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Biological membranes are complex, heterogeneous, and dynamic systems that play roles in the compartmentalization and protection of cells from the environment. It is still a challenge to elucidate kinetics and real-time transport routes for molecules through biological membranes in live cells. Currently, by developing and employing super-resolution microscopy; increasing evidence indicates channels and transporter nano-organization and dynamics within membranes play an important role in these regulatory mechanisms. Here we review recent advances and discuss the major advantages and disadvantages of using super-resolution microscopy to investigate protein organization and transport within plasma membranes.

Recently, the localization of amyloid precursor protein (APP) into reversible nanoscale supramolecular assembly or “nanodomains” has been highlighted as crucial towards understanding the onset of the molecular pathology of Alzheimer’s disease (AD). Surface expression of APP is regulated by proteins interacting with it, controlling its retention and lateral trafficking on the synaptic membrane. Here, we evaluated the involvement of a key risk factor for AD, PICALM, as a critical regulator of nanoscale dynamics of APP. Although it was enriched in the postsynaptic density, PICALM was also localized to the presynaptic active zone and the endocytic zone. PICALM colocalized with APP and formed nanodomains with distinct morphological properties in different subsynaptic regions. Next, we evaluated if this localization to subsynaptic compartments was regulated by the C-terminal sequences of APP, namely, the “Y682ENPTY687” domain. Towards this, we found that deletion of C-terminal regions of APP with partial or complete deletion of Y682ENPTY687, namely, APP–Δ9 and APP–Δ14, affected the lateral diffusion and nanoscale segregation of APP. Lateral diffusion of APP mutant APP–Δ14 sequence mimicked that of a detrimental Swedish mutant of APP, namely, APP–SWE, while APP–Δ9 diffused similar to wild-type APP. Interestingly, elevated expression of PICALM differentially altered the lateral diffusion of the APP C-terminal deletion mutants. These observations confirm that the C-terminal sequence of APP regulates its lateral diffusion and the formation of reversible nanoscale domains. Thus, when combined with autosomal dominant mutations, it generates distinct molecular patterns leading to onset of Alzheimer’s disease (AD). [ABSTRACT FROM AUTHOR]

ABSTRACTThe cell membrane is a fundamental biological structure, which is only 6–10 nm thick. It is composed of hundreds of lipid types, which form small and dynamic lipid domains or rafts. These rafts are thought to be a major aspect of cell organization, to provide support for various transmembrane proteins and are central to the communication of cells with their environs. Understanding the functions of lipid rafts presents an exciting opportunity to understand the molecular mechanisms of biologically important processes, as well as to uncover fundamental molecular mechanisms of membrane-associated diseases. Due to the high complexity of cell membranes, model membranes composed of synthetic lipids have been developed and are widely used to mimic biomembranes in an effort to study the structure and dynamics of lipid domains and their role in cell function. Atomic force microscopy (AFM), Kelvin probe force microscopy (KPFM) and atomic force spectroscopy (AFS) significantly advanced the study of nanodomains in model lipid membranes and monolayers. We review applications of these methods to the study of model membranes, which are widely used to mimic eukaryotic and bacterial cells, as well as neuronal cellular membranes in Alzheimer’s disease (AD).

The impact of microstructure on hardness in phase‐separated calcium aluminosilicate glasses is investigated. Changes in hardness are governed by microstructure deformations that occur during indentation. Phase separation leads to decreased hardness due to the incongruent yielding of the droplet and matrix phases. Moreover, the deformation of microstructures possessing dilute, spherical droplets did not have a significant impact on hardness. Microstructures characterized by concentrated, acicular droplets were found to deform through a process of droplet coalescence. This process absorbs additional energy during yielding and results in glasses that deform through droplet coalescence possessing improved hardness. [ABSTRACT FROM AUTHOR]

This paper deals with the problems encountered in the study of eukaryotic cell membranes. A discussion on the structure and composition of membranes, lateral heterogeneity of membranes, lipid raft formation, and involvement of actin and cytoskeleton networks in the maintenance of membrane structure is included. Modern methods for the study of membranes and their constituent domains are discussed. Various simplified models of biomembranes and lipid rafts are presented. Computer modelling is considered as one of the most important methods. This is stated that from the study of the plasma membrane structure, it is desirable to proceed to the diverse membranes of all organelles of the cell. The qualitative composition and molar content of individual classes of polar lipids, free sterols and proteins in each of these membranes must be considered. A program to create an open access electronic database including results obtained from the membrane modelling of individual cell organelles and the key sites of the membranes, as well as models of individual molecules composing the membranes, has been proposed. [ABSTRACT FROM AUTHOR]

Rapid release of calcium from internal stores via ryanodine receptors (RyRs) is one of the fastest types of cytoplasmic second messenger signalling in excitable cells. In the heart, rapid summation of the elementary events of calcium release, 'calcium sparks', determine the contraction of the myocardium. We adapted a correlative super-resolution microscopy protocol to correlate sub-plasmalemmal spontaneous calcium sparks in rat right ventricular myocytes with the local nanoscale RyR2 positions. This revealed a steep relationship between the integral of a calcium spark and the sum of the local RyR2s. Segmentation of recurring spark sites showed evidence of repeated and triggered saltatory activation of multiple local RyR2 clusters. In myocytes taken from failing right ventricles, RyR2 clusters themselves showed a dissipated morphology and fragmented (smaller) clusters. They also featured greater heterogeneity in both the spark properties and the relationship between the integral of the calcium spark and the local ensemble of RyR2s. While fragmented (smaller) RyR2 clusters were rarely observed directly underlying the larger sparks or the recurring spark sites, local interrogation of the channel-to-channel distances confirmed a clear link between the positions of each calcium spark and the tight, non-random clustering of the local RyR2 in both healthy and failing ventricles.

