A synergy between mechanosensitive calcium- and membrane-binding mediates tension-sensing by C2-like domains

When nuclear membranes are stretched, the peripheral membrane enzyme cytosolic phospholipase A2 (cPLA2) binds via its calcium-dependent C2 domain (cPLA2-C2) and initiates bioactive lipid signaling and tissue inflammation. More than 150 C2-like domains are encoded in vertebrate genomes. How many of them are mechanosensors and quantitative relationships between tension and membrane recruitment remain unexplored, leaving a knowledge gap in the mechanotransduction field. In this study, we imaged the mechanosensitive adsorption of cPLA2 and its C2 domain to nuclear membranes and artificial lipid bilayers, comparing it to related C2-like motifs. Stretch increased the Ca2+ sensitivity of all tested domains, promoting half-maximal binding of cPLA2 at cytoplasmic resting-Ca2+ concentrations. cPLA2-C2 bound up to 50 times tighter to stretched than to unstretched membranes. Our data suggest that a synergy of mechanosensitive Ca2+ interactions and deep, hydrophobic membrane insertion enables cPLA2-C2 to detect stretched membranes with antibody-like affinity, providing a quantitative basis for understanding mechanotransduction by C2-like domains.

Biophysical characteristics of TRIC-A and TRIC-B channels and their regulation of RYR2

Trimeric intracellular cation channels (TRIC-A and TRIC-B), found in the sarco/endoplasmic reticulum (SR/ER) and nuclear membranes, are thought to provide countercurrents to balance Ca2+-movements across the SR, but there is also evidence that they physically interact with ryanodine receptors (RYR). We therefore investigated if TRIC channels could modulate the single-channel function of RYR2 after incorporation of vesicles isolated from HEK293 cells expressing TRIC-A or TRIC-B with RYR2 into artificial membranes under voltage clamp. We also examined the gating and conductance properties of TRIC channels. Co-expression of RYR2 with either TRIC-A or TRIC-B significantly altered the gating behavior of RYR2; however, co-expression with TRIC-A was particularly effective at potentiating the activating effects of cytosolic Ca2+. Fusing membrane vesicles containing TRIC-A or TRIC-B together with RYR2 into bilayers produced large currents of rapidly gating current fluctuations of multiple amplitudes. In 740 cytosolic/210 luminal mM KCl gradient, current-voltage relationships of macroscopic currents revealed average reversal potentials (Erev) of −13.67 ± 9.02 (n = 7), −2.11 ± 3.84 (n = 11), and 13.19 ± 3.23 (n = 13, **, P = 0.0025) from vesicles from RYR2 only, RyR2 + TRIC-A, or RyR2 + TRIC-B cells, respectively. Thus, with the incorporation of TRIC channels, the Erevs depart further from the calculated Erev for ideally selective cation channels than occurs when vesicles from RYR2-only cells are incorporated, suggesting that TRIC channels are permeable to both K+ and Cl−. In conclusion, our results indicate that both TRIC-A and TRIC-B regulate the gating of RYR2, but that TRIC-A has greater capacity to stimulate the RYR2 opening. The results also suggest that TRIC channels may be relatively nonselective ion channels being permeable to both cations and anions. This property would enable TRIC channels to be versatile providers of counter-ion current throughout the SR of many cell types.

Fine structure of spermatogenesis in the spider crab Lissa chiragra (Fabricius, 1775) (Eubrachyura, Epialtidae)

This article uses the male spider crab Lissa chiragra, collected from Ras el Tin beach on the Mediterranean Sea at Alexandria, Egypt, between January and December 2017, as a model for a descriptive study of the functional morphology of the different structures of spermatogenesis with the aid of the transmission electron microscope. This study divides the seminiferous tubule into three zones, i.e., the germinal, transformation, and evacuation zone. The transformation or maturation zone occupies the central area. The evacuation zone lies at a pole opposite to the germinal zone. The anterior vas deferens is a tube where the formation of the spermatophore occurs by means of secretions from the epithelium. The ejaculatory duct is located between the muscles of the coxae of the fifth walking legs. This study concluded that the complex acrosome vesicle is formed during spermiogenesis and the cytoplasm includes the membrane system, constituted by the endoplasmic reticulum, Golgi apparatus, and vesicles associated with microtubules, and a few mitochondria. The Golgi apparatus is developed and is organized progressively to produce various vesicles. The cytoplasm forms a ring at the base of the nuclear extensions. The merger of the plasma and nuclear membranes, results in a pentalaminar membranous structure in some regions of the cell surface.

