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Mutations in activin-like kinase 2 (ALK2), e.g., ALK2-R206H, induce aberrant signaling to SMAD1/5/8, leading to Fibrodysplasia Ossificans Progressiva (FOP). In spite of extensive studies, the underlying mechanism is still unclear. Here, we quantified the homomeric and heteromeric interactions of ACVR2A, ACVR2B, ALK2-WT, and ALK2-R206H by combining IgG-mediated immobilization of one receptor with fluorescence recovery after photobleaching (FRAP) measurements on the lateral diffusion of a co-expressed receptor. ACVR2B formed stable homomeric complexes that were enhanced by Activin A (ActA), while ACVR2A required ActA for homodimerization. ALK2-WT, but not ALK2-R206H, exhibited homomeric complexes unaffected by ActA. ACVR2B formed ActA-enhanced heterocomplexes with ALK2-R206H or ALK2-WT, while ACVR2A interacted mainly with ALK2-WT. The extent of the homomeric complex formation of ACVR2A or ACVR2B was reflected in their ability to induce the oligomerization of ALK2-R206H and ALK2-WT. Thus, ACVR2B, which forms dimers without ligand, induced ActA-independent ALK2-R206H clustering but required ActA for enhancing the oligomerization of the largely dimeric ALK2-WT. In contrast, ACVR2A, which undergoes homodimerization in response to ActA, required ActA to induce ALK2-R206H oligomerization. To investigate whether these interactions are translated into signaling, we studied signaling by the FOP-inducing hyperactive ALK2-R206H mutant, with ALK2-WT signaling as control. The activation of SMAD1/5/8 signaling in cells expressing ALK2-R206H alone or together with ACVR2A or ACVR2B was measured by blotting for pSMAD1/5/8 and by transcriptional activation assays using BRE-Luc reporter. In line with the biophysical studies, ACVR2B activated ALK2-R206H without ligand, while activation by ACVR2A was weaker and required ActA. We propose that the homodimerization of ACVR2B or ACVR2A dictates their ability to recruit ALK2-R206H into higher complexes, enabling the homomeric interactions of ALK2-R206H receptors and, subsequently, their activation. [ABSTRACT FROM AUTHOR]

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]

Isothermal surfaces within the device [Display omitted] This paper presents a computational model of molecular diffusion through the interfollicular stratum corneum. Specifically, it extends an earlier two-dimensional microscopic model for the permeability in two ways: (1) a microporous leakage pathway through the intercellular lipid lamellae allows slow permeation of highly hydrophilic permeants through the tissue; and (2) the model yields explicit predictions of both lateral ( D ‾ ‖ sc ) and transdermal ( D ‾ ⊥ sc ) effective (average, homogenized) diffusivities of solutes within the tissue. We present here the mathematical framework for the analysis and a comparison of the predictions with experimental data on desorption of both hydrophilic and lipophilic solutes from human stratum corneum in vitro. Diffusion in the lipid lamellae is found to make the effective diffusivity highly anisotropic, with the predicted ratio D ‾ ‖ sc / D ‾ ⊥ sc ranging from 34 to 39 for fully hydrated skin and 150 to more than 1000 for partially hydrated skin. The diffusivities and their ratio are in accord with both experimental data and the results of mathematical analyses performed by others. [ABSTRACT FROM AUTHOR]

In living cells, the biochemical processes such as energy provision, molecule synthesis, gene expression, and cell division take place in a confined space where the internal chemical and physical conditions are different from those in dilute solutions. The concentrations of specific molecules and the specific reactions and interactions vary for different types of cells, but a number of factors are universal and kept within limits, which we refer to as physicochemical homeostasis. For instance, the internal pH of many cell types is kept within the range of 7.0 to 7.5, the fraction of macromolecules occupies 15%–20% of the cell volume (also known as macromolecular crowding) and the ionic strength is kept within limits to prevent salting-in or salting-out effects. In this article we summarize the generic physicochemical properties of the cytoplasm of bacteria, how they are connected to the energy status of the cell, and how they affect biological processes (Fig.  1). We describe how the internal pH and proton motive force are regulated, how the internal ionic strength is kept within limits, what the impact of macromolecular crowding is on the function of enzymes and the interaction between molecules, how cells regulate their volume (and turgor), and how the cytoplasm is structured. Physicochemical homeostasis is best understood in Escherichia coli , but pioneering studies have also been performed in lactic acid bacteria. [ABSTRACT FROM AUTHOR]

