The phylum Oomycota contains economically important pathogens of animals and plants, including Saprolegnia parasitica, the causal agent of the fish disease saprolegniasis. Due to intense fish farming and banning of the most effective control measures, saprolegniasis has re-emerged as a major challenge for the aquaculture industry. Oomycete cells are surrounded by a polysaccharide-rich cell wall matrix that, in addition to being essential for cell growth, also functions as a protective \"armor.\" Consequently, the enzymes responsible for cell wall synthesis provide potential targets for disease control. Oomycete cell wall biosynthetic enzymes are predicted to be plasma membrane proteins. To identify these proteins, we applied a quantitative (iTRAQ) mass spectrometry-based proteomics approach to the plasma membrane of the hyphal cells of S. parasitica, providing the first complete plasma membrane proteome of an oomycete species. Of significance is the identification of 65 proteins enriched in detergent-resistant microdomains (DRMs). In silico analysis showed that DRM-enriched proteins are mainly involved in molecular transport and β-1,3-glucan synthesis, potentially contributing to pathogenesis. Moreover, biochemical characterization of the glycosyltransferase activity in these microdomains further supported their role in β-1,3-glucan synthesis. Altogether, the knowledge gained in this study provides a basis for developing disease control measures targeting specific plasma membrane proteins in S. parasitica.IMPORTANCEThe significance of this research lies in its potential to combat saprolegniasis, a detrimental fish disease, which has resurged due to intensive fish farming and regulatory restrictions. By targeting enzymes responsible for cell wall synthesis in Saprolegnia parasitica, this study uncovers potential avenues for disease control. Particularly noteworthy is the identification of several proteins enriched in membrane microdomains, offering insights into molecular mechanisms potentially involved in pathogenesis. Understanding the role of these proteins provides a foundation for developing targeted disease control measures. Overall, this research holds promise for safeguarding the aquaculture industry against the challenges posed by saprolegniasis.

The migrasome is a newly discovered organelle of migrating cells. Migrasomes play diverse physiological roles including mitochondrial quality control, lateral transfer of material between cells, and delivery of signaling molecules to spatially defined locations. The formation of migrasomes is dependent on tetraspanins, a group of membrane proteins containing four transmembrane domains, which form membrane microdomains named tetraspanin-enriched microdomains (TEMs). In this review, we will discuss the mechanisms for migrasome biogenesis, with a focus on the role of TEMs and the organizing principles underlying the formation of TEMs.

Auxin is a versatile plant growth regulator that triggers multiple signalling pathways at different spatial and temporal resolutions. A plant cell is surrounded by the cell wall, a complex and dynamic network of polysaccharides. The cell wall needs to be rigid to provide mechanical support and protection and highly flexible to allow cell growth and shape acquisition. The modification of the pectin components, among other processes, is a mechanism by which auxin activity alters the mechanical properties of the cell wall. Auxin signalling precisely controls the transcriptional output of several genes encoding pectin remodelling enzymes, their local activity, pectin deposition, and modulation in different developmental contexts. This review examines the mechanism of auxin activity in regulating pectin chemistry at organ, cellular, and subcellular levels across diverse plant species. Moreover, we ask questions that remain to be addressed to fully understand the interplay between auxin and pectin in plant growth and development.

The investigation of the lipid-protein microdomains of the plasmalemma isolated with the aid of the non-detergent technique in the zones of the sucrose density gradient after high-speed centrifugation from the tissue pieces of beet roots, which underwent oxidative stress, was conducted. The microdomains, whose lipid composition - according to the definition - allowed us to classify them as rafts, were studied. After the exposure to oxidative stress (100 mM hydrogen peroxide), the variations in the composition of membrane lipids bound up mainly with the elevations of the content of raft-forming lipids (sterols, sterol esters). Oxidative stress provoked redistribution in the composition of sterols, which led to an elevation in the content of campesterol and in the ratio of stigmasterol/sitosterol. Furthermore, the variations were registered in the content of phospholipids and phosphoglycerolipids, which are capable of stabilizing the lamellar structure of membranes. The results obtained allow one to assume that under the oxidative stress, variations in the composition of lipids in microdomains of the plasma membrane can take place. These variations may influence the functioning of the membranes, and the membranes may participate in the protection of the plant cell.

