Cells respond to mechanical stress by rapid disassembly of caveolae.

The functions of caveolae, the characteristic plasma membrane invaginations, remain debated. Their abundance in cells experiencing mechanical stress led us to investigate their role in membrane-mediated mechanical response. Acute mechanical stress induced by osmotic swelling or by uniaxial stretching results in a rapid disappearance of caveolae, in a reduced caveolin/Cavin1 interaction, and in an increase of free caveolins at the plasma membrane. Tether-pulling force measurements in cells and in plasma membrane spheres demonstrate that caveola flattening and disassembly is the primary actin- and ATP-independent cell response that buffers membrane tension surges during mechanical stress. Conversely, stress release leads to complete caveola reassembly in an actin- and ATP-dependent process. The absence of a functional caveola reservoir in myotubes from muscular dystrophic patients enhanced membrane fragility under mechanical stress. Our findings support a new role for caveolae as a physiological membrane reservoir that quickly accommodates sudden and acute mechanical stresses.

The C terminus of p73 is essential for hippocampal development.

The p53 family member p73 has a complex gene structure, including alternative promoters and alternative splicing of the 3' UTR. This results in a complex range of isoforms whose biological relevance largely remains to be determined. By deleting exon 13 (which encodes a sterile α motif) from the Trp73 gene, we selectively engineered mice to replace the most abundantly expressed C-terminal isoform, p73α, with a shorter product of alternative splicing, p73ß. These mice (Trp73Δ13/Δ13 ) display severe neurodevelopmental defects with significant functional and morphological abnormalities. Replacement of p73α with p73ß results in the depletion of Cajal-Retzius (CR) cells in embryonic stages, thus depriving the developing hippocampus of the pool of neurons necessary for correct hippocampal architecture. Consequently, Trp73Δ13/Δ13 mice display severe hippocampal dysgenesis, reduced synaptic functionality and impaired learning and memory capabilities. Our data shed light on the relevance of p73 alternative splicing and show that the full-length C terminus of p73 is essential for hippocampal development.

Nitric oxide-mediated posttranslational modifications control neurotransmitter release by modulating complexin farnesylation and enhancing its clamping ability.

Nitric oxide (NO) regulates neuronal function and thus is critical for tuning neuronal communication. Mechanisms by which NO modulates protein function and interaction include posttranslational modifications (PTMs) such as S-nitrosylation. Importantly, cross signaling between S-nitrosylation and prenylation can have major regulatory potential. However, the exact protein targets and resulting changes in function remain elusive. Here, we interrogated the role of NO-dependent PTMs and farnesylation in synaptic transmission. We found that NO compromises synaptic function at the Drosophila neuromuscular junction (NMJ) in a cGMP-independent manner. NO suppressed release and reduced the size of available vesicle pools, which was reversed by glutathione (GSH) and occluded by genetic up-regulation of GSH-generating and de-nitrosylating glutamate-cysteine-ligase and S-nitroso-glutathione reductase activities. Enhanced nitrergic activity led to S-nitrosylation of the fusion-clamp protein complexin (cpx) and altered its membrane association and interactions with active zone (AZ) and soluble N-ethyl-maleimide-sensitive fusion protein Attachment Protein Receptor (SNARE) proteins. Furthermore, genetic and pharmacological suppression of farnesylation and a nitrosylation mimetic mutant of cpx induced identical physiological and localization phenotypes as caused by NO. Together, our data provide evidence for a novel physiological nitrergic molecular switch involving S-nitrosylation, which reversibly suppresses farnesylation and thereby enhances the net-clamping function of cpx. These data illustrate a new mechanistic signaling pathway by which regulation of farnesylation can fine-tune synaptic release.

Regulation of cargo-selective endocytosis by dynamin 2 GTPase-activating protein girdin.

In clathrin-mediated endocytosis (CME), specificity and selectivity for cargoes are thought to be tightly regulated by cargo-specific adaptors for distinct cellular functions. Here, we show that the actin-binding protein girdin is a regulator of cargo-selective CME. Girdin interacts with dynamin 2, a GTPase that excises endocytic vesicles from the plasma membrane, and functions as its GTPase-activating protein. Interestingly, girdin depletion leads to the defect in clathrin-coated pit formation in the center of cells. Also, we find that girdin differentially interacts with some cargoes, which competitively prevents girdin from interacting with dynamin 2 and confers the cargo selectivity for CME. Therefore, girdin regulates transferrin and E-cadherin endocytosis in the center of cells and their subsequent polarized intracellular localization, but has no effect on integrin and epidermal growth factor receptor endocytosis that occurs at the cell periphery. Our results reveal that girdin regulates selective CME via a mechanism involving dynamin 2, but not by operating as a cargo-specific adaptor.