Summary: In Arabidopsis, the receptor‐like kinase (RLK) FERONIA (FER) senses peptide ligands in the plasma membrane (PM), modulates plant growth and development, and integrates biotic and abiotic stress signaling for downstream adaptive responses. However, the molecular interplay of these diverse processes is largely unknown.Here, we show that FER, the receptor of Rapid Alkalinization Factor 1 (RALF1), physically interacts with C2 domain ABA‐related (CAR) proteins to control the nano‐organization of the PM. During this process, the RALF1‐FER pathway upregulates CAR protein translation, and then more CAR proteins are recruited to the PM. This acts as a rapid feedforward loop that stabilizes the PM liquid‐ordered phase. FER interacts with and phosphorylates CARs, thereby reducing their lipid‐binding ability and breaking the feedback regulation at later time points.The formation of the flg22‐induced FLS2‐BAK1 immune complex, which depends on the integrity of FER‐containing nanodomains, is impaired in fer and pentuple car14569 mutant.Together, we propose that the FER‐CAR module controls the formation of PM nano‐organization during RALF signaling through a self‐contained amplifying loop including both positive and negative feedback. [ABSTRACT FROM AUTHOR]

Cardiolipin (CL) is a key lipid for damaged mitochondrial recognition by the LC3/GABARAP human autophagy proteins. The role of ceramide (Cer) in this process is unclear, but CL and Cer have been proposed to coexist in mitochondria under certain conditions. Varela et al. showed that in model membranes composed of egg sphingomyelin (eSM), dioleoyl phosphatidylethanolamine (DOPE), and CL, the addition of Cer enhanced the binding of LC3/GABARAP proteins to bilayers. Cer gave rise to lateral phase separation of Cer-rich rigid domains but protein binding took place mainly in the fluid continuous phase. In the present study, a biophysical analysis of bilayers composed of eSM, DOPE, CL, and/or Cer was attempted to understand the relevance of this lipid coexistence. Bilayers were studied by differential scanning calorimetry, confocal fluorescence microscopy, and atomic force microscopy. Upon the addition of CL and Cer, one continuous phase and two segregated ones were formed. In bilayers with egg phosphatidylcholine instead of eSM, in which the binding of LC3/GABARAP proteins hardly increased with Cer in the former study, a single segregated phase was formed. Assuming that phase separation at the nanoscale is ruled by the same principles acting at the micrometer scale, it is proposed that Cer-enriched rigid nanodomains, stabilized by eSM:Cer interactions formed within the DOPE- and CL-enriched fluid phase, result in structural defects at the rigid/fluid nanointerfaces, thus hypothetically facilitatingLC3/GABARAP protein interaction. [ABSTRACT FROM AUTHOR]

Super-resolution microscopy allowed major improvements in our capacity to describe and explain biological organization at the nanoscale. Single-molecule localization microscopy (SMLM) uses the positions of molecules to create super-resolved images, but it can also provide new insights into the organization of molecules through appropriate pointillistic analyses that fully exploit the sparse nature of SMLM data. However, the main drawback of SMLM is the lack of analytical tools easily applicable to the diverse types of data that can arise from biological samples. Typically, a cloud of detections may be a cluster of molecules or not depending on the local density of detections, but also on the size of molecules themselves, the labeling technique, the photo-physics of the fluorophore, and the imaging conditions. We aimed to set an easy-to-use clustering analysis protocol adaptable to different types of data. Here, we introduce Diinamic, which combines different density-based analyses and optional thresholding to facilitate the detection of clusters. On simulated or real SMLM data, Diinamic correctly identified clusters of different sizes and densities, being performant even in noisy datasets with multiple detections per fluorophore. It also detected subdomains (“nanodomains”) in clusters with non-homogeneous distribution of detections.

Accumulation of aggregates of the microtubule‐binding protein Tau is a pathological hallmark of Alzheimer's disease. While Tau is thought to primarily associate with microtubules, it also interacts with and localizes to the plasma membrane. However, little is known about how Tau behaves and organizes at the plasma membrane of live cells. Using quantitative, single‐molecule imaging, we show that Tau exhibits spatial and kinetic heterogeneity near the plasma membrane of live cells, resulting in the formation of nanometer‐sized hot spots. The hot spots lasted tens of seconds, much longer than the short dwell time (∼ 40 ms) of Tau on microtubules. Pharmacological and biochemical disruption of Tau/microtubule interactions did not prevent hot spot formation, suggesting that these are different from the reported Tau condensation on microtubules. Although cholesterol removal has been shown to reduce Tau pathology, its acute depletion did not affect Tau hot spot dynamics. Our study identifies an intrinsic dynamic property of Tau near the plasma membrane that may facilitate the formation of assembly sites for Tau to assume its physiological and pathological functions. Synopsis: The cytosolic leaflet of the plasma membrane has long been suggested as a site for Tau to execute some of its functions. However, how this protein behaves and organizes at the plasma membrane of live cells remains largely unknown. Using single‐molecule super‐resolution imaging, this study quantifies Tau's spatiotemporal organization at a nanoscale level in this compartment. Tau exists in multiple spatial and kinetic subpopulations near the plasma membrane.Preventing Tau/microtubule interactions biochemically or pharmacologically increases the mobility of individual Tau molecules.Tau forms dynamic, nanometer‐sized hot spots lasting tens of seconds.Tau's mobility is reduced within the hot spots.Tau hot spots persist even after the disruption of Tau/microtubule interactions and cholesterol depletion. [ABSTRACT FROM AUTHOR]

Protein-protein interaction studies provide valuable insights into cellular signaling. Brassinosteroid (BR) signaling is initiated by the hormone-binding receptor Brassinosteroid Insensitive 1 (BRI1) and its co-receptor BRI1 Associated Kinase 1 (BAK1). BRI1 and BAK1 were shown to interact independently with the Receptor-Like Protein 44 (RLP44), which is implicated in BRI1/BAK1-dependent cell wall integrity perception. To demonstrate the proposed complex formation of BRI1, BAK1 and RLP44, we established three-fluorophore intensity-based spectral Förster resonance energy transfer (FRET) and FRET-fluorescence lifetime imaging microscopy (FLIM) for living plant cells. Our evidence indicates that RLP44, BRI1 and BAK1 form a ternary complex in a distinct plasma membrane nanodomain. In contrast, although the immune receptor Flagellin Sensing 2 (FLS2) also forms a heteromer with BAK1, the FLS2/BAK1 complexes are localized to other nanodomains. In conclusion, both three-fluorophore FRET approaches provide a feasible basis for studying the in vivo interaction and sub-compartmentalization of proteins in great detail. [ABSTRACT FROM AUTHOR]