H2S is a gaseous signaling molecule enzymatically produced in mammals and H2S-producing enzymes are expressed throughout the vascular wall. We previously reported that H2S-induced vasodilation is mediated through transient receptor potential cation channel subfamily V member 4 (TRPV4) and large conductance (BKCa) potassium channels; however, regulators of this pathway have not been defined. Previous reports have shown that membrane cholesterol limits activity of TRPV4 and BKCa potassium channels. The current study examined the ability of endothelial cell (EC) plasma membrane (PM) cholesterol to regulate H2S-induced vasodilation. We hypothesized that EC PM cholesterol hinders H2S-mediated vasodilation in large mesenteric arteries. In pressurized, U46619 pre-constricted mesenteric arteries, decreasing EC PM cholesterol in large arteries using methyl-β-cyclodextrin (MBCD, 100 µM) increased H2S-induced dilation (NaHS 10, 100 µM) but MBCD treatment had no effect in small arteries. Enface fluorescence showed EC PM cholesterol content is higher in large mesenteric arteries than in smaller arteries. The NaHS-induced vasodilation following MBCD treatment in large arteries was blocked by TRPV4 and BKCa channel inhibitors (GSK219384A, 300 nM and iberiotoxin, 100 nM, respectively). Immunohistochemistry of mesenteric artery cross-sections show that TRPV4 and BKCa are both present in EC of large and small arteries. Cholesterol supplementation into EC PM of small arteries abolished NaHS-induced vasodilation but the cholesterol enantiomer, epicholesterol, had no effect. Proximity ligation assay studies did not show a correlation between EC PM cholesterol content and the association of TRPV4 and BK. Collectively, these results demonstrate that EC PM cholesterol limits H2S-induced vasodilation through effects on EC TRPV4 and BKCa channels.

CONCLUSIONS: Variations in the content of tonoplast microdomains, isolated with the aid of a non-detergent technique, are induced by osmotic stress and may take part in plant cell adaptive mechanisms. Investigation of tonoplast microdomain lipids isolated with the aid of the non-detergent technique from beetroots (Beta vulgaris L.) subjected to either hyperosmotic or hypoosmotic stress was conducted. Earlier, an important role of tonoplast lipids in the protection of plant cells from stress was demonstrated (Ozolina et al. 2020a). In the present paper, we have put forward a hypothesis that lipids of microdomains of raft nature present in the tonoplast are responsible for this protective function. The variations in the content of lipids of the studied nondetergent-isolated microdomains (NIMs) under hyperosmotic and hypoosmotic stresses were different. Under hyperosmotic stress, in the scrutinized microdomains, some variations in the content of lipids were registered, which were characteristic of the already known protective anti-stress mechanisms. These variations were represented by an increase in sterols and polar lipids capable of stabilizing the bilayer structure of the membranes. The found variations in the content of sterols may be bound up with some intensification of the autophagy process under stress because sterols foster the formation of new membrane contacts necessary for this process. Under hypoosmotic stress, the pattern of redistribution of the lipids in the scrutinized membrane structures was different: the largest part of the lipids appeared to be represented by hydrocarbons, which fulfilled mainly a protective function in plants and could prevent the excess water influx into the vacuole. The results obtained not only demonstrate the possible functions of the vacuolar membrane microdomains but also put forward an assumption on the role of any membrane microdomain in the protection mechanisms of the plant cell.

SNARE proteins have been described as the effectors of fusion events in the secretory pathway more than two decades ago. The strong interactions between SNARE domains are clearly important in membrane fusion, but it is unclear whether they are involved in any other cellular processes. Here, we analyzed two classical SNARE proteins, syntaxin 1A and SNAP25. Although they are supposed to be engaged in tight complexes, we surprisingly find them largely segregated in the plasma membrane. Syntaxin 1A only occupies a small fraction of the plasma membrane area. Yet, we find it is able to redistribute the far more abundant SNAP25 on the mesoscale by gathering crowds of SNAP25 molecules onto syntaxin clusters in a SNARE-domain-dependent manner. Our data suggest that SNARE domain interactions are not only involved in driving membrane fusion on the nanoscale, but also play an important role in controlling the general organization of proteins on the mesoscale. Further, we propose these mechanisms preserve active syntaxin 1A-SNAP25 complexes at the plasma membrane.