NMDA receptors (NMDARs) are ionotropic glutamate receptors that play a key role in excitatory neurotransmission. The number and subtype of surface NMDARs are regulated at several levels, including their externalization, internalization, and lateral diffusion between the synaptic and extrasynaptic regions. Here, we used novel anti-GFP (green fluorescent protein) nanobodies conjugated to either the smallest commercially available quantum dot 525 (QD525) or the several nanometer larger (and thus brighter) QD605 (referred to as nanoGFP-QD525 and nanoGFP-QD605, respectively). Targeting the yellow fluorescent protein-tagged GluN1 subunit in rat hippocampal neurons, we compared these two probes to a previously established larger probe, a rabbit anti-GFP IgG together with a secondary IgG conjugated to QD605 (referred to as antiGFP-QD605). The nanoGFP-based probes allowed faster lateral diffusion of the NMDARs, with several-fold increased median values of the diffusion coefficient (D). Using thresholded tdTomato-Homer1c signals to mark synaptic regions, we found that the nanoprobe-based D values sharply increased at distances over 100 nm from the synaptic edge, while D values for antiGFP-QD605 probe remained unchanged up to a 400 nm distance. Using the nanoGFP-QD605 probe in hippocampal neurons expressing the GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits, we detected subunit-dependent differences in the synaptic localization of NMDARs, D value, synaptic residence time, and synaptic-extrasynaptic exchange rate. Finally, we confirmed the applicability of the nanoGFP-QD605 probe to study differences in the distribution of synaptic NMDARs by comparing to data obtained with nanoGFPs conjugated to organic fluorophores, using universal point accumulation imaging in nanoscale topography and direct stochastic optical reconstruction microscopy. [ABSTRACT FROM AUTHOR]

[Display omitted] Sodium oleate (NaOL) self-aggregates in water forming rodlike micelles with different length depending on NaOL concentration; when KCl is added wormlike micelles form, which entangle giving rise to a viscoelastic dispersion. It is expected that aggregates with different size and shape exhibit different internal and overall molecular motions and collective dynamics. Two low viscosity NaOL/water and two viscoelastic NaOL/KCl/water formulations with different NaOL concentration (0.23 and 0.43 M) were investigated by 1H fast field cycling NMR relaxometry over broad temperature and Larmor frequency ranges, after a first screening by 1H and 13C NMR spectroscopy at high frequency. The analysis of the collected data indicated that fast conformational isomerization and rotation of NaOL about its long molecular axis and lateral diffusion of NaOL around the axis of the cylindrical aggregates are slightly affected by the aggregate shape and length. On the other hand, fluctuations of the local order director are quite different in the fluid and viscoelastic systems, reflecting the shape and size of the aggregates. Quantitative information was obtained on activation energy for fast internal and overall motions, correlation times and activation energy for lateral diffusion, and coherence length for collective order fluctuations. [ABSTRACT FROM AUTHOR]

There is a growing interest in innovative products for eyebrow hair loss treatment with fewer adverse effects. Nevertheless, a fundamental formulation aspect of preventing the fragile skin from the ocular region from being irritated is that the formulations remain restricted to the application region and do not run off. Consequently, the methods and protocols in drug delivery scientific research must be adapted to fulfill such performance analysis demand. Thus, this work aimed to propose a novel protocol to evaluate the in vitro performance of a topical gel formulation with a reduced runoff for minoxidil (MXS) delivery to eyebrows. MXS was formulated with 16% poloxamer 407 (PLX) and 0.4% of hydroxypropyl methylcellulose (HPMC). The sol/gel transition temperature, viscosity at 25 °C, and formulation runoff distance on the skin were evaluated to characterize the formulation. The release profile and skin permeation were evaluated in Franz vertical diffusion cells for 12 h and compared to a control formulation (4% PLX and 0.7% HPMC). Then, the formulation's performance at promoting minoxidil skin penetration with minimum runoff was evaluated in a vertical custom-made permeation template (divided into three areas: superior, middle, and inferior). The MXS release profile from the test formulation was comparable to that from the MXS solution and the control formulation. There was also no difference in the MXS amount that penetrated the skin in the permeation experiments in Franz diffusion cells using the different formulations (p > 0.05). However, the test formulation demonstrated a localized MXS delivery at the application site in the vertical permeation experiment. In conclusion, the proposed protocol could differentiate the test formulation from the control, attesting to its better performance in efficiently delivering MXS to the site of interest (middle third of application). The vertical protocol can be easily employed to evaluate other gels with a drip-free appeal. [ABSTRACT FROM AUTHOR]

This study proposed a novel source/drain (S/D) extension scheme to increase the stress in nanosheet (NS) field-effect transistors (NSFETs) and investigated the scheme by using technology-computer-aided-design simulations. In three-dimensional integrated circuits, transistors in the bottom tier were exposed to subsequent processes; therefore, selective annealing, such as laser-spike annealing (LSA), should be applied. However, the application of the LSA process to NSFETs significantly decreased the on-state current (Ion) owing to diffusionless S/D dopants. Furthermore, the barrier height below the inner spacer was not lowered even under on-state bias conditions because ultra-shallow junctions between the NS and S/D were formed far from the gate metal. However, the proposed S/D extension scheme overcame these Ion reduction issues by adding an NS-channel-etching process before S/D formation. A larger S/D volume induced a larger stress in the NS channels; thus, the stress was boosted by over 25%. Additionally, an increase in carrier concentrations in the NS channels improved Ion. Therefore, Ion increased by approximately 21.7% (37.4%) in NFETs (PFETs) compared with NSFETs without the proposed scheme. Additionally, the RC delay was improved by 2.03% (9.27%) in NFETs (PFETs) compared with NSFETs using rapid thermal annealing. Therefore, the S/D extension scheme overcame the Ion reduction issues encountered in LSA and significantly enhanced the AC/DC performance. [ABSTRACT FROM AUTHOR]

The lateral diffusion of transmembrane proteins on plasma membranes is a fundamental process for various cellular functions. Diffusion properties specific for individual protein species have been extensively studied, but the common features among protein species are poorly understood. Here, we systematically studied the lateral diffusion of various transmembrane proteins in the lower eukaryote Dictyostelium discoideum cells using a hidden Markov model for single-molecule trajectories obtained experimentally. As common features, all membrane proteins that had from one to ten transmembrane regions adopted three free diffusion states with similar diffusion coefficients regardless of their structural variability. All protein species reduced their mobility similarly upon the inhibition of microtubule or actin cytoskeleton dynamics, or myosin II. The relationship between protein size and the diffusion coefficient was consistent with the Saffman--Delbruü ck model, meaning that membrane viscosity is a major determinant of lateral diffusion, but protein size is not. These protein species-independent properties of multistate free diffusion were explained simply and quantitatively by free diffusion on the three membrane regions with different viscosities, which is in sharp contrast to the complex diffusion behavior of transmembrane proteins in higher eukaryotes. [ABSTRACT FROM AUTHOR]

An upregulation of the Na+-K+-2Cl− cotransporter NKCC1, the main chloride importer in mature neurons, can lead to depolarizing/excitatory responses mediated by GABA type A receptors (GABAARs) and, thus, to hyperactivity. Understanding the regulatory mechanisms of NKCC1 would help prevent intra-neuronal chloride accumulation that occurs in pathologies with defective inhibition. The cell mechanisms regulating NKCC1 are poorly understood. Here, we report in mature hippocampal neurons that GABAergic activity controls the membrane diffusion and clustering of NKCC1 via the chloride-sensitive WNK lysine deficient protein kinase 1 (WNK1) and the downstream Ste20 Pro-line Asparagine Rich Kinase (SPAK) kinase that directly phosphorylates NKCC1 on key threonine residues. At rest, this signaling pathway has little effect on intracellular Cl− concentration, but it participates in the elevation of intraneuronal Cl− concentration in hyperactivity conditions associated with an up-regulation of NKCC1. The fact that the main chloride exporter, the K+-Cl− cotransporter KCC2, is also regulated in mature neurons by the WNK1 pathway indicates that this pathway will be a target of choice in the pathology. [ABSTRACT FROM AUTHOR]

Purpose: Deciphering salient features of biological tissue cellular microstructure in health and diseases is an ultimate goal of MRI. While most MRI approaches are based on studying MR properties of tissue "free" water indirectly affected by tissue microstructure, other approaches, such as magnetization transfer (MT), directly target signals from tissue‐forming macromolecules. However, despite three‐decades of successful applications, relationships between MT measurements and tissue microstructure remain elusive, hampering interpretation of experimental results. The goal of this paper is to develop microscopic theory connecting the structure of cellular and myelin membranes to their MR properties. Theory and Methods: Herein we introduce a lateral diffusion model (LDM) that explains the T2 (spin–spin) and T1 (spin–lattice) MRI relaxation properties of the macromolecular‐bound protons by their dipole–dipole interaction modulated by the lateral diffusion of long lipid molecules forming cellular and myelin membranes. Results: LDM predicts anisotropic T1 and T2 relaxation of membrane‐bound protons. Moreover, their T2 relaxation cannot be described in terms of a standard R2 = 1/T2 relaxation rate parameter, but rather by a relaxation rate function R2(t) that depends on time t after RF excitation, having, in the main approximation, a logarithmic behavior: R2(t) ∼ lnt. This anisotropic non‐linear relaxation leads to an absorption lineshape that is different from Super‐Lorentzian traditionally used in interpreting MT experiments. Conclusion: LDM‐derived analytical equations connect the membrane‐bound protons T1 and T2 relaxation with dynamic distances between protons in neighboring membrane‐forming lipid molecules and their lateral diffusion. This sheds new light on relationships between MT parameters and microstructure of cellular and myelin membranes. [ABSTRACT FROM AUTHOR]

Fullerene derivatives are of great interest in various fields of science and technology. Fullerene derivatives are known to have pronounced anticancer and antiviral activity. They have antibacterial properties. Their properties are largely determined by association processes. Understanding the nature and properties of associates in solvents of various types will make it possible to make significant progress in understanding the mechanisms of aggregation of molecules of fullerene derivatives in solutions. Thus, this work, aimed at studying the size and stability of associates, is relevant and promising for further research. The NMR method in a pulsed field gradient was used, which makes it possible to directly study the translational mobility of molecules. The sizes of individual molecules and associates were calculated based on the Stokes–Einstein model. The lifetime of associates was also estimated. The interaction of water-soluble C60 fullerene derivatives with erythrocytes was also evaluated. The values of self-diffusion coefficients and the lifetime of molecules of their compounds in cell membranes are obtained. It is concluded that the molecules of fullerene derivatives are fixed on the cell surface, and their forward movement is controlled by lateral diffusion. [ABSTRACT FROM AUTHOR]