The polarization difference (∆P) and the breakdown field strength (BDS) of ceramics are both key factors in achieving enhanced energy storage performance, such as energy storage density (W), recoverable energy storage density (Wrec), and energy storage efficiency (η). Using BaTiO3 (BT) as the main crystal phase and utilizing K0.5Na0.5NbO3 (KNN) as the coating agent, sintering aid, and additives, BT-KNN ceramics with grain sizes of 100 nm and 200 nm were synthesized by the self-assembly sintering method, respectively. The as-obtained BT-KNN ceramics showed obvious nanodomains, relaxor behaviors, and dielectric temperature stability, with high ∆P and large BDS. The BT-KNN ceramics with grain size of 200 nm have higher energy storage properties, including W (2.50 J/cm3), Wrec (2.08 J/cm3), and η (83.2%), than those of the BT-KNN ceramics with the grain size of 100 nm. This research may provide a theoretical basis for preparing BT-based ceramics with high energy storage performance. [ABSTRACT FROM AUTHOR]

Summary: Phosphatidylinositol 4‐phosphate (PI4P) is an anionic phospholipid which has been described as a master regulator of the Golgi apparatus in eukaryotic cells. However, recent evidence suggests that PI4P mainly accumulates at the plasma membrane in all plant cells analyzed so far. In addition, many functions that are typically attributed to phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P2) in animal and yeast cells are also supported by PI4P in plants. For example, PI4P is the key anionic lipid that powers the strong electrostatic properties of the plasma membrane. Phosphatidylinositol 4‐phosphate is also required for the establishment of stable membrane contacts between the endoplasmic reticulum and the plasma membrane, for exocytosis and to support signaling pathways. Thus, we propose that PI4P has a prominent role in specifying the identity of the plasma membrane and in supporting some of its key functions and should be considered a hallmark lipid of this compartment. [ABSTRACT FROM AUTHOR]

Strain glass is a new strain state discovered recently in ferroelastic systems that is characterized by nanoscale martensitic domains formed through a freezing transition. These nanodomains typically have mottled or tweed morphology depending on the elastic anisotropy of the system. Strain glass transition is a broadly smeared and high order–like transition, taking place within a wide temperature or stress range. It is accompanied by many unique properties, including linear superelasticity with high strength, low modulus, Invar and Elinvar anomalies, and large magnetostriction. In this review, we first discuss experimental characterization and testing that have led to the discovery of the strain glass transition and its unique properties. We then introduce theoretical models and computer simulations that have shed light on the origin and mechanisms underlying the unique characteristics and properties of strain glass transitions. Unresolved issues and challenges in strain glass study are also addressed. Strain glass transition can offer giant elastic strain and ultralow elastic modulus by well-controlled reversible structural phase transformations through defect engineering. [ABSTRACT FROM AUTHOR]

Despite its low abundance, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a key modulator of membrane-associated signaling events in eukaryotic cells. Temporal and spatial regulation of PI(4,5)P2 concentration can achieve localized increases in the levels of this lipid, which are crucial for the activation or recruitment of peripheral proteins to the plasma membrane. The recent observation of the dramatic impact of physiological divalent cation concentrations on PI(4,5)P2 clustering, suggests that protein anchoring to the plasma membrane through PI(4,5)P2 is likely not defined solely by a simple (monomeric PI(4,5)P2)/(protein bound PI(4,5)P2) equilibrium, but instead depends on complex protein interactions with PI(4,5)P2 clusters. The insertion of PI(4,5)P2-binding proteins within these clusters can putatively modulate protein–protein interactions in the membrane, but the relevance of such effects is largely unknown. In this work, we characterized the impact of Ca2+ on the organization and protein–protein interactions of PI(4,5)P2-binding proteins. We show that, in giant unilamellar vesicles presenting PI(4,5)P2, the membrane diffusion properties of pleckstrin homology (PH) domains tagged with a yellow fluorescent protein (YFP) are affected by the presence of Ca2+, suggesting direct interactions between the protein and PI(4,5)P2 clusters. Importantly, PH-YFP is found to dimerize in the membrane in the absence of Ca2+. This oligomerization is inhibited in the presence of physiological concentrations of the divalent cation. These results confirm that cation-dependent PI(4,5)P2 clustering promotes interactions between PI(4,5)P2-binding proteins and has the potential to dramatically influence the organization and downstream interactions of PI(4,5)P2-binding proteins in the plasma membrane. [ABSTRACT FROM AUTHOR]

The 5 known melanocortin receptors (MCs) have established physiological roles. With the exception of MC2, these receptors can behave unpredictably, and since they are more widely expressed than their established roles would suggest, it is likely that they have other poorly characterized functions. The aim of this review is to discuss some of the less well-explored aspects of the 4 enigmatic members of this receptor family (MC1,3-5) and describe how these are multifaceted G protein–coupled receptors (GPCRs). These receptors appear to be promiscuous in that they bind several endogenous agonists (products of the proopiomelanocortin [ POMC ] gene) and antagonists but with inconsistent relative affinities and effects. We propose that this is a result of posttranslational modifications that determine receptor localization within nanodomains. Within each nanodomain there will be a variety of proteins, including ion channels, modifying proteins, and other GPCRs, that can interact with the MCs to alter the availability of receptor at the cell surface as well as the intracellular signaling resulting from receptor activation. Different combinations of interacting proteins and MCs may therefore give rise to the complex and inconsistent functional profiles reported for the MCs. For further progress in understanding this family, improved characterization of tissue-specific functions is required. Current evidence for interactions of these receptors with a range of partners, resulting in modulation of cell signaling, suggests that each should be studied within the full context of their interacting partners. The role of physiological status in determining this context also remains to be characterized. [ABSTRACT FROM AUTHOR]