Membrane re-modelling by BAR domain superfamily proteins via molecular and non-molecular factors.

Lipid membranes are structural components of cell surfaces and intracellular organelles. Alterations in lipid membrane shape are accompanied by numerous cellular functions, including endocytosis, intracellular transport, and cell migration. Proteins containing Bin-Amphiphysin-Rvs (BAR) domains (BAR proteins) are unique, because their structures correspond to the membrane curvature, that is, the shape of the lipid membrane. BAR proteins present at high concentration determine the shape of the membrane, because BAR domain oligomers function as scaffolds that mould the membrane. BAR proteins co-operate with various molecular and non-molecular factors. The molecular factors include cytoskeletal proteins such as the regulators of actin filaments and the membrane scission protein dynamin. Lipid composition, including saturated or unsaturated fatty acid tails of phospholipids, also affects the ability of BAR proteins to mould the membrane. Non-molecular factors include the external physical forces applied to the membrane, such as tension and friction. In this mini-review, we will discuss how the BAR proteins orchestrate membrane dynamics together with various molecular and non-molecular factors.

The modeling of Alzheimer's disease by the overexpression of mutant Presenilin 1 in human embryonic stem cells.

Cellular disease models are useful tools for Alzheimer's disease (AD) research. Pluripotent stem cells, including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), are promising materials for creating cellular models of such diseases. In the present study, we established cellular models of AD in hESCs that overexpressed the mutant Presenilin 1 (PS1) gene with the use of a site-specific gene integration system. The overexpression of PS1 did not affect the undifferentiated status or the neural differentiation ability of the hESCs. We found increases in the ratios of amyloid-ß 42 (Aß42)/Aß40 and Aß43/Aß40. Furthermore, synaptic dysfunction was observed in a cellular model of AD that overexpressed mutant PS1. These results suggest that the AD phenotypes, in particular, the electrophysiological abnormality of the synapses in our AD models might be useful for AD research and drug discovery.

Thermosensitive Ion Channel Activation in Single Neuronal Cells by Using Surface-Engineered Plasmonic Nanoparticles.

Controlling cell functions using external photoresponsive nanomaterials has enormous potential for the development of cell-engineering technologies and intractable disease therapies, but the former currently requires genetic modification of the target cells. We present a method using plasma-membrane-targeted gold nanorods (pm-AuNRs) prepared with a cationic protein/lipid complex to activate a thermosensitive cation channel, TRPV1, in intact neuronal cells. Highly localized photothermal heat generation mediated by the pm-AuNRs induced Ca(2+) influx solely by TRPV1 activation. In contrast, the use of previously reported cationic AuNRs that are coated with a conventional synthetic polymer also led to photoinduced Ca(2+) influx, but this influx resulted from membrane damage. Our method provides an optogenetic platform without the need for prior genetic engineering of the target cells and might be useful for novel TRPV1-targeted phototherapeutic approaches.

Membrane mechanisms for signal transduction: the coupling of the meso-scale raft domains to membrane-skeleton-induced compartments and dynamic protein complexes.

Virtually all biological membranes on earth share the basic structure of a two-dimensional liquid. Such universality and peculiarity are comparable to those of the double helical structure of DNA, strongly suggesting the possibility that the fundamental mechanisms for the various functions of the plasma membrane could essentially be understood by a set of simple organizing principles, developed during the course of evolution. As an initial effort toward the development of such understanding, in this review, we present the concept of the cooperative action of the hierarchical three-tiered meso-scale (2-300 nm) domains in the plasma membrane: (1) actin membrane-skeleton-induced compartments (40-300 nm), (2) raft domains (2-20 nm), and (3) dynamic protein complex domains (3-10nm). Special attention is paid to the concept of meso-scale domains, where both thermal fluctuations and weak cooperativity play critical roles, and the coupling of the raft domains to the membrane-skeleton-induced compartments as well as dynamic protein complexes. The three-tiered meso-domain architecture of the plasma membrane provides an excellent perspective for understanding the membrane mechanisms of signal transduction.

Diffusion-coupled molecular assembly: structuring of coordination polymers across multiple length scales.

Porous coordination polymers (PCPs) are an intriguing class of molecular-based materials because of the designability of framework scaffolds, pore sizes and pore surface functionalities. Besides the structural designability at the molecular scale, the structuring of PCPs into mesoscopic/macroscopic morphologies has attracted much attention due to the significance for the practical applications. The structuring of PCPs at the mesoscopic/macroscopic scale has been so far demonstrated by the spatial localization of coordination reactions on the surface of templates or at the phase boundaries. However, these methodologies have never been applied to the fabrication of solid-solution or multivariate metal-organic frameworks (MOFs), in which multiple components are homogeneously mixed. Herein, we demonstrate the structuring of a box-type superstructure comprising of a solid-solution PCP by integrating a bidirectional diffusion of multiple organic ligands into molecular assembly. The parent crystals of [Zn2(ndc)2(bpy)]n were placed in the DMF solution of additional organic component of H2bdc, and the temperature was rapidly elevated up to 80 °C (ndc = 1,4-naphthalenedicarboxylate, bpy = 4,4'-bipyridyl, bdc = 1,4-benzenedicarboxylate). The dissolution of the parent crystals induced the outward diffusion of components; contrariwise, the accumulation of the other organic ligand of H2bdc induced the inward diffusion toward the surface of the parent crystals. This bidirectional diffusion of multiple components spatially localized the recrystallization at the surface of cuboid parent crystals; therefore, the nanocrystals of a solid-solution PCP ([Zn2(bdc)1.5(ndc)0.5(bpy)]n) were organized into a mesoscopic box superstructure. Furthermore, we demonstrated that the box superstructures enhanced the mass transfer kinetics for the separation of hydrocarbons.

Induced pluripotent stem cells from CINCA syndrome patients as a model for dissecting somatic mosaicism and drug discovery.

Chronic infantile neurologic cutaneous and articular (CINCA) syndrome is an IL-1-driven autoinflammatory disorder caused mainly by NLRP3 mutations. The pathogenesis of CINCA syndrome patients who carry NLRP3 mutations as somatic mosaicism has not been precisely described because of the difficulty in separating individual cells based on the presence or absence of the mutation. Here we report the generation of NLRP3-mutant and nonmutant-induced pluripotent stem cell (iPSC) lines from 2 CINCA syndrome patients with somatic mosaicism, and describe their differentiation into macrophages (iPS-MPs). We found that mutant cells are predominantly responsible for the pathogenesis in these mosaic patients because only mutant iPS-MPs showed the disease relevant phenotype of abnormal IL-1ß secretion. We also confirmed that the existing anti-inflammatory compounds inhibited the abnormal IL-1ß secretion, indicating that mutant iPS-MPs are applicable for drug screening for CINCA syndrome and other NLRP3-related inflammatory conditions. Our results illustrate that patient-derived iPSCs are useful for dissecting somatic mosaicism and that NLRP3-mutant iPSCs can provide a valuable platform for drug discovery for multiple NLRP3-related disorders.

Development of a reentrant arrhythmia model in human pluripotent stem cell-derived cardiac cell sheets.

AIMS: Development of a human cell-derived reentrant arrhythmia model is needed for studying the mechanisms of disease and accurate drug response. METHODS AND RESULTS: We differentiated human pluripotent stem cells (hPSCs) into cardiomyocytes, and then re-plated them into cell sheets that proved capable of forming electrically coupled assemblies. We monitored the function of these re-plated sheets optically with the Ca(2+) sensitive dye Fluo-4, and found that they generated characteristic waves of activity whose velocity and patterns of propagation depended upon the concentration of sodium channel blockers; lidocaine and tetrodotoxin, and also the time after re-plating, as well as the applied stimulation frequency. Importantly, reentrant spiral-wave propagation could be generated in these sheets by applying high-frequency stimulation, particularly when cell-density in the sheets was relatively low. This was because cardiac troponin T-positive cells were more non-homogeneously distributed at low cell densities. Especially in such sheets, we could terminate spiral waves by administering the anti-arrhythmic drugs; nifekalant, E-4031, sotalol, and quinidine. We also found that in these sheets, nifekalant showed a clear dose-dependent increase in the size of the unexcitable 'cores' of these induced spiral waves, an important parallel with the treatment for ventricular tachycardia in the clinical situation, which was not shown properly in cardiac-cell sheets derived from dissociated rodent hearts. CONCLUSIONS: We have succeeded in creating from hPSCs a valuable type of cardiomyocyte sheet that is capable of generating reentrant arrhythmias, and thus is demonstrably useful for screening and testing all sorts of drugs with anti-arrhythmic potential.