[Display omitted] Based on the structure of the Stratum corneum (SC) the potential penetration/diffusion pathways of drugs and cosmetic actives through the SC are presented and discussed. The well-known lipophilic pathway across the SC is presented and relevant examples are used to show that highly lipophilic molecules such as glucocorticoids, coenzyme Q10 etc. are accumulated in the SC and penetrate into the inner liquid like layer of the SC lipid bilayer by lateral diffusion . The diffusion into and across the SC of highly hydrophilic drugs and active substances such as urea, amino acids and peptides is still under discussion. Another diffusion pathway for the highly hydrophilic molecules via the corneocytes and the corneodesmosomes is presented and discussed, the corneocytary diffusion pathway . [ABSTRACT FROM AUTHOR]

Oligomerization and complex formation play a key role for many membrane proteins and has been described to influence ion channel function in both neurons and the heart. In this study, we observed clustering of single KcsA channels in planar lipid bilayer using single molecule fluorescence, while simultaneously measuring single channel currents. Clustering coincided with cooperative opening of KcsA. We demonstrate that clustering was not caused by direct protein-protein interactions or hydrophobic mismatch with the lipid environment, as suggested earlier, but was mediated via microdomains induced by the channel in the lipid matrix. We found that single channel activity of KcsA requires conically-shaped lipids in the lamellar liquid-crystalline (Lα) phase, and the need for a negative spontaneous curvature seem to lead to the deformations in the membrane that cause the clustering. The method introduced here will be applicable to follow oligomerization of a wide range of membrane proteins. [ABSTRACT FROM AUTHOR]

Oligomerization and complex formation play a key role for many membrane proteins and has been described to influence ion channel function in both neurons and the heart. In this study, we observed clustering of single KcsA channels in planar lipid bilayer using single molecule fluorescence, while simultaneously measuring single channel currents. Clustering coincided with cooperative opening of KcsA. We demonstrate that clustering was not caused by direct protein-protein interactions or hydrophobic mismatch with the lipid environment, as suggested earlier, but was mediated via microdomains induced by the channel in the lipid matrix. We found that single channel activity of KcsA requires conically-shaped lipids in the lamellar liquid-crystalline (Lα) phase, and the need for a negative spontaneous curvature seem to lead to the deformations in the membrane that cause the clustering. The method introduced here will be applicable to follow oligomerization of a wide range of membrane proteins.

According to the design requirements of high-temperature combustion chamber, an advanced shaped hole structure was designed for film cooling. Numerical method was applied in this study to investigate the flow and heat transfer characteristics of shaped holes and compared with those of cylindrical holes. The influence of the forward expansion angle of shaped holes on the flow and heat transfer was studied. The results show that compared to cylindrical holes, the diffused structure of shaped holes decreases the momentum of jet flow, improves the adhesion characteristics of the cooling air film, increases the diffusion of the coolant air outflow and improves the cooling efficiency between adjacent columns of holes in the lateral direction. When the forward expansion angle increases, the expansion section induced the flow vortex, which reduces the radial velocity of coolant flow and enhances the diffusion of cooling air film both in streamwise and spanwise directions. However, as the forward expansion angle increases further, the scale of vortex inside the shaped hole grows. Too large vortex inside the shaped hole increases the coolant eject angle, which weakens the film covering effect. Additionally, the shaped hole results in an increase in lateral spreading and enhances the cooling effect between adjacent columns of the film hole. The enhancement of the film cooling characteristics is due to the change in the shape of the film hole, resulting in the enhancement of the flow vortex, which induces complicated secondary flow.

There is a growing interest in innovative products for eyebrow hair loss treatment with fewer adverse effects. Nevertheless, a fundamental formulation aspect of preventing the fragile skin from the ocular region from being irritated is that the formulations remain restricted to the application region and do not run off. Consequently, the methods and protocols in drug delivery scientific research must be adapted to fulfill such performance analysis demand. Thus, this work aimed to propose a novel protocol to evaluate the in vitro performance of a topical gel formulation with a reduced runoff for minoxidil (MXS) delivery to eyebrows. MXS was formulated with 16% poloxamer 407 (PLX) and 0.4% of hydroxypropyl methylcellulose (HPMC). The sol/gel transition temperature, viscosity at 25 °C, and formulation runoff distance on the skin were evaluated to characterize the formulation. The release profile and skin permeation were evaluated in Franz vertical diffusion cells for 12 h and compared to a control formulation (4% PLX and 0.7% HPMC). Then, the formulation’s performance at promoting minoxidil skin penetration with minimum runoff was evaluated in a vertical custom-made permeation template (divided into three areas: superior, middle, and inferior). The MXS release profile from the test formulation was comparable to that from the MXS solution and the control formulation. There was also no difference in the MXS amount that penetrated the skin in the permeation experiments in Franz diffusion cells using the different formulations (p > 0.05). However, the test formulation demonstrated a localized MXS delivery at the application site in the vertical permeation experiment. In conclusion, the proposed protocol could differentiate the test formulation from the control, attesting to its better performance in efficiently delivering MXS to the site of interest (middle third of application). The vertical protocol can be easily employed to evaluate other gels with a drip-free appeal.

