, in stark contrast to extracellular ABs, and (iv) the iron in the Fe-rich layer is derived from the particle itself. Internalized particles and ABs follow two distinct formation mechanisms reaching different physicochemical end-states.

An oomycete NLP cytolysin forms transient small pores in lipid membranes.

Microbial plant pathogens secrete a range of effector proteins that damage host plants and consequently constrain global food production. Necrosis and ethylene-inducing peptide 1-like proteins (NLPs) are produced by numerous phytopathogenic microbes that cause important crop diseases. Many NLPs are cytolytic, causing cell death and tissue necrosis by disrupting the plant plasma membrane. Here, we reveal the unique molecular mechanism underlying the membrane damage induced by the cytotoxic model NLP. This membrane disruption is a multistep process that includes electrostatic-driven, plant-specific lipid recognition, shallow membrane binding, protein aggregation, and transient pore formation. The NLP-induced damage is not caused by membrane reorganization or large-scale defects but by small membrane ruptures. This distinct mechanism of lipid membrane disruption is highly adapted to effectively damage plant cells.

Silicon: The key element in early stages of biocalcification.

Biocalcification is a widespread process of forming hard tissues like bone and teeth in vertebrates. It is also a topic connecting life sciences and earth sciences: calcified skeletons and shells deposited as sediments represent the earth's fossil record and are of paramount interest for biogeochemists trying to get an insight into the past of our planet. This study reports on the role of silicon in the early biocalcification steps, where silicon and calcium were detected on the surface of cyanobacteria (initial stage of lacustrine calcite precipitation) and in crustacean cuticles. By using innovative methodological approaches of correlative microscopy (AFM in combination with analytical TEM: EFTEM, EELS) the chemical form of silicon in biocalcifying matrices and organic-inorganic particles is determined. Previously, silicon was reported to be localized in active growth areas in the young bone of vertebrates. We have found evidence that biocalcification in evolutionarily distant organisms involves very similar initial phases with silicon as a key element at the organic-inorganic interface.

Ultrastructure and mineral distribution in the tergal cuticle of the terrestrial isopod Titanethes albus. Adaptations to a karst cave biotope.

Composition and spatial distribution of organic and inorganic materials within the cuticle of isopods vary between species. These variations are related to the behaviour and habitat of the animal. The troglobiotic isopod Titanethes albus lives in the complete darkness of caves in the Slovenian Karst. This habitat provides constant temperature and saturated humidity throughout the year and inconsistent food supply. These conditions should have lead to functional adaptations of arthropod cuticles. However, studies on structure and composition of cave arthropod cuticles are rare and lacking for terrestrial isopods. We therefore analysed the tergite cuticle of T. albus using transmission and field-emission electron microscopy, confocal micro-Raman spectroscopic imaging, quantitative X-ray diffractometry, thermogravimetric analysis and atomic absorption spectroscopy. The ultrastructure of the epicuticle suggests a poor resistance against water loss. A weak interconnection between the organic and mineral phase within the endo- and exocuticle, a comparatively thin apical calcite layer, and almost lack of magnesium within the calcite crystal lattice suggest that the mechanical strength of the cuticle is low in the cave isopod. This may possibly be of advantage in maintaining high cuticle flexibility and reducing metabolic expenditures.

Ultrastructural differentiation of plasma membrane and cell junctions in the hindgut cells is synchronized with key developmental transitions in Porcellio scaber.

Differentiation of transporting epithelial cells during development of animal organisms includes remodelling of apical and basal plasma membranes to increase the available surface for transport and formation of occluding junctions, which maintain a paracellular diffusion barrier. This study provides a detailed ultrastructural analysis of apical and basal plasma membrane remodelling and cell junction formation in hindgut cells during late embryonic and early postembryonic development of the crustacean Porcellio scaber. Hindgut cells in late-stage embryos are columnar with flat apical and basal plasma membranes. In early-stage marsupial mancae the hindgut cells begin to acquire their characteristic dome shape, the first apical membrane folding is evident and the septate junctions expand considerably, all changes being probably associated with the onset of active feeding. In postmarsupial mancae the apical labyrinth is further elaborated and the septate junctions are expanded. This coincides with the transition to an external environment and food sources. First basal infoldings appear in the anterior chamber of early-stage marsupial mancae, but in the papillate region they are mostly formed in postmarsupial mancae. In molting late-stage marsupial mancae, the plasma membrane acquires a topology characteristic of cuticle-producing arthropod epithelia and the septate junctions are considerably reduced.

Intra-annual dynamics of phloem formation and ultrastructural changes in sieve tubes in Fagus sylvatica.

Despite increased interest in the timing and dynamics of phloem formation, seasonal changes in the structure of phloem sieve elements remain largely unexplored. To understand better the dynamics of phloem formation and the functioning of sieve tubes in the youngest phloem in Fagus sylvatica L., we investigated repeatedly taken phloem samples during the growing season of 2017 by means of light microscopy, and transmission and scanning electron microscopy. Phloem formation started with the expansion of the overwintered early phloem sieve tubes adjacent to the cambium and concurrent cambial cell production. The highest phloem growth rate was observed in general 1 week after the onset of cambial cell production, whereas the transition from early to late phloem occurred at the end of May. Cambial cell production ceased at the end of July. The final width of the phloem increment was 184 ± 10 µm, with an early phloem proportion of 59%. Collapse of older phloem tissue is a progressive process, which continuously occurred during the sampling period. Collapse of early phloem sieve tubes started shortly after the cessation of cambial cell production. Prior to the onset of radial growth, late phloem from the previous year represented 80% of the total non-collapsed part; during the growth period, this percentage decreased to 20%. Differences were observed in both sieve tube ultrastructure and sieve plate geometry between the youngest and older phloem. However, sieve plates were never completely occluded by callose, suggesting that processes affecting the functionality of sieve tubes may differ in the case of regular collapse or injury. The youngest parts of the phloem increment from the previous year (i.e., previous late phloem) continue functioning for some time in the current growing season, but the two-step development of overwintered phloem cells also ensures a sufficient translocation pathway for photosynthates to the actively growing tissues.