Lead-free piezoelectric actuators emerge as promising substitutes for their lead-containing counterparts to address environmental concerns. However, they often confront a trade-off between low driving electric fields and high electro-strain. Herein, a novel strategy to boost electro-strain under low electric fields is proposed by doping high-electronegativity B-site atoms into perovskite potassium sodium niobate-based ceramics. Our findings reveal that high-electronegativity B-site atoms elevate the covalency of B-O bonding, softening the short-range repulsion and introducing local multiphase coexistence. This leads to more nanoscale domain structures and lower coercive field, thereby enabling large strains to be produced at lower electric fields. Notably, a substantial 0.2 % bipolar electro-strain and 0.1 % unipolar electro-strain under 10 kV cm-1 is achieved in Sr, Sb co-doped potassium sodium niobate ceramics, with a broad working frequency and temperature range, as well as excellent fatigue resistance. This study unveils innovative insights into designing lead-free piezoelectric ceramics with remarkable electro-strain performance and low driving electric field, promising a significant advancement in lead-free piezoelectric materials science and piezoelectric actuators. [Display omitted] [ABSTRACT FROM AUTHOR]

Driven by the increasing demands in advanced aerospace engineering, the integrated structural and functional materials are explored. In present study, we fabricate the TiB w /Ti–V–Al lightweight shape memory alloy composites with large recoverable strain (>5 %), high specific strength (>200 MPa cm3/g) and good elongation (>20 %). The satisfied structural and functional performances are attributed to the unique gradient microstructure, including TiB whiskers, short-range martensitic nanodomains and long-range martensitic microdomains. TiB w with the optimized orientation exhibits high load-bearing capacity. The transition area between TiB w and matrix is composed of short-range martensitic nanodomains. Nanodomains are affected by the diffused interstitial B atoms and local internal stress field regulated by TiB w. The elastic interaction energy between nanodomains and TiB w are calculated according to the Eshelby method. Upon deformation, nanodomains grow to long-range martensitic laths. The long-range martensitic laths keep stable after unloading. The microstructure evolution ties well with the Landau free energy model. It achieves effective loading transfer from matrix to reinforcement phase, resulting in less irreversible defects and better shape memory effect. In addition, grain refinement strengthening, loading transfer strengthening and solution strengthening are utilized to achieve the improvement of the strength and plasticity. The finding offers a promising inspiration for the development of new type shape memory alloy composites with integrated structural and functional properties. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are secreted from all cell types and are intimately involved in tissue homeostasis. They are being explored as vaccine and gene therapy platforms, as well as potential biomarkers. As their size is below the diffraction limit of light microscopy, direct visualizations have been daunting and single‐particle studies under physiological conditions have been hampered. Here, direct stochastic optical reconstruction microscopy (dSTORM) was employed to visualize EVs in three‐dimensions and to localize molecule clusters such as the tetraspanins CD81 and CD9 on the surface of individual EVs. These studies demonstrate the existence of membrane microdomains on EVs. These were confirmed by Cryo‐EM. Individual particle visualization provided insights into the heterogeneity, structure, and complexity of EVs not previously appreciated [ABSTRACT FROM AUTHOR]

Glasses with nanoscale phase separation have the potential to possess improved hardness and fracture toughness while maintaining their optical transparency. Here we present the results of isothermal heat treatments of phase‐separated calcium aluminosilicate glasses. Our results indicate that a transition from Lifshitz–Slozof–Wagner (LSW)‐type kinetics to a diffusion‐controlled pseudo‐coalescence mechanism occurs at ~17% droplet volume fraction, which results in the droplets becoming increasingly elongated and interconnected. The activation barrier for both mechanisms suggests that calcium diffusion is the underlying means for the coarsening of the silica‐rich domains. Simple approximations show the transition cannot be explained by Brownian motion or Van der Waals attraction between domains, and instead suggest various osmotic forces may be responsible. [ABSTRACT FROM AUTHOR]

α2δ proteins (CACNA2D1–4) are required for normal neurological function and contribute to membrane trafficking of voltage‐gated calcium channels, through which calcium entry initiates numerous physiological processes. However, it remains unclear how α2δ proteins influence calcium‐mediated signalling to control neuronal output. Using whole‐cell recordings of mouse Purkinje cells, we show that α2δ‐2 is required for functional coupling of postsynaptic voltage‐dependent calcium entry with calcium‐dependent effector mechanisms controlling two different outputs, depolarization‐induced suppression of excitation and spike afterhyperpolarization. Our findings indicate an important role for α2δ‐2 proteins in regulating functional postsynaptic calcium channel coupling in neurons, providing new context for understanding the effects of α2δ mutations on neuronal circuit function and presenting additional potential avenues to manipulate α2δ‐mediated signalling for therapeutic gain. Key points: Calcium influx, via voltage‐dependent calcium channels, drives numerous neuronal signalling processes with precision achieved in part by tight coupling between calcium entry and calcium‐dependent effectors.α2δ proteins are important for neurological function and contribute to calcium channel membrane trafficking, although how α2δ proteins influence postsynaptic calcium‐dependent signalling is largely unexplored.Here it is shown that loss of α2δ‐2 proteins disrupts functional calcium coupling to two different postsynaptic calcium‐dependent signals in mouse Purkinje cell neurons, retrograde endocannabinoid signalling and the action potential afterhyperpolarization.The findings provide new insights into the control of calcium coupling as well as new roles for α2δ‐2 proteins in neurons. [ABSTRACT FROM AUTHOR]

Abstract Extracellular vesicles (EVs) are secreted from all cell types and are intimately involved in tissue homeostasis. They are being explored as vaccine and gene therapy platforms, as well as potential biomarkers. As their size is below the diffraction limit of light microscopy, direct visualizations have been daunting and single‐particle studies under physiological conditions have been hampered. Here, direct stochastic optical reconstruction microscopy (dSTORM) was employed to visualize EVs in three‐dimensions and to localize molecule clusters such as the tetraspanins CD81 and CD9 on the surface of individual EVs. These studies demonstrate the existence of membrane microdomains on EVs. These were confirmed by Cryo‐EM. Individual particle visualization provided insights into the heterogeneity, structure, and complexity of EVs not previously appreciated

Biological membranes are complex, heterogeneous, and dynamic systems that play roles in the compartmentalization and protection of cells from the environment. It is still a challenge to elucidate kinetics and real-time transport routes for molecules through biological membranes in live cells. Currently, by developing and employing super-resolution microscopy; increasing evidence indicates channels and transporter nano-organization and dynamics within membranes play an important role in these regulatory mechanisms. Here we review recent advances and discuss the major advantages and disadvantages of using super-resolution microscopy to investigate protein organization and transport within plasma membranes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zulueta Diaz and Arnspang.)

