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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.

Dysregulated mitochondrial fusion and fission has been implicated in the pathogenesis of numerous diseases. We have identified a novel function of the p53 family protein TAp73 in regulating mitochondrial dynamics. TAp73 regulates the expression of Optic atrophy 1, a protein responsible for controlling mitochondrial fusion, cristae biogenesis and electron transport chain function. Disruption of this axis results in a fragmented mitochondrial network and an impaired capacity for energy productionviaoxidative phosphorylation. Owing to the role of OPA1 in modulating cytochromecrelease, TAp73-/-cells also display an increased sensitivity to apoptotic cell death, e.g.,viaBH3-mimetics. We also show that the TAp73/OPA1 axis has functional relevance in the upper airway, where TAp73 expression is essential for multiciliated cell differentiation and function. Consistently, ciliated epithelial cells ofTrp73-/-(global p73 KO) mice display decreased expression of OPA1 and perturbations of the mitochondrial network, which may drive multiciliated cell loss. In support of this,Trp73andOPA1gene expression is decreased in COPD patients, a disease characterised by alterations in mitochondrial dynamics. We therefore highlight a potential mechanism involving the loss of p73 in COPD pathogenesis. This work also adds to the growing body of evidence for growth-promoting roles of TAp73 isoforms.

Exposure to brake abrasion dust (BAD) reduces the phagocytic capacity of macrophages, possibly contributing to increased risk of airway infections in polluted regions. The mechanisms that underlie this toxicity remain to be characterised and, considering that pre-exposure of respiratory bacteria to alternative traffic-related particulates enhances their dissemination into the lungs of mice, may include changes in the virulence of pathogens themselves. Dual proteomic screening (TMT-labelled UPLC-MS) was performed for RAW 264.7 monocyte-derived macrophages and S.aureus (USA300) following 24h exposures to BAD (0-16 ug/ml). In S.aureus, expression of agr-regulated, anti-leukocytic virulence factors (lipases, staphopains, hemolysins) increased in a dose-dependent manner as well as proteins required for epithelial invasion (serine proteases) and antibiotic resistance. RAW 264.7 exhibited a predominantly adaptive response following exposures, enhancing expression of proteins required for metal homeostasis, NFkB-mediated inflammation and endosomal maturation. However, disrupted expression of proteins required for electron transport chain activity and Fe-S cluster synthesis accompanied significant dose-dependent reductions in ATP production (60-82%, p< 0.001) while transition electron microscopy (TEM) detected disrupted mitochondrial membrane morphology. TEM also identified reductions in pseudopodia length in exposed macrophages (54% shorter than control, p< 0.05), highlighting a mechanism by which BAD exposure may decrease the incidence of host-pathogen interactions. These data highlight dual mechanisms by which BAD emissions could impair bacterial phagocytosis and enhance susceptibility to airway infections. Through avoidance and suppression of host anti-microbial mechanisms by the bacteria and, as actin polymerisation depends on ATP availability, loss of mitochondrial function and downstream pseudopodia projection in macrophages.

Single-molecule intramolecular dynamics were successfully measured for three variants of the SARS-CoV-2 spike protein, alpha: B.1.1.7, delta: B.1.617, omicron: B.1.1.529, with a time resolution of 100 microseconds using X-rays and we compared the results in respects to the magnitude and directions of motions for three variants. The largest 3-D intramolecular movement (=40 mrad2/ms) were observed for the omicron variant with and without ACE2 receptor binding. The smallest movement (=20 mrad2/ms) was observed in the alpha variant. A more detailed analysis of the intramolecular motions revealed that the intramolecular motion distribution state of the three variants showed a complete difference in binding and nonbinding to the ACE2 receptor. Interestingly, in the spike variant without ACE2 receptors, the alpha and delta variants showed a clockwise bias in the direction of rotational motions, while only the omicron variant showed an anticlockwise bias in rotational motion. However, when the ACE2 receptor was bound, all variants exhibited an anticlockwise-biased rotational motion. This information is very important for early assessment of the cell invasion process and the infectivity of the variants.

The p53 family transcriptional factor p73 plays a pivotal role in development. Ablation of p73 results in severe neurodevelopmental defects, chronic infections, inflammation and infertility. In addition to this, Trp73(−\-) mice display severe alteration in the ciliated epithelial lining and the full-length N-terminal isoform TAp73 has been implicated in the control of multiciliogenesis transcriptional program. With our recently generated Trp73(Δ13/Δ13) mouse model, we interrogate the physiological role of p73 C-terminal isoforms in vivo. Trp73(Δ13/Δ13) mice lack exon 13 in Trp73 gene, producing an ectopic switch from the C-terminal isoforms p73α to p73β. Trp73(Δ13/Δ13) mice show a pattern of expression of TAp73 comparable to the wild-type littermates, indicating that the α to β switch does not significantly alter the expression of the gene in this cell type. Moreover, Trp73(Δ13/Δ13) do not display any significant alteration in the airway ciliated epithelium, suggesting that in this context p73β can fully substitute the function of the longer isoform p73α. Similarly, Trp73(Δ13/Δ13) ciliated epithelium of the brain ependyma also does appear defective. In this district however expression of TAp73 is not detectable, indicating that expression of the gene might be compensated by alternative mechanisms. Overall our work indicates that C-terminus p73 is dispensable for the multiciliogenesis program and suggests a possible tissue-specific effect of p73 alternative splicing.

Summary The transcriptional corepressor complex CoREST is one of seven histone deacetylase complexes that regulate the genome through controlling chromatin acetylation. The CoREST complex is unique in containing both histone demethylase and deacetylase enzymes, LSD1 and HDAC1, held together by the RCOR1 scaffold protein. To date, it has been assumed that the enzymes function independently within the complex. Now, we report the assembly of the ternary complex. Using both structural and functional studies, we show that the activity of the two enzymes is closely coupled and that the complex can exist in at least two distinct states with different kinetics. Electron microscopy of the complex reveals a bi-lobed structure with LSD1 and HDAC1 enzymes at opposite ends of the complex. The structure of CoREST in complex with a nucleosome reveals a mode of chromatin engagement that contrasts with previous models., Graphical Abstract, Highlights • The activities of LSD1 and HDAC1 are closely coupled in the CoREST complex • Both LSD1 and HDAC1 exist in two different kinetic states • CoREST has a bi-lobed, flexible structure with the two enzymes located at opposite ends • CoREST interacts with methylated nucleosomes via LSD1, but not HDAC1 or RCOR1, