Plane Of Membrane Research Articles

Plant Membrane-On-A-Chip: A Platform for Studying Plant Membrane Proteins and Lipids.

The cell plasma membrane is a two-dimensional, fluid mosaic material composed of lipids and proteins that create a semipermeable barrier defining the cell from its environment. Compared with soluble proteins, the methodologies for the structural and functional characterization of membrane proteins are challenging. An emerging tool for studies of membrane proteins in mammalian systems is a "plasma membrane on a chip," also known as a supported lipid bilayer. Here, we create the "plant-membrane-on-a-chip,″ a supported bilayer made from the plant plasma membranes of Arabidopsis thaliana, Nicotiana benthamiana, or Zea mays. Membrane vesicles from protoplasts containing transgenic membrane proteins and their native lipids were incorporated into supported membranes in a defined orientation. Membrane vesicles fuse and orient systematically, where the cytoplasmic side of the membrane proteins faces the chip surface and constituents maintain mobility within the membrane plane. We use plant-membrane-on-a-chip to perform fluorescent imaging to examine protein-protein interactions and determine the protein subunit stoichiometry of FLOTILLINs. We report here that like the mammalian FLOTILLINs, FLOTILLINs expressed in Arabidopsis form a tetrameric complex in the plasma membrane. This plant-membrane-on-a-chip approach opens avenues to studies of membrane properties of plants, transport phenomena, biophysical processes, and protein-protein and protein-lipid interactions in a convenient, cell-free platform.

The neutral Red probe location in large unilamellar vesicles: Influence of membrane composition and phase states

Biological membranes are complex and dynamic self-assembled structures composed of lipids and proteins. They depict emergent properties that are heterogeneous in the membrane plane. Therefore, their study is currently one of the greatest challenges in biophysics.The versatility of spectroscopic techniques has encouraged the development of new fluorescent probes capable of sensing different membrane properties. The Neutral Red (NR) molecule is a dye that protonates at pH < 6.8 (pKa = 6.8), leading to the charged species (NRH+). NR is widely employed in molecular biology to assess cell viability and mitochondrial function, and to sense intracellular pH. However, the photophysical behavior of NR in complex media such as membranes were not systematically characterized even though of the wide application in molecular biology.In this work, the photophysical behavior of NNR (pH 8 neutral specie) and NRH+ (pH 4) was studied by employing large unilamellar vesicles (LUVs) with different compositions, membrane phase states.The results of this study show that the spectroscopic behavior of NNR is more sensitive to the membrane composition than that of NRH+. The wavelength of the maximum emission of NNR was at shorter wavelengths in liquid-disordered (Ld) membranes compared to gel or liquid-ordered (Lo) membranes. The Red-edge excitation shifts (REES) and Anisotropy magnitudes were lower in membranes in the gel or Lo, compared to membranes in Ld. The same tendency was observed for NRH+, although the spectral changes in this case were smaller, as well as the changes in the REES effect, and anisotropy magnitudes were lower related to the NR species. We suggest that NNR and NRH+ are localized in different regions of the membrane depending on the membrane phase estate. NR and NRH+ are located in more internal regions of the interface in membranes in Ld phase state, whereas in gel or Lo phase membranes, NR is located in a more external region, and NRH+ is adsorbed on the membrane surface. Consequently, the spectral signals of both, NNR and NRH+, correspond to the species distributed in different locations of the membrane in Ld compared to gel and Lo phase. Therefore, the information provided by the probe inserted in different membranes is not directly comparable.

How AlphaFold2 shaped the structural coverage of the human transmembrane proteome

AlphaFold2 (AF2) provides a 3D structure for every known or predicted protein, opening up new prospects for virtually every field in structural biology. However, working with transmembrane protein molecules pose a notorious challenge for scientists, resulting in a limited number of experimentally determined structures. Consequently, algorithms trained on this finite training set also face difficulties. To address this issue, we recently launched the TmAlphaFold database, where predicted AlphaFold2 structures are embedded into the membrane plane and a quality assessment (plausibility of the membrane-embedded structure) is provided for each prediction using geometrical evaluation. In this paper, we analyze how AF2 has improved the structural coverage of membrane proteins compared to earlier years when only experimental structures were available, and high-throughput structure prediction was greatly limited. We also evaluate how AF2 can be used to search for (distant) homologs in highly diverse protein families. By combining quality assessment and homology search, we can pinpoint protein families where AF2 accuracy is still limited, and experimental structure determination would be desirable.

