Specialized mechanical connection between exoskeleton and underlying muscles in arthropods is a complex network of interconnected matrix constituents, junctions

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.

The distribution and orientation of microtubules were investigated in cells of distinct shapes from different hindgut regions of adult Porcellio scaber Latreille, 1804 and during hindgut morphogenesis in late embryonic and early postembryonic development. All hindgut cells of adult P. scaber contain abundant apico-basal microtubules organized in extensive bundles, but the architecture of bundles is specific for distinct cells. In the anterior chamber the architecture of microtubule bundles closely coincides with different shapes of the cells in this hindgut region and are most prominent in hindgut cells associated with extensive muscles. The shape of cells that form the typhlosole and typhlosole channels is particularly complex. In the papillate region the microtubule bundles protrude between the infoldings of apical plasma membrane and the mitochondria are closely aligned along the microtubules, thus the microtubule bundles in the papillate region are likely involved in the stabilization of the apical labyrinth and positioning of mitochondria. During hindgut morphogenesis the apico-basal microtubule bundles are established relatively late, mainly during early postembryonic development. Morphogenesis of the typhlosole is characterized by coinciding changes in cell shape and microtubule arrangement.

Biomineralized structures consist of an organic matrix and mineral constituents. Imaging of the mineralized biological tissues is demanding due to specific requirements for the preservation and visualization of chemically different constituents and due to sectioning difficulties. In this study, a characterization of the cuticular matrix of the crustacean exoskeleton was performed by a combination of microscopic methods, aiming to obtain spatial information on the matrix composition. Histochemical procedures were performed and compared in artificially decalcified and non-decalcified samples, in paraffin and resin sections. Wheat germ agglutinin (WGA) lectin-gold conjugate and a fluorescent chitin-binding probe were used to localize chitin in paraffin and resin sections of samples prepared by different fixations. Calcified regions of the matrix were determined by histochemical staining of aldehyde-fixed, methanol-fixed, and resin-embedded samples and by scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM–EDXS) of “intact” cuticle transverse profiles that were not subjected to any surface processing. We show that the spatial distribution of cuticular organic matrix components is not homogenous, as a differential staining of exocuticular and endocuticular matrix was obtained by histochemistry. Chitin localization, performed by two different methods, shows a localization pattern of horizontal lines of alternating intensity in the transversely cut endocuticle. Histochemical demonstration of calcified cuticular matrix in resin sections was successful in 2.5 μm thick resin sections of methanol-fixed samples. Methanol-fixed, dried, and fractured samples displayed the characteristic elemental composition and are useful to obtain qualitative composition data from the non-processed surface profiles of the cuticle.