Helical DNA origami tubular structures with various sizes and arrangements.

We developed a novel method to design various helical tubular structures using the DNA origami method. The size-controlled tubular structures which have 192, 256, and 320 base pairs for one turn of the tube were designed and prepared. We observed the formation of the expected short tubes and unexpected long ones. Detailed analyses of the surface patterns of the tubes showed that the short tubes had mainly a left-handed helical structure. The long tubes mainly formed a right-handed helical structure and extended to the directions of the double helical axes as structural isomers of the short tubes. The folding pathways of the tubes were estimated by analyzing the proportions of short and long tubes obtained at different annealing conditions. Depending on the number of base pairs involved in one turn of the tube, the population of left-/right-handed and short/long tubes changed. The bending stress caused by the stiffness of the bundled double helices and the non-natural helical pitch determine the structural variety of the tubes.

A Novel Sandwich Method for Serial Block Face SEM Imaging of Airway Multiciliated Epithelium.

Volume electron microscopy technologies such as serial block face scanning electron microscopy (SBF-SEM) allow the characterization of tissue organization and cellular content in three dimensions at nanoscale resolution. Here, we describe the procedure to process and image an air-liquid interface culture of human or mouse airway epithelial cells for visualization of the multiciliated epithelium by SBF-SEM in vertical or horizontal cross section.

Micro-second time-resolved X-ray single-molecule internal motions of SARS-CoV-2 spike variants.

Single-molecule intramolecular dynamics were successfully measured for three variants of SARS-CoV-2 spike protein, alpha: B.1.1.7, delta: B.1.617, and omicron: B.1.1.529, with a time resolution of 100 µs using X-rays. The results were then compared with respect to the magnitude and directions of motions for the three variants. The largest 3-D intramolecular movement was observed for the omicron variant irrespective of ACE2 receptor binding. A more detailed analysis of the intramolecular motions revealed that the distribution state of intramolecular motion for the three variants was completely different with and without ACE2 receptor binding. The molecular dynamics for the trimeric spike protein of the omicron variant increased when ACE2 binding occurred. At that time, the diffusion constant increased from 71.0 [mrad2/ms] to 91.1 [mrad2/ms].

VAMP2 regulates phase separation of α-synuclein.

α-Synuclein (αSYN), a pivotal synaptic protein implicated in synucleinopathies such as Parkinson's disease and Lewy body dementia, undergoes protein phase separation. We reveal that vesicle-associated membrane protein 2 (VAMP2) orchestrates αSYN phase separation both in vitro and in cells. Electrostatic interactions, specifically mediated by VAMP2 via its juxtamembrane domain and the αSYN C-terminal region, drive phase separation. Condensate formation is specific for R-SNARE VAMP2 and dependent on αSYN lipid membrane binding. Our results delineate a regulatory mechanism for αSYN phase separation in cells. Furthermore, we show that αSYN condensates sequester vesicles and attract complexin-1 and -2, thus supporting a role in synaptic physiology and pathophysiology.

Cholesterol- and actin-centered view of the plasma membrane: updating the Singer-Nicolson fluid mosaic model to commemorate its 50th anniversary†.

Two very polarized views exist for understanding the cellular plasma membrane (PM). For some, it is the simple fluid described by the original Singer-Nicolson fluid mosaic model. For others, due to the presence of thousands of molecular species that extensively interact with each other, the PM forms various clusters and domains that are constantly changing and therefore, no simple rules exist that can explain the structure and molecular dynamics of the PM. In this article, we propose that viewing the PM from its two predominant components, cholesterol and actin filaments, provides an excellent and transparent perspective of PM organization, dynamics, and mechanisms for its functions. We focus on the actin-induced membrane compartmentalization and lipid raft domains coexisting in the PM and how they interact with each other to perform PM functions. This view provides an important update of the fluid mosaic model.

The antipsychotic medications aripiprazole, brexpiprazole and cariprazine are off-target respiratory chain complex I inhibitors.