The soybean root system is complex. In addition to being composed of various cell types, the soybean root system includes the primary root, the lateral roots, and the nodule, an organ in which mutualistic symbiosis with N-fixing rhizobia occurs. A mature soybean root nodule is characterized by a central infection zone where atmospheric nitrogen is fixed and assimilated by the symbiont, resulting from the close cooperation between the plant cell and the bacteria. To date, the transcriptome of individual cells isolated from developing soybean nodules has been established, but the transcriptomic signatures of cells from the mature soybean nodule have not yet been characterized. Using single-nucleus RNA-seq and Molecular Cartography technologies, we precisely characterized the transcriptomic signature of soybean root and mature nodule cell types and revealed the co-existence of different sub-populations of B. diazoefficiens-infected cells in the mature soybean nodule, including those actively involved in nitrogen fixation and those engaged in senescence. Mining of the single-cell-resolution nodule transcriptome atlas and the associated gene co-expression network confirmed the role of known nodulation-related genes and identified new genes that control the nodulation process. For instance, we functionally characterized the role of GmFWL3, a plasma membrane microdomain-associated protein that controls rhizobial infection. Our study reveals the unique cellular complexity of the mature soybean nodule and helps redefine the concept of cell types when considering the infection zone of the soybean nodule., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Protein-protein interaction studies provide valuable insights into cellular signaling. Brassinosteroid (BR) signaling is initiated by the hormone-binding receptor Brassinosteroid Insensitive 1 (BRI1) and its co-receptor BRI1 Associated Kinase 1 (BAK1). BRI1 and BAK1 were shown to interact independently with the Receptor-Like Protein 44 (RLP44), which is implicated in BRI1/BAK1-dependent cell wall integrity perception. To demonstrate the proposed complex formation of BRI1, BAK1 and RLP44, we established three-fluorophore intensity-based spectral Förster resonance energy transfer (FRET) and FRET-fluorescence lifetime imaging microscopy (FLIM) for living plant cells. Our evidence indicates that RLP44, BRI1 and BAK1 form a ternary complex in a distinct plasma membrane nanodomain. In contrast, although the immune receptor Flagellin Sensing 2 (FLS2) also forms a heteromer with BAK1, the FLS2/BAK1 complexes are localized to other nanodomains. In conclusion, both three-fluorophore FRET approaches provide a feasible basis for studying the in vivo interaction and sub-compartmentalization of proteins in great detail.

[Display omitted] • DiphenylPhosphate(DPP)/bis(2-ethylhexyl)Amine(BEEA) mixtures show optical birefringence if subjected to a magnetic field. • Magnetically-induced birefringence data suggests that DPP-BEEA anisotropy contrasts that shown by unassociated species. • Around DPP molar ratio 0.1–0.2, a probable percolation of DPP/BEEA ionic liquid domains takes place. Diphenyl phosphate (DPP)/bis(2-ethylhexyl) amine (BEEA) liquid mixtures are expected to show peculiar self-assembly behavior due to their, respectively, acidic and basic nature which can trigger acid-base reaction. The properties of DPP/BEEA mixtures of different compositions are explored by a combined theoretical (Ab initio calculations) and experimental (rheology, X-ray and light scattering) approach. (i) a proton transfer from DPP to BEEA takes place with formation of cationic and anionic species in liquid phase (ion pair formation); (ii) due to the strong and long-range electrostatic interactions, each of the two charged species is preferentially surrounded by the other one, in a picture resembling the structure of ionic materials; (iii) this gives rise to a striking viscosity increase takes place as DPP concentration increases; (iv) the composition dependencies of all measured parameters show clear deviations around DPP molar fraction of 0.2, indicating unequivocally the formation of isolated domains, formed by DPP-BEEA pairs which coalescence/percolate into a unique, extended, network at higher DPP concentrations. [ABSTRACT FROM AUTHOR]

Compartmentalization, together with transbilayer and lateral asymmetries, provide the structural foundation for functional specializations at the cell surface, including the active role of the lipid microenvironment in the modulation of membrane-bound proteins. The chemical synapse, the site where neurotransmitter-coded signals are decoded by neurotransmitter receptors, adds another layer of complexity to the plasma membrane architectural intricacy, mainly due to the need to accommodate a sizeable number of molecules in a minute subcellular compartment with dimensions barely reaching the micrometer. In this review, we discuss how nature has developed suitable adjustments to accommodate different types of membrane-bound receptors and scaffolding proteins via membrane microdomains, and how this “effort-sharing” mechanism has evolved to optimize crosstalk, separation, or coupling, where/when appropriate. We focus on a fast ligand-gated neurotransmitter receptor, the nicotinic acetylcholine receptor, and a second-messenger G-protein coupled receptor, the cannabinoid receptor, as a paradigmatic example.