CD28 and 41BB Costimulatory Domains Alone or in Combination Differentially Influence Cell Surface Dynamics and Organization of Chimeric Antigen Receptors and Early Activation of CAR T Cells

Chimeric antigen receptor (CAR)-modified T cells brought a paradigm shift in the treatment of chemotherapy-resistant lymphomas. Conversely, clinical experience with CAR T cells targeting solid tumors has been disheartening, indicating the necessity of their molecular-level optimization. While incorporating CD28 or 41BB costimulatory domains into CARs in addition to the CD3z signaling domain improved the long-term efficacy of T cell products, their influence on early tumor engagement has yet to be elucidated. We studied the antigen-independent self-association and membrane diffusion kinetics of first- (.z), second- (CD28.z, 41BB.z), and third- (CD28.41BB.z) generation HER2-specific CARs in the resting T cell membrane using super-resolution AiryScan microscopy and fluorescence correlation spectroscopy, in correlation with RoseTTAFold-based structure prediction and assessment of oligomerization in native Western blot. While .z and CD28.z CARs formed large, high-density submicron clusters of dimers, 41BB-containing CARs formed higher oligomers that assembled into smaller but more numerous membrane clusters. The first-, second-, and third-generation CARs showed progressively increasing lateral diffusion as the distance of their CD3z domain from the membrane plane increased. Confocal microscopy analysis of immunological synapses showed that both small clusters of highly mobile CD28.41BB.z and large clusters of less mobile .z CAR induced more efficient CD3ζ and pLck phosphorylation than CD28.z or 41BB.z CARs of intermediate mobility. However, electric cell-substrate impedance sensing revealed that the CD28.41BB.z CAR performs worst in sequential short-term elimination of adherent tumor cells, while the .z CAR is superior to all others. We conclude that the molecular structure, membrane organization, and mobility of CARs are critical design parameters that can predict the development of an effective immune synapse. Therefore, they need to be taken into account alongside the long-term biological effects of costimulatory domains to achieve an optimal therapeutic effect.

A novel force transduction pathway from a tension sensor to the gate in the mechano-gating of MscL channel.

The bacterial mechanosensitive channel of large conductance MscL is activated exclusively by increased tension in the membrane bilayer. Despite many proposed models for MscL opening, its precise mechano-gating mechanism, particularly how the received force at the tension sensor transmits to the gate remains incomplete. Previous studies have shown that along with amphipathic N-terminus located near the cytoplasmic surface of the membrane, Phe78 residue near the outer surface also acts as a "tension sensor," while Gly22 is a central constituent of the "hydrophobic gate." Present study focused on elucidating the force transmission mechanism from the sensor Phe78 in the outer transmembrane helix (TM2) to the gate in the inner transmembrane helix (TM1) of MscL by applying the patch clamp and molecular dynamics (MD) simulations to the wild type MscL channel and its single mutants at the sensor (F78N), the gate (G22N) and their combination (G22N/F78N) double mutant. F78N MscL resulted in a severe loss-of-function, while G22N MscL caused a gain-of-function channel exhibiting spontaneous openings at the resting membrane tension. We initially speculated that the spontaneous opening in G22N mutant might occur without tension acting on Phe78 residue. To test this hypothesis, we examined the (G22N/F78N) double mutant, which unexpectedly exhibited neither spontaneous activity nor activity by a relatively high membrane tension. To understand the underlying mechanism, we conducted MD simulations and analyzed the force transduction pathway. Results showed that the mutation at the tension sensor (F78N) in TM2 caused decreased interaction of this residue not only with lipids, but also with a group of amino acids (Ile32-Leu36-Ile40) in the neighboring TM1 helix, which resulted in an inefficient force transmission to the gate-constituting amino acids on TM1. This change also induced a slight tilting of TM1 towards the membrane plane and decreased the size of the channel pore at the gate, which seems to be the major mechanism for the inhibition of spontaneous opening of the double mutant channel. More importantly, the newly identified interaction between the TM2 (Phe78) and adjacent TM1 (Ile32-Leu36-Ile40) helices seems to be an essential force transmitting mechanism for the stretch-dependent activation of MscL given that substitution of any one of these four amino acids with Asn resulted in severe loss-of-function MscL as reported in our previous work.

Solar water splitting Pt-nanoparticle photosystem I thylakoid systems: Catalyst identification, location and oligomeric structure

Photosynthetic conversion of light energy into chemical energy occurs in sheet-like membrane-bound compartments called thylakoids and is mediated by large integral membrane protein-pigment complexes called reaction centers (RCs). Oxygenic photosynthesis of higher plants, cyanobacteria and algae requires the symbiotic linking of two RCs, photosystem II (PSII) and photosystem I (PSI), to split water and assimilate carbon dioxide. Worldwide there is a large research investment in developing RC-based hybrids that utilize the highly evolved solar energy conversion capabilities of RCs to power catalytic reactions for solar fuel generation. Of particular interest is the solar-powered production of H2, a clean and renewable energy source that can replace carbon-based fossil fuels and help provide for ever-increasing global energy demands. Recently, we developed thylakoid membrane hybrids with abiotic catalysts and demonstrated that photosynthetic Z-scheme electron flow from the light-driven water oxidation at PSII can drive H2 production from PSI. One of these hybrid systems was created by self-assembling Pt-nanoparticles (PtNPs) with the stromal subunits of PSI that extend beyond the membrane plane in both spinach and cyanobacterial thylakoids. Using PtNPs as site-specific probe molecules, we report the electron microscopic (EM) imaging of oligomeric structure, location and organization of PSI in thylakoid membranes and provide the first direct visualization of photosynthetic Z-scheme solar water-splitting biohybrids for clean H2 production.

A LRRC8 chimera with native functional properties is a heptamer with a large lipid-blocked pore.

The volume-regulated anion channel VRAC mediates volume regulatory Cl- and organic solute efflux from vertebrate cells. VRACs are heteromeric assemblies of LRRC8A-E proteins with an undefined, variable, and experimentally uncontrollable stoichiometry. The heteromeric structure of VRACs greatly complicates structure-function analyses. We circumvented this serious limitation by developing homomeric LRRC8 chimeric channels that recapitulate the functional properties of native heteromeric VRACs. Cryo-EM structures of abnormally functioning LRRC8A and non-functional LRRC8D homomeric channels demonstrated that they have a narrow-pore hexameric structure. In striking contrast, the 8C-8A(IL125) chimera comprising LRRC8C and 25 amino acids unique to the first intracellular loop (IL1) of LRRC8A is a heptamer of asymmetrically arranged subunits. Five conformationally distinct structures from a single dataset exhibit diverse subunit arrangements and interactions, especially in the cytoplasmic LRR domains. The narrowest region of the 8C-8A(IL125) pore has a solvent-accessible radius of 4.7 Å. This limiting radius is similar to that estimated for native VRACs and is more than twice that of the limiting radii of homohexameric LRRC8A and LRRC8D channels. The 8C-8A(IL125) chimera is strongly inhibited by DCPIB, similar to that of native VRACs. In contrast, homohexameric LRRC8A channels exhibit grossly abnormal DCPIB pharmacology indicating a non-native pore structure. Within the 8C-8A(IL125) pore, we observe two layers of lipids running parallel to the membrane plane. Gaps between protomers are occupied by lipid molecules providing a potential route for lipids to move in and out of the channel pore. Like native VRACs, 8C-8A(IL125) chimeric channels are activated strongly by cell swelling and low intracellular ionic strength. We suggest that VRACs are heptamers similar to closely related pannexin channels and that movement of lipids into and out of the channel pore plays a central role in channel gating.

Molecular mechanisms of integrin αvβ8 activation regulated by graphene, boron nitride and black phosphorus nanosheets

Integrin αvβ8 is a heterodimeric transmembrane protein on macrophages. Nanosheets can activate the integrin and elicit immune responses, exhibiting adverse immunotoxicity. Understanding the mechanism of integrin activation regulated by nanosheets is crucial for safe and effective use of nanosheets in biomedical applications. Herein, we performed all-atom molecular dynamics simulations to clarify the interactions between integrin αvβ8 in the cell membrane and three types of nanosheets, graphene (GRA), hexagonal boron nitride (BN), and black phosphorus (BP). We observed that BP could adsorb the intracellular end of αv monomer and thus break the inner membrane clasp, an important hydrophobic cluster for maintaining the inactive state of integrin. The association between αv and β8 subunit is weakened, promoting the integrin activation. By contrast, GRA and BN exert little influence on the association state of the integrin. Interestingly, the puckered structure of BP affects the integrin activation, where BP with the armchair direction perpendicular to the membrane plane cannot unpack the integrin. Moreover, the perturbation effect of nanosheets on the membrane was also evaluated. BP shows a milder effect on membrane structures and lipid properties than GRA and BN. This work unravels the molecular basis on the activation of integrin mediated by three nanosheets, and suggests the toxicity and therapeutic effect of well-established nanomaterials in the immune system.

A major conformational change of N-terminal helices of Bacillus thuringiensis Cry1Ab insecticidal protein is necessary for membrane insertion and toxicity.

Pore forming toxins rely on oligomerization for membrane insertion to kill their targets. Bacillus thuringiensis produces insecticidal Cry-proteins composed of three domains that form pores that kill the insect larvae. Domain I is involved in oligomerization and membrane insertion, whereas Domains II and III participate in receptor binding and specificity. However, the structural changes involved in membrane insertion of these proteins remain unsolved. The most widely accepted model for membrane insertion, the 'umbrella model', proposed that the α-4/α-5 hairpin of Domain I swings away and is inserted into the membrane. To determine the topology of Cry1Ab in the membrane, disulfide bonds linking α-helices of Domain I were introduced to restrict their movement. Disulfide bonds between helices α-2/α-3 or α-3/α-4 lost oligomerization and toxicity, indicating that movement of these helices is needed for insecticidal activity. By contrast, disulfide bonds linking helices α-5/α-6 did not affect toxicity, which contradicts the 'umbrella model'. Additionally, Föster resonance energy transfer closest approach analyses measuring distances of different points in the toxin to the membrane plane and collisional quenching assays analysing the protection of specific fluorescent-labeled residues to the soluble potassium iodide quencher in the membrane inserted state were performed. Overall, the data show that Domain I from Cry1Ab may undergo a major conformational change during its membrane insertion, where the N-terminal region (helices α-1 to α-4) participates in oligomerization and toxicity, probably forming an extended helix. These data break a paradigm, showing a new 'folding white-cane model', which better explains the structural changes of Cry toxins during insertion into the membrane.

A multipoint guidance mechanism for β-barrel folding on the SAM complex.

Mitochondrial β-barrel proteins are essential for the transport of metabolites, ions and proteins. The sorting and assembly machinery (SAM) mediates their folding and membrane insertion. We report the cryo-electron microscopy structure of the yeast SAM complex carrying an early eukaryotic β-barrel folding intermediate. The lateral gate of Sam50 is wide open and pairs with the last β-strand (β-signal) of the substrate-the 19-β-stranded Tom40 precursor-to form a hybrid barrel in the membrane plane. The Tom40 barrel grows and curves, guided by an extended bridge with Sam50. Tom40's first β-segment (β1) penetrates into the nascent barrel, interacting with its inner wall. The Tom40 amino-terminal segment then displaces β1 to promote its pairing with Tom40's last β-strand to complete barrel formation with the assistance of Sam37's dynamic α-protrusion. Our study thus reveals a multipoint guidance mechanism for mitochondrial β-barrel folding.

Many-body interactions between curvature-inducing membrane inclusions with arbitrary cross-sections.

By means of a multipolar expansion, we study analytically and numerically the interaction, in tensionless membranes, between multiple identical curvature-inducing membrane inclusions having arbitrary cross sections but uniform small detachment angles. In particular, for N circular inclusions forming regular polygons, we obtain analytical expressions for the total asymptotic interaction, up to N = 6, and we numerically compute the different multi-body contributions at arbitrary separations. We find that the latter are comparable to the sum of the two-body contributions. For N = 5 inclusions, the analytical asymptotic interaction scales as the inverse sixth power of the nearest neighbors distance d, weaker than the d-4 power for N ≠ 5. The analytical interactions are always repulsive and in good agreement with the numerical results. In the case of noncircular cross sections, we consider the case of two identical inclusions having a given number of equally shaped lobes. Depending on the number of lobes and their amplitude, we find that the interaction is asymptotically either repulsive as d-4 or attractive as d-2, and always repulsive at short distances. We also characterize how the interaction depends on the inclusion rotation angles in the membrane plane.

Bergamottin: location, aggregation and interaction with the plasma membrane

Bioactive furanocoumarins, a group of natural secondary metabolites common in higher plants, are recognized for their benefits to human health and have been shown to have numerous biological properties. However, the knowledge of its biomolecular mechanism is not known. One of the main furanocoumarins is bergamottin (BGM), which is characterized by a planar three-ringed structure and a hydrocarbon chain, which give BGM its high lipid/water partition coefficient. Because of that, and although the biological mechanism of BGM is not known, BGM bioactive properties could be ascribed to its potential to interact with the biological membrane, modulating its structure, changing its dynamics and at the same time that it might interact with lipids. For our goal, we have applied molecular dynamics to determine the position of BGM in a complex membrane and discern the possibility of certain interactions with membrane lipids. Our findings establish that BGM tends to locate in the middle of the hydrocarbon layer of the membrane, inserts in between the hydrocarbon chains of the phospholipids in an oblique position with respect to the membrane plane, increasing the fluidity of the membrane. Significantly, BGM tends to be surrounded by POPC molecules but exclude the molecule of CHOL. Outstandingly, BGM molecules associate spontaneously creating aggregates, which does not preclude them from interacting with and inserting into the membrane. The bioactive properties of BGM could be ascribed to its membranotropic effects and support the improvement of these molecules as therapeutic molecules, giving place to new opportunities for potential medical improvements. Communicated by Ramaswamy H. Sarma

Discrete localization patterns of PIP5Kγ and PLCβ3 working sequentially in phosphoinositide-cycle within mouse sensory neuron somata.

It is known that phosphatidylinositol phosphate 5 kinase (PIP5K) γ and phospholipase C (PLC) β3, working sequentially in the phosphoinositide cycle, are localized in dorsal root ganglion (DRG) somata and are involved in the regulation of pain and related sensations. However, the sites of their involvement have remained to be clarified. In the present study, immunoreactivity for PLCβ3 was distinct only in the central process of mouse DRG, but not in its peripheral process, in contrast to distinct PIP5Kγ-immunoreactivity in both peripheral and central DRG processes. No nerve terminals showing immunoreactivity for PLCβ3 were detected in any peripheral sensory fields, similar to PIP5Kγ-immunoreactivity. In DRG somata, PIP5Kγ-immunoreactivity was rather confined to the neurolemma in which dots and threads were discerned in 3D bright field light microscopy. This feature well corresponded to its discontinuous localization along the plasma membranes in immuno-electron microscopy. In contrast, PLCβ3-immunoreactivity occurred diffusely throughout the somata, but did not take distinct appearance of immunoreaction on neurolemma or plasma membranes, unlike PIP5Kγ-immunoreactivity. In addition, satellite glial cells were immunonegative for PLCβ3, but immunopositive for PIP5Kγ. The involvement of PLCβ3 in regulation of pain and related sensations is thus suggested to be mainly exerted at levels of the DRG soma and its upstream, but to be less significant in the peripheral sensory fields, similar to PIP5Kγ. The possibility is also suggested that PIP, PIP5Kγ-target, is localized heterogeneously, but PIP2, PLCβ3-target, is localized homogenously over the plane of the neuronal plasma membranes. RESEARCH HIGHLIGHTS: PIP5Kγ, different from PLCβ3, was localized heterogeneously on neuronal membranes, and this difference was demonstrated in 3D-bright field immuno-light and electron microscopy. Either PIP5Kγ or PLCβ3 was not detected in peripheral nerve terminals.

Spatiotemporal modelling reveals geometric dependence of AMPAR dynamics on dendritic spine morphology.

The modification of neural circuits depends on the strengthening and weakening of synaptic connections. Synaptic strength is often correlated to the density of the ionotropic, glutamatergic receptors, AMPARs, (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors) at the postsynaptic density (PSD). While AMPAR density is known to change based on complex biological signalling cascades, the effect of geometric factors such as dendritic spine shape, size and curvature remain poorly understood. In this work, we developed a deterministic, spatiotemporal model to study the dynamics of AMPARs during long-term potentiation (LTP). This model includes a minimal set of biochemical events that represent the upstream signalling events, trafficking of AMPARs to and from the PSD, lateral diffusion in the plane of the spine membrane, and the presence of an extrasynaptic AMPAR pool. Using idealized and realistic spine geometries, we show that the dynamics and increase of bound AMPARs at the PSD depends on a combination of endo- and exocytosis, membrane diffusion, the availability of free AMPARs and intracellular signalling interactions. We also found non-monotonic relationships between spine volume and the change in AMPARs at the PSD, suggesting that spines restrict changes in AMPARs to optimize resources and prevent runaway potentiation. KEY POINTS: Synaptic plasticity involves dynamic biochemical and physical remodelling of small protrusions called dendritic spines along the dendrites of neurons. Proper synaptic functionality within these spines requires changes in receptor number at the synapse, which has implications for downstream neural functions, such as learning and memory formation. In addition to being signalling subcompartments, spines also have unique morphological features that can play a role in regulating receptor dynamics on the synaptic surface. We have developed a spatiotemporal model that couples biochemical signalling and receptor trafficking modalities in idealized and realistic spine geometries to investigate the role of biochemical and biophysical factors in synaptic plasticity. Using this model, we highlight the importance of spine size and shape in regulating bound AMPA receptor dynamics that govern synaptic plasticity, and predict how spine shape might act to reset synaptic plasticity as a built-in resource optimization and regulation tool.