This study proposed a novel source/drain (S/D) extension scheme to increase the stress in nanosheet (NS) field-effect transistors (NSFETs) and investigated the scheme by using technology-computer-aided-design simulations. In three-dimensional integrated circuits, transistors in the bottom tier were exposed to subsequent processes; therefore, selective annealing, such as laser-spike annealing (LSA), should be applied. However, the application of the LSA process to NSFETs significantly decreased the on-state current (Ion) owing to diffusionless S/D dopants. Furthermore, the barrier height below the inner spacer was not lowered even under on-state bias conditions because ultra-shallow junctions between the NS and S/D were formed far from the gate metal. However, the proposed S/D extension scheme overcame these Ion reduction issues by adding an NS-channel-etching process before S/D formation. A larger S/D volume induced a larger stress in the NS channels; thus, the stress was boosted by over 25%. Additionally, an increase in carrier concentrations in the NS channels improved Ion. Therefore, Ion increased by approximately 21.7% (37.4%) in NFETs (PFETs) compared with NSFETs without the proposed scheme. Additionally, the RC delay was improved by 2.03% (9.27%) in NFETs (PFETs) compared with NSFETs using rapid thermal annealing. Therefore, the S/D extension scheme overcame the Ion reduction issues encountered in LSA and significantly enhanced the AC/DC performance.

An upregulation of the Na+-K+-2Cl− cotransporter NKCC1, the main chloride importer in mature neurons, can lead to depolarizing/excitatory responses mediated by GABA type A receptors (GABAARs) and, thus, to hyperactivity. Understanding the regulatory mechanisms of NKCC1 would help prevent intra-neuronal chloride accumulation that occurs in pathologies with defective inhibition. The cell mechanisms regulating NKCC1 are poorly understood. Here, we report in mature hippocampal neurons that GABAergic activity controls the membrane diffusion and clustering of NKCC1 via the chloride-sensitive WNK lysine deficient protein kinase 1 (WNK1) and the downstream Ste20 Pro-line Asparagine Rich Kinase (SPAK) kinase that directly phosphorylates NKCC1 on key threonine residues. At rest, this signaling pathway has little effect on intracellular Cl− concentration, but it participates in the elevation of intraneuronal Cl− concentration in hyperactivity conditions associated with an up-regulation of NKCC1. The fact that the main chloride exporter, the K+-Cl− cotransporter KCC2, is also regulated in mature neurons by the WNK1 pathway indicates that this pathway will be a target of choice in the pathology.

Proteasome activity at the excitatory synapse plays an important role in neuronal communication. The proteasome translocation to synapses is mediated by neuronal activity, in particular the activation of N‐methyl‐d‐aspartate receptors (NMDARs). These receptors are composed of different subunits with distinct trafficking properties that provide various signalling and plasticity features to the synapse. Yet whether the interplay between the proteasome and NMDAR relies on specific subunit properties remain unclear. Using a combination of single molecule and immunocytochemistry imaging approaches in rat hippocampal neurons, we unveil a specific interplay between GluN2B‐containing NMDARs (GluN2B‐NMDARs) and the synaptic proteasome. Sustained proteasome activation specifically increases GluN2B‐NMDAR (not GluN2A‐NMDAR) lateral diffusion. In addition, when GluN2B‐NMDAR expression is downregulated, the proteasome localization decreases at glutamatergic synapses. Collectively, our data fuel a model in which the cellular dynamics and location of GluN2B‐NMDARs and proteasome are intermingled, shedding new lights on the NMDAR‐dependent regulation of synaptic adaptation. [ABSTRACT FROM AUTHOR]

Epidermal growth factor receptor (EGFR)-mediated signal transduction controls cell growth and proliferation. The signaling pathway is regulated so that it is activated only by external EGF stimuli, but the mechanisms that prevent EGF-independent spontaneous activation of EGFR-mediated signaling are unknown. Here we report cholesterol depletion activates EGFR-mediated signaling without EGF. We applied automated single-molecule imaging to EGFR and characterized the lateral diffusion and cluster formation on cholesterol-depleted and cholesterol-supplemented membranes. In cells in which cholesterol was depleted by methyl-β-cyclodextrin (MβCD) treatment, EGFR exhibited a reduction in lateral diffusion, an acceleration of cluster formation, and autophosphorylation without EGF. Concurrently, extracellular signal-regulated kinase (ERK), which is regulated by EGFR-mediated signaling, exhibited phosphorylation and nuclear translocation without EGF. These cholesterol depletion-induced changes were similar, albeit less efficient, to those that occurred with EGF stimulation in normal cells without MβCD, indicating the spontaneous activation of EGFR signaling. The exogenous supplementation of cholesterol suppressed the MβCD-induced spontaneous activation of EGFR and ERK nuclear translocation. Single-molecule imaging of EGFR in a large number of cells revealed cell-to-cell heterogeneity, with a sub-population showing a high ability for spontaneous activation. These results provide evidence that EGFR-mediated signaling is properly regulated by cholesterol metabolism to prevent uncontrolled spontaneous activation. [Display omitted] • EGFR-mediated signaling is activated spontaneously with cholesterol depletion. • The phosphorylation, slow diffusion, and clusters of EGFR are induced without EGF. • Nuclear transport of ERK is induced by cholesterol depletion without EGF. • Cellular heterogeneity found in spontaneously activated EGFR-mediated signaling. [ABSTRACT FROM AUTHOR]