Imaging of intracellular spherical lamellar structures and tissue gross morphology by a focused ion beam/scanning electron microscope (FIB/SEM).

We report the use of a focused ion beam/scanning electron microscope (FIB/SEM) for simultaneous investigation of digestive gland epithelium gross morphology and ultrastructure of multilamellar intracellular structures. Digestive glands of a terrestrial isopod (Porcellio scaber, Isopoda, Crustacea) were examined by FIB/SEM and by transmission electron microscopy (TEM). The results obtained by FIB/SEM and by TEM are comparable and complementary. The FIB/SEM shows the same ultrastructural complexity of multilamellar intracellular structures as indicated by TEM. The term lamellar bodies was used for the multillamellar structures in the digestive glands of P. scaber due to their structural similarity to the lamellar bodies found in vertebrate lungs. Lamellar bodies in digestive glands of different animals vary in their abundance, and number as well as the thickness of concentric lamellae per lamellar body. FIB/SEM revealed a connection between digestive gland gross morphological features and the structure of lamellar bodies. Serial slicing and imaging of cells enables easy identification of the contact between a lamellar body and a lipid droplet. There are frequent reports of multilamellar intracellular structures in different vertebrate as well as invertebrate cells, but laminated cellular structures are still poorly known. The FIB/SEM can significantly contribute to the structural knowledge and is always recommended when a link between gross morphology and ultrastructure is investigated, especially when cells or cellular inclusions have a dynamic nature due to normal, stressed or pathological conditions.

Epithelial thickness and lipid droplets in the hepatopancreas of Porcellio scaber (Crustacea: Isopoda) in different physiological conditions.

We investigated the morphometric characteristics of the hepatopancreatic epithelium in the terrestrial isopod Porcellio scaber during acclimatization to laboratory conditions, during the daily cycle, the molt cycle, and fasting. The hepatopancreatic epithelium was analyzed using computer-assisted microscopy of serial sections of the hepatopancreatic tubes. In addition, the abundance, the distribution, and the size of lipid droplets in the hepatopancreatic epithelium were recorded. The experimental animals were collected in the field and transferred to the laboratory. The hepatopancreatic epithelium was thinner and lipid droplets reduced after 2 months of acclimatization to laboratory conditions. The daily cycle and the molt cycle affected neither the epithelial thickness nor the abundance of lipid droplets. But in animals fasted for 2 weeks, these two parameters were significantly reduced. Based on both the epithelial thickness and the abundance of lipid droplets in B cells, we propose criteria for estimating the stress status of the animals. With the possibility to determine the stress status, many studies on isopods gain in relevance.

Comparative ultrastructure of cells and cuticle in the anterior chamber and papillate region of Porcellioscaber (Crustacea, Isopoda) hindgut.

Isopod hindgut consists of two anatomical and functional parts, the anterior chamber, and the papillate region. This study provides a detailed ultrastructural comparison of epithelial cells in the anterior chamber and the papillate region with focus on cuticle ultrastructure, apical and basal plasma membrane labyrinths, and cell junctions. Na+/K+-ATPase activity in the hindgut epithelial cells was demonstrated by cytochemical localisation. The main difference in cuticle ultrastructure is in the thickness of epicuticle which is almost as thick as the procuticle in the papillate region and only about one sixth of the thickness of procuticle in the anterior chamber. The apical plasma membrane in both hindgut regions forms an apical plasma membrane labyrinth of cytoplasmic strands and extracellular spaces. In the papillate region the membranous infoldings are deeper and the extracellular spaces are wider. The basal plasma membrane is extensively infolded and associated with numerous mitochondria in the papillate region, while it forms relatively scarce basal infoldings in the anterior chamber. The junctional complex in both hindgut regions consists of adherens and septate junctions. Septate junctions are more extensive in the papillate region. Na+/K+-ATPase was located mostly in the apical plasma membranes in both hindgut regions. The ultrastructural features of hindgut cuticle are discussed in comparison to exoskeletal cuticle and to cuticles of other arthropod transporting epithelia from the perspective of their mechanical properties and permeability. The morphology of apical and basal plasma membranes and localisation of Na+/K+-ATPase are compared with other arthropod-transporting epithelia according to different functions of the anterior chamber and the papillate region.

Cuticle morphogenesis in crustacean embryonic and postembryonic stages.

The crustacean cuticle is a chitin-based extracellular matrix, produced in general by epidermal cells and ectodermally derived epithelial cells of the digestive tract. Cuticle morphogenesis is an integrative part of embryonic and postembryonic development and it was studied in several groups of crustaceans, but mainly with a focus on one selected aspect of morphogenesis. Early studies were focused mainly on in vivo or histological observations of embryonic or larval molt cycles and more recently, some ultrastructural studies of the cuticle differentiation during development were performed. The aim of this paper is to review data on exoskeletal and gut cuticle formation during embryonic and postembryonic development in crustaceans, obtained in different developmental stages of different species and to bring together and discuss different aspects of cuticle morphogenesis, namely data on the morphology, ultrastructure, composition, connections to muscles and molt cycles in relation to cuticle differentiation. Based on the comparative evaluation of microscopic analyses of cuticle in crustacean embryonic and postembryonic stages, common principles of cuticle morphogenesis during development are discussed. Additional studies are suggested to further clarify this topic and to connect the new knowledge to related fields.