TmAlphaFold database: membrane localization and evaluation of AlphaFold2 predicted alpha-helical transmembrane protein structures.

AI-driven protein structure prediction, most notably AlphaFold2 (AF2) opens new frontiers for almost all fields of structural biology. As traditional structure prediction methods for transmembrane proteins were both complicated and error prone, AF2 is a great help to the community. Complementing the relatively meager number of experimental structures, AF2 provides 3Dpredictions for thousands of new alpha-helical membrane proteins. However, the lack of reliable structural templates and the fact that AF2 was not trained to handle phase boundaries also necessitates a delicate assessment of structural correctness. In our new database, Transmembrane AlphaFold database (TmAlphaFold database), we apply TMDET, a simple geometry-based method to visualize the likeliest position of the membrane plane. In addition, we calculate several parameters to evaluate the location of the protein into the membrane. This also allows TmAlphaFold database to show whether the predicted 3D structure is realistic or not. The TmAlphaFold database is available at https://tmalphafold.ttk.hu/.

Spatial organization and early signaling of the B-cell receptor in CLL.

Most chronic lymphocytic leukemia (CLL) clones express B-cell receptors (BcR) of both IgM/IgD isotypes; however, 5%–10% of CLL cases express isotype-switched immunoglobulin G (IgG). The early signaling and spatial patterning of the various BcRs at steady state and after activation are still fully unresolved. Herein, we show higher expression of the BcR signalosome elements and a more robust constitutive cell-intrinsic proximal BcR signaling in CLL with unmutated IGHV expressing IgM isotype (IgM U-CLL), compared with IGHV-mutated CLL (M-CLL) expressing either IgM or IgG isotypes. IgM in U-CLL is frequently located in the membrane plane in polarized patches, occasionally in caps, and sometimes inside the cells. Among M-CLL, IgM is scattered laterally in the membrane plane in a similar pattern as seen in normal B cells, whereas IgG is dispersed around the cell membrane in smaller clusters than in IgM U-CLL. Upon BcR engagement, both IgG and IgM expressing M-CLL showed attenuated signaling and only slight spatial reorganization dynamics of BcR microclusters and internalization, compared with the extensive reorganization and internalization of the BcR in IgM expressing U-CLL. The global gene signature of IgG M-CLL was closely related to that of IgM M-CLL rather than IgM U-CLL. Overall, we report fundamental differences in the basal composition, biochemical status, and spatial organization of the BcR in the three examined immunogenetic CLL subtypes that correlate with their clinical behavior. On the basis of our findings, IgG class-switched M-CLL likely represents the same disease as IgM M-CLL rather than a different biological and/or clinical entity.

Understanding the Interaction between Flow Field Geometries and Porous Electrode Microstructures in Redox Flow Batteries