Beta-adrenoceptors (βAR) are often viewed as archetypal G-protein coupled receptors. Over the past fifteen years, investigations in cardiovascular biology have provided remarkable insights into this receptor family. These studies have shifted pharmacological dogma, from one which centralized the receptor to a new focus on structural micro-domains such as caveolae and t-tubules. Important studies have examined, separately, the structural compartmentation of ion channels and βAR. Despite links being assumed, relatively few studies have specifically examined the direct link between structural remodeling and electrical remodeling with a focus on βAR. In this review, we will examine the nature of receptor and ion channel dysfunction on a substrate of cardiomyocyte microdomain remodeling, as well as the likely ramifications for cardiac electrophysiology. We will then discuss the advances in methodologies in this area with a specific focus on super-resolution microscopy, fluorescent imaging, and new approaches involving microdomain specific, polymer-based agonists. The advent of powerful computational modelling approaches has allowed the science to shift from purely empirical work, and may allow future investigations based on prediction. Issues such as the cross-reactivity of receptors and cellular heterogeneity will also be discussed. Finally, we will speculate as to the potential developments within this field over the next ten years.

Electrical activity and oscillations of cytosolic Ca2+ concentrations ([Ca2+]i) that trigger insulin release in response to glucose are key functions of pancreatic β cells. Although oscillatory Ca2+ signals have been intensively studied in β cells, their lower frequency did not match that of electrical activity. In addition, the measured peak [Ca2+]i did not reach levels that are typically required by synaptotagmins to elicit the release of insulin-containing vesicles in live-cell experiments. We therefore sought to resolve the Ca2+ dynamics in the subplasmalemmal microdomain that is critical for triggering fast exocytosis. Applying total internal reflection fluorescence (TIRF) microscopy in insulin-producing INS-1E and primary mouse β cells, we resolved extraordinary fast trains of Ca2+ spiking (frequency > 3 s-1) in response to glucose exposure. Using a low-affinity [Ca2+]i indicator dye, we provide experimental evidence that Ca2+ spikes reach low micromolar apparent concentrations in the vicinity of the plasma membrane. Analysis of Ca2+ spikes evoked by repeated depolarization for 10 ms closely matched the Ca2+ dynamics observed upon glucose application. To our knowledge, this is the first study that experimentally demonstrates Ca2+ spikes in β cells with velocities that resemble those of bursting or continuously appearing trains of action potentials (APs) in non-patched cells.

Light plays an essential role in photosynthesis; however, its excess can cause damage to cellular components. Photosynthetic organisms thus developed a set of photoprotective mechanisms (e.g., non-photochemical quenching, photoinhibition) that can be studied by a classic biochemical and biophysical methods in cell suspension. Here, we combined these bulk methods with single-cell identification of microdomains in thylakoid membrane during high-light (HL) stress. We used Synechocystis sp. PCC 6803 cells with YFP tagged photosystem I. The single-cell data pointed to a three-phase response of cells to acute HL stress. We defined: (1) fast response phase (0-30 min), (2) intermediate phase (30-120 min), and (3) slow acclimation phase (120-360 min). During the first phase, cyanobacterial cells activated photoprotective mechanisms such as photoinhibition and non-photochemical quenching. Later on (during the second phase), we temporarily observed functional decoupling of phycobilisomes and sustained monomerization of photosystem II dimer. Simultaneously, cells also initiated accumulation of carotenoids, especially ɣ-carotene, the main precursor of all carotenoids. In the last phase, in addition to ɣ-carotene, we also observed accumulation of myxoxanthophyll and more even spatial distribution of photosystems and phycobilisomes between microdomains. We suggest that the overall carotenoid increase during HL stress could be involved either in the direct photoprotection (e.g., in ROS scavenging) and/or could play an additional role in maintaining optimal distribution of photosystems in thylakoid membrane to attain efficient photoprotection.