Short-term cell death in tissues of Pulsatilla vernalis seeds from natural and ex situ conserved populations

Pulsatilla vernalis is a IUCN listed species that occurs in mountain and lowland habitats. The seeds collected from different populations are remarkably diverse in their viability depending on locality or year of collection. We aim to analyse seed viability, among others, by investigation of the percentage of alive, dying, and dead cells in embryos and endosperm when comparing the seeds from a wild lowland population and ex situ cultivation of plants of lowland and Alpine origin. The cell death was detected by staining with two fluorescence probes, one penetrating only the changed nuclear membranes, the other penetrating also the unchanged cells. 54.5% of Alpine origin seeds were presumably capable of germination if they were sown after collection, however, four months later only 36.4% had healthy embryos. In the case of lowland wild plants it was 31.8% and 18.2%, and from ex situ, 27.3% and 13.6%, respectively. 27.3% of Alpine origin seeds had embryo in torpedo stage (9.1% in the case of lowland seeds). Mean weight of the former was 2.9 mg (2.0 mg in lowland ones). Our results confirm the significance of seed origin and seed weight on viability, and that Pulsatilla seeds have a short ‘germination time window’.

New Activities of the Nuclear Pore Complexes

Nuclear pore complexes (NPCs) at the surface of nuclear membranes play a critical role in regulating the transport of both small molecules and macromolecules between the cell nucleus and cytoplasm via their multilayered spiderweb-like central channel. During mitosis, nuclear envelope breakdown leads to the rapid disintegration of NPCs, allowing some NPC proteins to play crucial roles in the kinetochore structure, spindle bipolarity, and centrosome homeostasis. The aberrant functioning of nucleoporins (Nups) and NPCs has been associated with autoimmune diseases, viral infections, neurological diseases, cardiomyopathies, and cancers, especially leukemia. This Special Issue highlights several new contributions to the understanding of NPC proteostasis.

Comprehensive histological imaging of native microbiota in human glioma

Mounting evidence suggests that distinct microbial communities reside in tumors and play important roles in tumor physiology. Recently, Nejman et al. profiled the composition and localization of intratumoral bacteria using 16S DNA sequencing and histological visualization methods across seven tumor types, including human glioblastoma. However, considering potential contamination in their sample origins and processing, the results based on traditional histological methods need to be validated. Here, we propose a three-dimensional (3D) intratumoral microbiota visualization and quantification protocol to observe microbiota in intact tumor tissues on the premise of avoiding possible contamination in the surface of tissues, based on tissue clearing, immunofluorescent labeling, microscopy imaging, and image processing. For the first time, we have achieved 3D quantitative imaging of bacterial LPS fluorescent signals deep in gliomas in a contamination-free manner, which was founded mostly localized near nuclear membranes or in the intercellular space. Through an automated statistical algorithm, reliable signals can be distinguished for further analysis of their sizes, distribution, and fluorescence intensities. Combining two-dimensional images from multiple thin-section histological methods, including immunochemistry and fluorescence in situ hybridization, we provide a comprehensive histological investigation of the morphology and distribution of these signals on human glioma samples. We expect that this multi-evidence chain will provide supporting proof for the presence of intratumoral bacteria in human glioma and that the integrated pipeline can be applied to investigate the native bacteria within diverse tumors and contribute to the interpretation of their direct roles in the tumor microenvironment.