Redox flow batteries are a promising technological option to integrate the growing supply of renewable energies into the electricity grid, however their deployment is hampered by high costs. To increase cost competitiveness, research efforts have targeted design of new electrolytes, high performance materials, and alternative electrochemical reactor concepts [1]. One powerful strategy is to increase the overall efficiency of the electrochemical stack, which can be achieved by improving the electrochemical performance and reducing the pumping power requirements. Selecting and optimizing the flow field design and electrode microstructure is crucial to accomplish an optimum trade-off. Drawing inspiration from polymer electrolyte fuel cells, current flow battery technologies leverage flow-through, interdigitated and serpentine flow field designs [2]. However, while functional, these designs have not been tailored for the specific requirements of redox flow batteries where single-phase reactive flows are sustained. Recent studies have investigated the influence of the channels and ribs dimensions [3], branched channel geometries [4], as well as the electrode microstructure on the reactor performance [5]. However, the interaction between the flow field geometries and the electrode microstructure determines the accessible surface area, mass transfer phenomena, and pressure drop; but remains poorly understood. With this in mind, we are poised to answer the following scientific question: What is the optimal combination of flow field and electrodes in redox flow batteries? In this work, we evaluate the interaction between geometrically diverse flow field geometries and porous electrode microstructures. We study seven different flow field designs in combination with two commonly used fibrous electrode structures – a carbon paper and a cloth (Figure 1). Flow-through, serpentine, and multiple variations of interdigitated flow fields were designed and fabricated by graphite milling. We employ a suite of polarization, electrochemical impedance spectroscopy, capacitance, and pressure drop measurements to elucidate structure-property-performance relationships. We find that the interdigitated designs perform better with high density of channels (i.e. shorter rib-channel width), even though this leads to higher pressure losses. Interestingly, pressure drop measurements show a similar relative contribution of the flow field and the electrode to the pumping losses, which motivates engineering of flow field geometries and electrode structures in tandem. Mass transfer overpotentials and pressure losses in cloth electrodes are reduced when using flow field geometries that force electrolyte flow into an in-plane direction in the electrode (i.e. parallel to the membrane plane), such as flow-through and interdigitated designs with wider ribs between channels. On the contrary, carbon paper electrodes perform better with interdigitated designs that force electrolyte flow into the through-plane direction (i.e. perpendicular to the membrane plane). Based on these findings, we have undertaken the engineering of innovative flow fields designs by combining interdigitated and branched patterns using 3D-printing, obtaining promising results that will be discussed in the final part of my talk.Figure 1. Polarization results for the combination of cloth and paper electrodes with three different flow field designs, at 5 cm·s-1 in the electrode. References Sánchez-Díez, E. Ventosa, M. Guarnieri, A. Trovò, C. Flox, R. Marcilla, F. Soavi, P. Mazur, E. Aranzabe, R. Ferret, Journal of Power Sources, 481, 228804 (2021).D.Milshtein, K.M.Tenny, J.L.Barton, J.Drake, R.M.Darling and F.R.Brushett, J. Electrohcem. Soc., 164, E3265-E3275 (2017).R.Gerhardt, A.A. Wong, M.J. Aziz, J. Electrohcem. Soc., 165, A2625-A2643 (2018).Zeng, F. Li, F. Lu, X. Zhou, Y. Yuan, X. Cao, B. Xiang, Applied Energy, 238, 435-441 (2019).Forner-Cuenca, E.E. Penn, A.M. Oliveira, F.R.Brushett, J. Electrohcem. Soc., 166, A2230-A2241 (2019). Acknowledgments This work has been partially funded by the Agencia Estatal de Investigación (PID2019-106740RB-I00 and RTC-2017-5955-3/AEI/10.13039/501100011033). Figure 1

Human Claudin-7 cis-Interactions Are Not Crucial for Membrane-Membrane (Trans-) Interactions.

Human Claudin-7 (Cldn7) is a member of the Claudin (Cldn) superfamily. In vivo, these proteins form tight junctions, which establish constricted connections between cells. Cldns oligomerize within the membrane plane (= cis-interaction), and also interact with Cldns from adjacent cells (= trans-interaction). Interactions of Cldns are typically studied in vivo and structural analyses of isolated Cldns are limited. Here, we describe heterologous expression in E. coli and purification of human Cldn7, enabling in vitro analyses of the isolated protein using detergent and model membrane systems. Cldn7 exists as a monomer, hexamer, and various higher oligomers in micelles. While only limited unfolding of the protein was observed in the presence of the anionic detergent sodium dodecyl sulfate, decreased ionic strength did affect Cldn7 cis-interactions. Furthermore, we identified two amino acids which mediate electrostatic cis-interactions and analyzed the impact of disturbed cis-interaction on trans-contacts via atomic force microscopy and monitoring Förster resonance energy transfer between fluorescently labeled Cldn7-containing proteoliposomes. Our results indicate that Cldn7 cis-oligomerization might not be a prerequisite for establishing trans-contacts.