Calcium handling is vital to normal physiological function in the heart. Human atrial arrhythmias, eg. atrial fibrillation, are a major morbidity and mortality burden, yet major gaps remain in our understanding of how calcium signaling pathways function and interact. Inositol trisphosphate (IP3) is a Ca2+-mobilizing second messenger and its agonist-induced effects have been observed in many tissue types. In the atria IP3 receptors (IR3Rs) residing on junctional sarcoplasmic reticulum augment cellular Ca2+ transients and, when over-stimulated, lead to arrhythmogenesis. Recent studies have demonstrated that the predominant pathway for IP3 actions in atrial myocytes depends on stimulation of calcium-dependent forms of adenylyl cyclase (AC8 and AC1) by IP3-evoked Ca2+ release from the sarcoplasmic reticulum. AC8 shows co-localisation with IP3Rs and AC1 appears to be nearby. These observations support crosstalk between Ca2+ and cAMP pathways in nanodomains in atria. Similar mechanisms also appear to operate in the pacemaker region of the sinoatrial node. Here we discuss these significant advances in our understanding of atrial physiology and pathology, together with implications for the identification of potential novel targets and modulators for the treatment of atrial arrhythmias. [ABSTRACT FROM AUTHOR]

Calcium handling is vital to normal physiological function in the heart. Human atrial arrhythmias, eg. atrial fibrillation, are a major morbidity and mortality burden, yet major gaps remain in our understanding of how calcium signaling pathways function and interact. Inositol trisphosphate (IP3) is a calcium-mobilizing second messenger and its agonist-induced effects have been observed in many tissue types. In the atria IP3 receptors (IR3Rs) residing on junctional sarcoplasmic reticulum augment cellular calcium transients and, when over-stimulated, lead to arrhythmogenesis. Recent studies have demonstrated that the predominant pathway for IP3 actions in atrial myocytes depends on stimulation of calcium-dependent forms of adenylyl cyclase (AC8 and AC1) by IP3-evoked calcium release from the sarcoplasmic reticulum. AC8 shows co-localisation with IP3Rs and AC1 appears to be nearby. These observations support crosstalk between calcium and cAMP pathways in nanodomains in atria. Similar mechanisms also appear to operate in the pacemaker region of the sinoatrial node. Here we discuss these significant advances in our understanding of atrial physiology and pathology, together with implications for the identification of potential novel targets and modulators for the treatment of atrial arrhythmias. [ABSTRACT FROM AUTHOR]

Despite its low abundance, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a key modulator of membrane-associated signaling events in eukaryotic cells. Temporal and spatial regulation of PI(4,5)P2 concentration can achieve localized increases in the levels of this lipid, which are crucial for the activation or recruitment of peripheral proteins to the plasma membrane. The recent observation of the dramatic impact of physiological divalent cation concentrations on PI(4,5)P2 clustering, suggests that protein anchoring to the plasma membrane through PI(4,5)P2 is likely not defined solely by a simple (monomeric PI(4,5)P2)/(protein bound PI(4,5)P2) equilibrium, but instead depends on complex protein interactions with PI(4,5)P2 clusters. The insertion of PI(4,5)P2-binding proteins within these clusters can putatively modulate protein–protein interactions in the membrane, but the relevance of such effects is largely unknown. In this work, we characterized the impact of Ca2+ on the organization and protein–protein interactions of PI(4,5)P2-binding proteins. We show that, in giant unilamellar vesicles presenting PI(4,5)P2, the membrane diffusion properties of pleckstrin homology (PH) domains tagged with a yellow fluorescent protein (YFP) are affected by the presence of Ca2+, suggesting direct interactions between the protein and PI(4,5)P2 clusters. Importantly, PH-YFP is found to dimerize in the membrane in the absence of Ca2+. This oligomerization is inhibited in the presence of physiological concentrations of the divalent cation. These results confirm that cation-dependent PI(4,5)P2 clustering promotes interactions between PI(4,5)P2-binding proteins and has the potential to dramatically influence the organization and downstream interactions of PI(4,5)P2-binding proteins in the plasma membrane.

Keywords: -adrenergic receptors; AKAPs; nanodomains; signaling EN -adrenergic receptors AKAPs nanodomains signaling 1 3 3 08/11/22 20220501 NES 220501 A Perspective on "Subcellular Propagation of Cardiomyocyte -Adrenergic Activation of Calcium... The least one expects from an anchor is to stay put (freely adapted from Salvador Dali's opinion about Alexander Calder's sculptures). Phosphorylation state-dependent interaction between AKAP7delta/gamma and phospholamban increases phospholamban phosphorylation. They show that the PLN-targeted intracellular 1AR-PKA-AKAP7 complex they recently identified[9] is endowed with considerable mobility, exceeding that of PLN itself. [Extracted from the article]

Plasmodesmata are intercellular pores connecting together most plant cells. These structures consist of a central constricted form of the endoplasmic reticulum, encircled by some cytoplasmic space, in turn delimited by the plasma membrane, itself ultimately surrounded by the cell wall. The presence and structure of plasmodesmata create multiple routes for intercellular trafficking of a large spectrum of molecules (encompassing RNAs, proteins, hormones and metabolites) and also enable local signalling events. Movement across plasmodesmata is finely controlled in order to balance processes requiring communication with those necessitating symplastic isolation. Here, we describe the identities and roles of the molecular components (specific sets of lipids, proteins and wall polysaccharides) that shape and define plasmodesmata structural and functional domains. We highlight the extensive and dynamic interactions that exist between the plasma/endoplasmic reticulum membranes, cytoplasm and cell wall domains, binding them together to effectively define plasmodesmata shapes and purposes. [ABSTRACT FROM AUTHOR]

Action potentials arriving at a nerve terminal activate voltage-gated calcium channels and set the electrical driving force for calcium entry which affects the amount and duration of neurotransmitter release. During propagation, the duration, amplitude, and shape of action potentials often changes. This affects calcium entry, and can cause large changes in neurotransmitter release. Here, we have used a series of amplitude and area matched stimuli to examine how the shape and amplitude of a stimulus affect calcium influx at a presynaptic nerve terminal in the mammalian brain. We identify fundamental differences in calcium entry following calcium channel activation by a standard voltage jump vs. an action potential-like stimulation. We also tested a series of action potential-like stimuli with the same amplitude, duration, and stimulus area, but differing in their rise and decay times. We find that a stimulus that matches the rise and decay times of a physiological action potential produces a calcium channel response that is optimized over a range of peak amplitudes. Next, we determined the relative number of calcium channels that are active at different times during an action potential, which is important in the context of how local calcium domains trigger neurotransmitter release. We find the depolarizing phase of an AP-like stimulus only opens ~20% of the maximum number of calcium channels that can be activated. Channels continue to activate during the falling phase of the action potential, with peak calcium channel activation occurring near 0 mV. Although less than 25% of calcium channels are active at the end of the action potential, these calcium channels will generate a larger local calcium concentration that will increase the release probability for nearby vesicles. Determining the change in open probability of presynaptic calcium channels, and taking into account how local calcium concentration also changes throughout the action potential are both necessary to fully understand how the action potential triggers neurotransmitter release.