Ceramide From Sphingomyelin Hydrolysis Induces Neuronal Differentiation, Whereas De Novo Ceramide Synthesis And Sphingomyelin Hydrolysis Initiate Apoptosis After NGF Withdrawal In PC12 Cells

Background: Ceramide, important for both neuronal differentiation and dedifferentiation, resides in several membranes, is synthesized in the endoplasmic reticulum, mitochondrial, and nuclear membranes, and can be further processed into glycosphingolipids or sphingomyelin. Ceramide may also be generated by hydrolysis of sphingomyelin by neutral or acidic sphingomyelinases in lysosomes and other membranes. Here we asked whether the differing functions of ceramide derived from different origins. Methods: We added NGF to PC12 cells and to TrkA cells. These latter overexpress NGF receptors and are partially activated to differentiate, whereas NGF is required for PC12 cells to differentiate. We differentiated synthesis from hydrolysis by the use of appropriate inhibitors. Results: When NGF is added, the kinetics and amounts of ceramide and sphingomyelin indicate that the ceramide comes primarily from hydrolysis but, when hydrolysis is inhibited, can also come from neosynthesis. When NGF is removed, the ceramide comes from both neosynthesis and hydrolysis. Conclusion: We conclude that the function of ceramide depends heavily on its intracellular location, and that further understanding of its function will depend on resolving its location during changes of cell status.

Norepinephrine activates β1-adrenergic receptors localized to the inner nuclear membrane in cortical astrocytes

Studies in cardiomyocytes have established that adrenergic receptors, conventionally thought to initiate signaling events exclusively from the plasma membrane, can also localize to and signal from the nuclear membrane. Activation of these receptors by their endogenous cationic ligands requires transmembrane uptake mediated by organic cation transporter 3 (OCT3). We have demonstrated that OCT3 is densely localized to outer nuclear membranes in neurons and astrocytes, suggesting that nuclear adrenergic signaling is also present in the central nervous system. In this study, we examined the subcellular localization of β1-adrenergic receptors, their G-protein signaling partners, and catecholamine transporters in mouse astrocytes. We identified a population of β1-adrenergic receptors localized to astrocyte inner nuclear membranes. We demonstrated that key components of Gs-mediated signaling are localized to the nuclear compartment and identified OCT3 and other catecholamine transporters localized to plasma and nuclear membranes. Treatment of astrocytes with NE induced rapid increases in nuclear PKA activity which were blocked by pretreatment with inhibitors of catecholamine transport. These data indicate that nuclear adrenergic receptors are functionally coupled to Gs-coupled signaling mediators and that their activation by norepinephrine requires transporter-mediated uptake. These receptors represent a powerful mechanism by which norepinephrine may alter astrocyte gene expression and brain function.

Atg39 links and deforms the outer and inner nuclear membranes in selective autophagy of the nucleus

In selective autophagy of the nucleus (hereafter nucleophagy), nucleus-derived double membrane vesicles (NDVs) are formed, sequestered within autophagosomes, and delivered to lysosomes or vacuoles for degradation. In Saccharomyces cerevisiae, the nuclear envelope (NE) protein Atg39 acts as a nucleophagy receptor, which interacts with Atg8 to target NDVs to forming autophagosomal membranes. In this study, we revealed that Atg39 is anchored to the outer nuclear membrane (ONM) via its transmembrane domain and also associated with the inner nuclear membrane (INM) via membrane-binding amphipathic helices (APHs) in its perinuclear space region, thereby linking these membranes. We also revealed that overaccumulation of Atg39 causes the NE to protrude towards the cytoplasm, and the tips of the protrusions are pinched off to generate NDVs. The APHs of Atg39 are crucial for Atg39 assembly in the NE and subsequent NE protrusion. These findings suggest that the nucleophagy receptor Atg39 plays pivotal roles in NE deformation during the generation of NDVs to be degraded by nucleophagy.

Cholesterol and Sphingolipid Enriched Lipid Rafts as Therapeutic Targets in Cancer

Lipid rafts are critical cell membrane lipid platforms enriched in sphingolipid and cholesterol content involved in diverse cellular processes. They have been proposed to influence membrane properties and to accommodate receptors within themselves by facilitating their interaction with ligands. Over the past decade, technical advances have improved our understanding of lipid rafts as bioactive structures. In this review, we will cover the more recent findings about cholesterol, sphingolipids and lipid rafts located in cellular and nuclear membranes in cancer. Collectively, the data provide insights on the role of lipid rafts as biomolecular targets in cancer with good perspectives for the development of innovative therapeutic strategies.