Protein mobility in the cytoplasm is essential for cellular functions, and slow diffusion may limit the rates of biochemical reactions in the living cell. Here, we determined the apparent lateral diffusion coefficient (DL) of GFP in Listeria monocytogenes as a function of osmotic stress, temperature, and media composition. We find that DL is much less affected by hyperosmotic stress in L. monocytogenes than under similar conditions in Lactococcus lactis and Escherichia coli. We find a temperature optimum for protein diffusion in L. monocytogenes at 30°C, which deviates from predicted trends from the generalized Stokes-Einstein equation under dilute conditions and suggests that the structure of the cytoplasm and macromolecular crowding vary as a function of temperature. The turgor pressure of L. monocytogenes is comparable to other Gram-positive bacteria like Bacillus subtilis and L. lactis but higher in a knockout strain lacking the stress-inducible sigma factor SigB. We discuss these findings in the context of how L. monocytogenes survives during environmental transmission and interaction with the human host.

Protein mobility in the cytoplasm is essential for cellular functions, and slow diffusion may limit the rates of biochemical reactions in the living cell. Here, we determined the apparent lateral diffusion coefficient (D L ) of GFP in Listeria monocytogenes as a function of osmotic stress, temperature, and media composition. We find that D L is much less affected by hyperosmotic stress in L. monocytogenes than under similar conditions in Lactococcus lactis and Escherichia coli. We find a temperature optimum for protein diffusion in L. monocytogenes at 30°C, which deviates from predicted trends from the generalized Stokes-Einstein equation under dilute conditions and suggests that the structure of the cytoplasm and macromolecular crowding vary as a function of temperature. The turgor pressure of L. monocytogenes is comparable to other Gram-positive bacteria like Bacillus subtilis and L. lactis but higher in a knockout strain lacking the stress-inducible sigma factor SigB. We discuss these findings in the context of how L. monocytogenes survives during environmental transmission and interaction with the human host. [ABSTRACT FROM AUTHOR]

Synaptic strength depends on the number of cell‐surface neurotransmitter receptors in dynamic equilibrium with intracellular pools. Dysregulation of this homeostatic balance occurs, for example in myasthenia gravis, an autoimmune disease characterized by a decrease in the number of postsynaptic nicotinic acetylcholine receptors (nAChRs). Monoclonal antibody mAb35 mimics this effect. Here we use STORM nanoscopy to characterize the individual and ensemble dynamics of monoclonal antibody‐crosslinked receptors in the clonal cell line CHO‐K1/A5, which robustly expresses adult muscle‐type nAChRs. Antibody labeling of live cells results in 80% receptor immobilization. The remaining mobile fraction exhibits a heterogeneous combination of Brownian and anomalous diffusion. Single‐molecule trajectories exhibit a two‐state switching behavior between free Brownian walks and anticorrelated walks within confinement areas. The latter act as permeable fences (~34 nm radius, ~400 ms lifetime). Dynamic clustering, trapping, and immobilization also occur in larger nanocluster zones (120–180 nm radius) with longer lifetimes (11 ± 1 s), in a strongly cholesterol‐sensitive manner. Cholesterol depletion increases the size of the clustering phenomenon; cholesterol enrichment has the opposite effect. The disclosed high proportion of monoclonal antibody‐crosslinked immobile receptors, together with their anomalous, cholesterol‐sensitive diffusion and clustering, provides new insights into the antibody‐enhanced antigenic modulation that leads to physiopathological internalization and degradation of receptors in myasthenia. [ABSTRACT FROM AUTHOR]

The uniformity of secondary air (SA) in large-scale CFB boilers has an important influence on gas-solid flow and combustion, but was seldom considered in previous studies. Numerical simulation based on the Eulerian–Eulerian and RNG k-ε turbulence models was conducted to explore the influence of SA uniformity and load variation on jet penetration, diffusion characteristics and gas-solid mixing in the first 600 MW supercritical CFB boiler. The results showed that better SA uniformity was conductive to the uniformity of SA penetration and gas-solid mixing along the furnace height, although the penetration depth and diffusion distance showed an opposite trend. In addition, the penetration depth and diffusion distance got enhanced with higher boiler load. The inner and outer SA jets could not cover the furnace width, and the uneven SA uniformity led to a huge deviation of the solid concentration within 10 m of the air distributor. Eventually, a calculation model was successfully established for predicting the penetration depth of inclined thermal SA jets during boiler operation.