The plasma membrane is a complex system, consisting of two layers of lipids and proteins compartmentalized into small structures called nanodomains. Despite the asymmetric composition of both leaflets, coupling between the layers is surprisingly strong. This can be evidenced, for example, by recent experimental studies performed on phospholipid giant unilamellar vesicles showing that nanodomains formed in the outer layer are perfectly registered with those in the inner leaflet. Similarly, microscopic phase separation in one leaflet can induce phase separation in the opposing leaflet that would otherwise be homogeneous. In this review, we summarize the current theoretical and experimental knowledge that led to the current view that domains are – irrespective of their size – commonly registered across the bilayer. Mechanisms inducing registration of nanodomains suggested by theory and calculations are discussed. Furthermore, domain coupling is evidenced by experimental studies based on the sparse number of methods that can resolve registered from independent nanodomains. Finally, implications that those findings using model membrane studies might have for cellular membranes are discussed.

In response to the invasion of microorganisms, plants actively balance their resources for growth and defence, thus ensuring their survival. The regulatory mechanisms underlying plant immunity and growth operate through complex networks, in which the brassinosteroid phytohormone is one of the central players. In the past decades, a growing number of studies have revealed a multi-layered crosstalk between brassinosteroid-mediated growth and plant immunity. In this Review, by means of the tango metaphor, we immerse ourselves into the intimate relationship between brassinosteroid and plant immune signalling pathways that is tailored by the lifestyle of the pathogen and modulated by other phytohormones. The plasma membrane is the unique stage where brassinosteroid and immune signals are dynamically integrated and where compartmentalization into nanodomains that host distinct protein consortia is crucial for the dance. Shared downstream signalling components and transcription factors relay the tango play to the nucleus to activate the plant defence response and other phytohormonal signalling pathways for the finale. Understanding how brassinosteroid and immune signalling pathways are integrated in plants will help develop strategies to minimize the growth–defence trade-off, a key challenge for crop improvement. [ABSTRACT FROM AUTHOR]

Gangliosides are glycosphingolipids consisting of a ceramide base and a bulky sugar chain that contains one or more sialic acids. This unique structure endows gangliosides with a strong tendency to self‐aggregate in solution, as well as in cellular membranes, where they can form nanoscopic assemblies called ganglioside nanodomains. As gangliosides are important biological molecules involved in a number of physiological processes, characterization of their lateral organization in membranes is essential. This review aims at providing comprehensive information about the nanoscale organization of gangliosides in various synthetic models. To this end, the impact of the hydrophobic backbone and the headgroup on the segregation of gangliosides into nanodomains are discussed in detail, as well as the way in which the properties of nanodomains are affected by ligand binding. Small size makes the characterization of ganglioside nanodomains challenging, and we thus highlight the biophysical methods that have advanced this research, such as Monte Carlo Förster resonance energy transfer, atomic force microscopy and approaches based on molecular diffusion. [ABSTRACT FROM AUTHOR]

The requirement for ferroelectric ceramics with a high Curie temperature and a high piezoelectric coefficient remains an important research task for high‐temperature sensors and actuators applications. (0.76‐x)BiFeO3‐0.24PbTiO3‐xBa(Sn0.2Ti0.8)O3 (BF‐PT‐BST) piezoelectric ceramics were fabricated using the solid‐state reaction method. XRD analysis indicated that the incorporation of large ionic radius Ba2+ at A‐site and nonferroelectric‐active Sn4+ at B‐site generated a decrease in the tetragonality degree c/a. A wide multiphase coexistence region was formed with the content of BST ranging from 0.13 to 0.28. The enhanced piezoelectric coefficient (d33 ~ 200pC/N) was achieved while maintaining a high Curie temperature (TC ~ 500°C) and a high depolarization temperature (Td ~ 450°C) for the composition of 0.6BF‐24PT‐0.16BST. TEM patterns provided clear evidence for the presence of nanodomains (~2nm) would be the predominant source for the enhanced piezoelectricity for the composition x = 0.20. The designed BF‐PT‐BST ternary system provides great potential for high‐temperature piezoelectric applications. [ABSTRACT FROM AUTHOR]

Pseudobinary Gd 5 (Si,Ge) 4 compounds show a giant magnetocaloric effect due to an intriguing structure-magnetism interplay. Here, we report a high resolution transmission electron microscopic study of microstructure in a melt-grown compound of Gd 5 Si 1.5 Ge 2.5. Electron diffraction showed that mesoscopic microstructure of the compound consists of crystals of a monoclinic structure and two kinds of orthorhombic structure. However, atomic-level imaging revealed a coexistence of mosaic-like nanodomains of these structures in each crystal. The nanodomains have twin-like interfaces along the b axis of the crystal but have semicoherent interfaces along a perpendicular axis due to stacking faults. Such a nanoscale structural heterogeneity is due to covalent bond-controlled phase separation at elevated temperatures. This finding offers insights into phase relations and abnormal magnetic behavior of Gd 5 (Si,Ge) 4 compounds and points out a route of synthesizing bulk nanocomposites via nanoscale phase separation. Image, graphical abstract [ABSTRACT FROM AUTHOR]