Antipsychotic drugs are the mainstay of treatment for schizophrenia and provide adjunct therapies for other prevalent psychiatric conditions, including bipolar disorder and major depressive disorder. However, they also induce debilitating extrapyramidal syndromes (EPS), such as Parkinsonism, in a significant minority of patients. The majority of antipsychotic drugs function as dopamine receptor antagonists in the brain while the most recent 'third'-generation, such as aripiprazole, act as partial agonists. Despite showing good clinical efficacy, these newer agents are still associated with EPS in ~ 5 to 15% of patients. However, it is not fully understood how these movement disorders develop. Here, we combine clinically-relevant drug concentrations with mutliscale model systems to show that aripiprazole and its primary active metabolite induce mitochondrial toxicity inducing robust declines in cellular ATP and viability. Aripiprazole, brexpiprazole and cariprazine were shown to directly inhibit respiratory complex I through its ubiquinone-binding channel. Importantly, all three drugs induced mitochondrial toxicity in primary embryonic mouse neurons, with greater bioenergetic inhibition in ventral midbrain neurons than forebrain neurons. Finally, chronic feeding with aripiprazole resulted in structural damage to mitochondria in the brain and thoracic muscle of adult Drosophila melanogaster consistent with locomotor dysfunction. Taken together, we show that antipsychotic drugs acting as partial dopamine receptor agonists exhibit off-target mitochondrial liabilities targeting complex I.

Utilization of photoinduced charge-separated state of donor-acceptor-linked molecules for regulation of cell membrane potential and ion transport.

The control of ion transport across cell membranes by light is an attractive strategy that allows targeted, fast control of precisely defined events in the biological membrane. Here we report a novel general strategy for the control of membrane potential and ion transport by using charge-separation molecules and light. Delivery of charge-separation molecules to the plasma membrane of PC12 cells by a membranous nanocarrier and subsequent light irradiation led to depolarization of the membrane potential as well as inhibition of the potassium ion flow across the membrane. Photoregulation of the cell membrane potential and ion transport by using charge-separation molecules is highly promising for control of cell functions.

Fractal dimension analysis and mathematical morphology of structural changes in actin filaments imaged by electron microscopy.

In this work, we examined structural changes of actin filaments interacting with myosin visualized by quick freeze deep-etch replica electron microscopy (EM) by using a new method of image processing/analysis based on mathematical morphology. In order to quantify the degree of structural changes, two characteristic patterns were extracted from the EM images. One is the winding pattern of the filament shape (WP) reflecting flexibility of the filament, and the other is the surface pattern of the filament (SP) reflecting intra-molecular domain-mobility of actin monomers constituting the filament. EM images were processed by morphological filtering followed by box-counting to calculate the fractal dimensions for WP (D(WP)) and SP (D(SP)). The result indicates that D(WP) was larger than D(SP) irrespective of the state of the filament (myosin-free or bound) and that both parameters for myosin-bound filaments were significantly larger than those for myosin-free filaments. Overall, this work provides the first quantitative insight into how conformational disorder of actin monomers is correlated with the myosin-induced increase in flexibility of actin filaments along their length as suggested by earlier studies with different techniques. Our method is yet to be improved in details, but promising as a powerful tool for studying the structural change of protein molecules and their assemblies, which can potentially be applied to a wide range of biological and biomedical images.

Three-dimensional reconstruction of the membrane skeleton at the plasma membrane interface by electron tomography.

Three-dimensional images of the undercoat structure on the cytoplasmic surface of the upper cell membrane of normal rat kidney fibroblast (NRK) cells and fetal rat skin keratinocytes were reconstructed by electron tomography, with 0.85-nm-thick consecutive sections made approximately 100 nm from the cytoplasmic surface using rapidly frozen, deeply etched, platinum-replicated plasma membranes. The membrane skeleton (MSK) primarily consists of actin filaments and associated proteins. The MSK covers the entire cytoplasmic surface and is closely linked to clathrin-coated pits and caveolae. The actin filaments that are closely apposed to the cytoplasmic surface of the plasma membrane (within 10.2 nm) are likely to form the boundaries of the membrane compartments responsible for the temporary confinement of membrane molecules, thus partitioning the plasma membrane with regard to their lateral diffusion. The distribution of the MSK mesh size as determined by electron tomography and that of the compartment size as determined from high speed single-particle tracking of phospholipid diffusion agree well in both cell types, supporting the MSK fence and MSK-anchored protein picket models.