Structure of cytoplasmic ring of nuclear pore complex by integrative cryo-EM and AlphaFold

INTRODUCTION The nuclear pore complex (NPC) is the molecular conduit in the nuclear membrane of eukaryotic cells that regulates import and export of biomolecules between the nucleus and the cytosol, with vertebrate NPCs ~110 to 125 MDa in molecular mass and ~120 nm in diameter. NPCs are organized into four main rings: the cytoplasmic ring (CR) at the cytosolic side, the inner ring and the luminal ring on the plane of the nuclear membrane, and the nuclear ring facing the nucleus. Each ring possesses an approximate eightfold symmetry and is composed of multiple copies of different nucleoporins. NPCs have been implicated in numerous biological processes, and their dysfunctions are associated with a growing number of serious human diseases. However, despite pioneering studies from many groups over the past two decades, we still lack a full understanding of NPCs' organization, dynamics, and complexity. RATIONALE We used the Xenopus laevis oocyte as a model system for the structural characterization because each oocyte possesses a large number of NPC particles that can be visualized on native nuclear membranes without the aid of detergent extraction. We used single-particle cryo-electron microscopy (cryo-EM) analysis on data collected at different stage tilt angles for three-dimensional reconstruction and structure prediction with AlphaFold for model building. RESULTS We reconstructed the CR map of X. laevis NPC at 6.9 and 6.7 Å resolutions for the full CR protomer and a core region, respectively, and predicted the structures of the individual nucleoporins using AlphaFold because no high-resolution models of X. laevis Nups were available. For any ambiguous subunit interactions, we also predicted complex structures, which further guided model fitting of the CR protomer. We placed the nucleoporin or complex structures into the CR density to obtain an almost full CR atomic model, composed of the inner and outer Y-complexes, two copies of Nup205, two copies of the Nup214-Nup88-Nup62 complex, one Nup155, and five copies of Nup358. In particular, we predicted the largest protein in the NPC, Nup358, as having an S-shaped globular domain, a coiled-coil domain, and a largely disordered C-terminal region containing phenylalanine-glycine (FG) repeats previously shown to form a gel-like condensate phase for selective cargo passage. Four of the Nup358 copies clamp around the inner and outer Y-complexes to stabilize the CR, and the fifth Nup358 situates in the center of the cluster of clamps. AlphaFold also predicted a homo-oligomeric, likely specifically pentameric, coiled-coil structure of Nup358 that may provide the avidity for Nup358 recruitment to the NPC and for lowering the threshold for Nup358 condensation in NPC biogenesis. CONCLUSION Our studies offer an example of integrative cryo-EM and structure prediction as a general approach for attaining more precise models of megadalton protein complexes from medium-resolution density maps. The more accurate and almost complete model of the CR presented here expands our understanding of the molecular interactions in the NPC and represents a substantial step forward toward the molecular architecture of a full NPC, with implications for NPC function, biogenesis, and regulation. [Figure: see text].

Proportion-dependent osteogenic activity of electrospun nano-hydroxyapatite/polylactic acid fiber membrane in vitro and in vivo

The effect of proportion on the physicochemical and osteogenic properties of electrospun nano-hydroxyapatite/polylactic acid (nHAP/PLA) fiber membrane was investigated from 0/100 to 20/80 (nHAP/PLA mass ratio), aiming to optimise the proportion and integrate mechanical support, biodegradability, cell infiltration and osteogenic activity for better bone repair. The results showed the incorporated nHAP increased the hydrophilicity of the fiber surface, and 6–10 wt% nHAP/PLA membrane showed the enhanced modulus in the direction perpendicular to membrane plane. Biological experiments demonstrated the nHAP proportion-dependent osteogenic activity of nHAP/PLA membranes. The addition of 6–10 wt% nHAP in the fiber membrane significantly promoted cell proliferation and differentiation. In vivo experiments of stacked implantation of the membrane displayed multilayer growth in the pure PLA group owing to the hydrophobicity of PLA and single-layer growth in the nHAP/PLA group because the hydrophilicity of the nHAP/PLA membrane improved the adhesion of the layers. The addition of 6–10 wt% nHAP/PLA also promoted cell recruitment and infiltration and increased osteogenesis, with enhancements of 41% in bone tissue volume/total tissue volume and 37% in bone mineral density. Our study provides helpful support for the preparation of composite electrospun membranes with improved osteogenic activity by optimising the organic–inorganic proportion.