The nanoscale organization of biological membranes into structurally and compositionally distinct lateral domains is believed to be central to membrane function. The nature of this organization has remained elusive due to a lack of methods to directly probe nanoscopic membrane features. We show here that cryogenic electron micros-copy (cryo-EM) can be used to directly image coexisting nanoscopic domains in synthetic and bioderived membranes without extrinsic probes. Analyzing a series of single-component liposomes composed of synthetic lipids of varying chain lengths, we demonstrate that cryo-EM can distinguish bilayer thickness differences as small as 0.5 Å, comparable to the resolution of small-angle scattering methods. Simulated images from computational models reveal that features in cryo-EM images result from a complex interplay between the atomic distribution normal to the plane of the bilayer and imaging parameters. Simulations of phase-separated bilayers were used to predict two sources of contrast between coexisting ordered and disordered phases within a single liposome, namely differences in membrane thickness and molecular density. We observe both sources of contrast in biomimetic membranes composed of saturated lipids, unsaturated lipids, and cholesterol. When extended to isolatedmammalian plasma membranes, cryo-EM reveals similar nanoscale lateral heterogeneities. The methods reported here for direct, probe-free imaging of nanodomains in unperturbed membranes open new avenues for investigation of nanoscopic membrane organization. [ABSTRACT FROM AUTHOR]

Chemical doping is an indispensable tool to tailor the properties of the commercial piezoelectric materials. However, a high piezoelectric coefficient with enhanced thermal stability is rarely achieved by one dopant in some high-performance ferroelectrics, e.g., the recently discovered eco-friendly (Ba 0.85 Ca 0.15)(Zr 0.1 Ti 0.9)O 3 (BCZT) ceramics. In order to optimize the piezoelectric property in BCZT system by a simple way, we investigated the doping effect of Fe3+, Nb5+ and Bi3+ cations in BCZT ceramics respectively. The results indicate that only Nb5+-doped BCZT ceramics display a combination of large piezoelectric coefficient and enhanced thermal stability, compared with others. Moreover, the established phase diagrams and in-situ transmission electron microscope (TEM) observations reveal that such optimized piezoelectric properties after Nb5+ doping originates from (i) the low polarization anisotropy near the ambient tetragonal (T)-orthorhombic (O) phase transition and (ii) the easy domain wall motion of persistent miniaturized ferroelectric domains upon heating. [ABSTRACT FROM AUTHOR]

• The superparaelectric phase engineering is employed in BNT RFE ceramics to realize high energy storage performance. • A novel approach has been introduced to assess the linear relaxation characteristics. • A remarkable W rec value of 7.2 J⋅cm−3 together with a high η of 86 % can be obtained. The utilization of electrostatic energy storage technology, which relies on dielectrics, is of utmost importance in the realm of advanced electronics and high-power electrical systems. However, there is still a lack of a universally applicable method to simultaneously attain elevated recoverable energy density (W rec) and high efficiency (η). Herein, based on the design of superparaelectric regulation, SmFeO 3 was introduced into the typical 0.6Bi 0.5 Na 0.5 TiO 3 –0.4Sr 0.7 Bi 0.2 TiO 3 relaxor. A nearly linear P – E loop with near-zero remanent polarization and excellent comprehensive performance were realized in the 0.9(0.6Bi 0.5 Na 0.5 TiO 3 –0.4Sr 0.7 Bi 0.2 TiO 3)–0.1SmFeO 3 ceramic. A remarkable W rec value of 7.2 J⋅cm−3 together with a high η of 86% was obtained at an improved Weibull breakdown electric field (E b) of 430 kV⋅cm−1. Wherein the ultra-small nanodomain size and the increased linearity of the P – E loop based on superparaelectric regulation strategy at largely enhanced E b are responsible for the improvement of W rec and η. Therefore, the method of superparaelectric regulation realized on relaxor ferroelectrics opens a new avenue for the progress of cutting-edge ceramic capacitors. [ABSTRACT FROM AUTHOR]

Strain glass transition attracts increasing attention in ferroelastic alloys in the context that martensitic transformation serves as the dominant mechanism for shape memory alloys. However, observing the coexistence of strain glass transition and martensitic transformation has not been reported. In this work, we introduce the strain glass transition by doping element to form highly nickel-rich Ni 55 Ti 45 at.% alloys and in the meantime attain the characteristics of martensitic transformation in the alloys. We confirm the existence of strain glass transition by 1) no exothermic/endothermic peaks in heat flow curves, 2) two deviations in electric resistivity curves, 3) two frequency-dependent dips of storage modulus, and 4) successive formation and constrained growth of nanodomains over a wide temperature range. We observe the martensitic transformation characterized by 1) two pronounced exothermic peaks upon cooling and one endothermic peak upon heating in heat flow curves, 2) thermal hysteresis in electric resistivity curves, and 3) macroscopic martensitic twins in transmission electron microscopy images. With these two phase transformations, the quasilinear superelasticity, high strength, and large elastocaloric Δ T ad of up to 9.7 K are achieved. By designing the strain maps and microstructural evolution pathways, we have provided a temperature-composition transformation path diagram to offer mechanistic insights and tuning strategies for the development of advanced alloys. [Display omitted] [ABSTRACT FROM AUTHOR]

Compartmentalization of the membrane is essential for cells to perform highly specific tasks and spatially constrained biochemical functions in topographically defined areas. These membrane lateral heterogeneities range from nanoscopic dimensions, often involving only a few molecular constituents, to micron-sized mesoscopic domains resulting from the coalescence of nanodomains. Short-lived domains lasting for a few milliseconds coexist with more stable platforms lasting from minutes to days. This panoply of lateral domains subserves the great variety of demands of cell physiology, particularly high for those implicated in signaling. The dendritic spine, a subcellular structure of neurons at the receiving (postsynaptic) end of central nervous system excitatory synapses, exploits this compartmentalization principle. In its most frequent adult morphology, the mushroom-shaped spine harbors neurotransmitter receptors, enzymes, and scaffolding proteins tightly packed in a volume of a few femtoliters. In addition to constituting a mesoscopic lateral heterogeneity of the dendritic arborization, the dendritic spine postsynaptic membrane is further compartmentalized into spatially delimited nanodomains that execute separate functions in the synapse. This review discusses the functional relevance of compartmentalization and nanodomain organization in synaptic transmission and plasticity and exemplifies the importance of this parcelization in various neurotransmitter signaling systems operating at dendritic spines, using two fast ligand-gated ionotropic receptors, the nicotinic acetylcholine receptor and the glutamatergic receptor, and a second-messenger G-protein coupled receptor, the cannabinoid receptor, as paradigmatic examples.