Biological membranes play a vital role in cell functioning, providing structural integrity, controlling signal transduction, and controlling the transport of various chemical species. Owing to the complex nature of biomembranes, the self-assembly of lipids in aqueous media has been utilized to develop model systems mimicking the lipid bilayer structure, paving the way to elucidate the mechanisms underlying various biological processes, as well as to develop a number of biomedical and technical applications. The hydration properties of lipid bilayers are crucial for their activity in various cellular processes. Of particular interest is the local membrane dehydration, which occurs in membrane fusion events, including neurotransmission, fertilization, and viral entry. The lack of universal technique to evaluate the local hydration state of the membrane components hampers understanding of the molecular-level mechanisms of these processes. Here, we present a new approach to quantify the hydration state of lipid bilayers. It takes advantage of the change in the lateral diffusion of lipids that depends on the number of water molecules hydrating them. Using fluorescence recovery after photobleaching technique, we applied this approach to planar single and multicomponent supported lipid bilayers. The method enables the determination of the hydration level of a biomimetic membrane down to a few water molecules per lipid.

The main inhibitory neurotransmitter receptors in the adult central nervous system (CNS) are type A γ-aminobutyric acid receptors (GABAARs) and glycine receptors (GlyRs). Synaptic responses mediated by GlyR and GABAAR display a hyperpolarizing shift during development. This shift relies mainly on the developmental up-regulation of the K+-Cl− co-transporter KCC2 responsible for the extrusion of Cl−. In mature neurons, altered KCC2 function—mainly through increased endocytosis—leads to the re-emergence of depolarizing GABAergic and glycinergic signaling, which promotes hyperexcitability and pathological activities. Identifying signaling pathways and molecular partners that control KCC2 surface stability thus represents a key step in the development of novel therapeutic strategies. Here, we present our current knowledge on the cellular and molecular mechanisms governing the plasma membrane turnover rate of the transporter under resting conditions and in response to synaptic activity. We also discuss the notion that KCC2 lateral diffusion is one of the first parameters modulating the transporter membrane stability, allowing for rapid adaptation of Cl− transport to changes in neuronal activity.

Lateral proton transport (PT) on the surface of biological membranes is a fundamental biochemical process in the bioenergetics of living cells, but a lack of available experimental techniques has resulted in a limited understanding of its mechanism. Here, we present a molecular protonics experimental approach to investigate lateral PT across membranes by measuring long-range (70 μm) lateral proton conduction via a few layers of lipid bilayers in a solid-state-like environment, i.e., without having bulk water surrounding the membrane. This configuration enables us to focus on lateral proton conduction across the surface of the membrane while decoupling it from bulk water. Hence, by controlling the relative humidity of the environment, we can directly explore the role of water in the lateral PT process. We show that proton conduction is dependent on the number of water molecules and their structure and on membrane composition, where we explore the role of the headgroup, the tail saturation, the membrane phase, and membrane fluidity. The measured PT as a function of temperature shows an inverse temperature dependency, which we explain by the desorption and adsorption of water molecules into the solid membrane platform. We explain our findings by discussing the role of percolating hydrogen bonding within the membrane structure in a Grotthuss-like mechanism.

The time that it takes a diffusing particle to find a small target has emerged as a critical quantity in many systems in molecular and cellular biology. In this paper, we extend the theory for calculating this time to account for several ubiquitous biological features which have largely been ignored in the mathematics and physics literature on this problem. In particular, we allow (i) targets to diffuse on the two-dimensional boundary of the three-dimensional domain, (ii) targets to diffuse in the interior of the domain, (iii) the diffusivities of the searcher particle and the targets to stochastically fluctuate, (iv) targets to be stochastically gated, and (v) the transition times between fluctuations in diffusivity and gating to have effectively any probability distribution. In this general framework, we analytically calculate the leading order behavior of the mean first passage time and splitting probability for the searcher to reach a target as the target size decays, which is the so-called narrow escape limit. To make these extensions, we use a generalized Itô's formula to derive a system of coupled partial differential equations which are satisfied by statistics of the process, where the size of the system and its spatial dimension can be arbitrarily large. We apply matched asymptotic analysis to this system and verify our analytical results by numerical simulation. Our results reveal several new features and generic principles of diffusive search for small targets. [ABSTRACT FROM AUTHOR]

A reliable modelling approach is required for simultaneous characterisation of static and dynamic properties of bulk and interfacial ionic liquids (ILs). This is a prerequisite for a successful investigation of experimentally inaccessible, yet important properties, including those that change significantly with the distance from both vacuum and solid interfaces. We perform molecular dynamics simulations of bulk [C 2 Mim][NTf 2 ], and thick IL films in contact with vacuum and hydroxylated sapphire surface, using the charge methods CHelpG, RESP-HF and RESP-B3LYP with charge scaling factors 1.0, 0.9 and 0.85. By determining and employing appropriate system sizes and simulations lengths, and by benchmarking against self-diffusion coefficients, surface tension, X-ray reflectivity, and structural data, we identify RESP-HF/0.9 as the best non-polarizable force field for this IL. We use this optimal parametrisation to predict novel physical properties of confined IL films. First we fully characterise the internal configurations and orientations of IL molecules relative to, and as a function of the distance from the solid and vacuum interfaces. Second, we evaluate densities together with mobilities in-plane and normal to the interfaces and find that strong correlations between the IL's stratification and diffusive transport in the interfacial layers persist for several nanometres deep into IL films. [ABSTRACT FROM AUTHOR]

Many biological processes are initiated when diffusing extracellular reactants reach receptors on a cell membrane. Calculating this arrival rate has therefore attracted theoretical interest for decades. However, previous work has largely ignored the fact that receptors diffuse on the two-dimensional cell membrane in a process called surface or lateral diffusion. In this work, we derive an analytical formula for this arrival rate that takes into account receptor surface diffusion and cell rotational diffusion. Our theory predicts that the impact of these diffusive processes can be quantitatively described in terms of the relative size of the cell and the reactant. As applications, our theory predicts that surface and rotational diffusion have a negligible impact on bacterial chemoreception and a moderate impact on bacteriophage adsorption. Mathematically, our model is a three-dimensional anisotropic diffusion equation coupled to boundary conditions that are described by stochastic differential equations. We first apply matched asymptotic analysis to this stochastic partial differential equation and then use probabilistic methods to show that the solution can be represented by a Brownian particle in a stochastic environment. This representation enables efficient numerical computation of solution statistics and validation of our analytical results. [ABSTRACT FROM AUTHOR]

The main inhibitory neurotransmitter receptors in the adult central nervous system (CNS) are type A γ-aminobutyric acid receptors (GABAARs) and glycine receptors (GlyRs). Synaptic responses mediated by GlyR and GABAAR display a hyperpolarizing shift during development. This shift relies mainly on the developmental up-regulation of the K+-Cl− co-transporter KCC2 responsible for the extrusion of Cl−. In mature neurons, altered KCC2 function—mainly through increased endocytosis—leads to the re-emergence of depolarizing GABAergic and glycinergic signaling, which promotes hyperexcitability and pathological activities. Identifying signaling pathways and molecular partners that control KCC2 surface stability thus represents a key step in the development of novel therapeutic strategies. Here, we present our current knowledge on the cellular and molecular mechanisms governing the plasma membrane turnover rate of the transporter under resting conditions and in response to synaptic activity. We also discuss the notion that KCC2 lateral diffusion is one of the first parameters modulating the transporter membrane stability, allowing for rapid adaptation of Cl− transport to changes in neuronal activity. [ABSTRACT FROM AUTHOR]

Here, we uncover a mechanism for regulating the number of active presynaptic GABAB receptors (GABABRs) at nerve terminals, an important determinant of neurotransmitter release. We find that GABABRs gain access to axon terminals by lateral diffusion in the membrane. Their relative accumulation is dependent upon agonist activation and the presence of the two distinct sushi domains that are found only in alternatively spliced GABABR1a subunits. Following brief activation of NMDA receptors (NMDARs) using glutamate, GABABR diffusion is reduced, causing accumulation at presynaptic terminals in a Ca2+-dependent manner that involves phosphorylation of GABABR2 subunits at Ser783. This signaling cascade indicates how synaptically released glutamate can initiate, via a feedback mechanism, increased levels of presynaptic GABABRs that limit further glutamate release and excitotoxicity.

Long-term potentiation (LTP) of excitatory synaptic strength, which has long been considered a synaptic correlate for learning and memory, requires a fast recruitment of additional α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors (AMPARs) to the postsynaptic sites. As cell biological concepts have been applied to the field and genetic manipulation and microscopic imaging technologies have been advanced, visualization of the trafficking of AMPARs to synapses for LTP has been investigated intensively over the last decade. Recycling endosomes have been reported as intracellular storage organelles to supply AMPARs for LTP through the endocytic recycling pathway. In addition, exocytic domains in the spine plasma membrane, where AMPARs are inserted from the intracellular compartment, and nanodomains, where diffusing AMPARs are trapped and immobilized inside synapses for LTP, have been described. Furthermore, cell surface lateral diffusion of AMPARs from extrasynaptic to synaptic sites has been reported as a key step for AMPAR location to the synaptic sites for LTP. This review article will discuss recent findings and views on the reservoir(s) of AMPARs and their trafficking for LTP expression by focusing on the exocytosis and lateral diffusion of AMPARs, and provide some future directions that need to be addressed in the field of LTP.

An electro-optic device in silicon such as a photodetector and a modulator requires doping to realize desired junctions. Doping Si is typically done using ion-implantation or the thermal diffusion process. Both processes rely on high-temperature anneal to either activate or drive the dopant into the bulk. As diffusion of dopants is a thermally driven process, knowledge of actual diffusion lengths is essential for efficient device designs. Unlike other methods, we present an integrated-silicon-photonic-device-based lateral diffusion length characterization technique. A silicon microring resonator is used as a test device for the demonstration in a silicon-on-insulator substrate. Lateral diffusion lengths of 0.35 and $0.69~\mu \text{m}$ have been obtained for the drive-in temperatures of 950 °C and 1100 °C, respectively. We also present process simulation results to validate the experimental result. [ABSTRACT FROM AUTHOR]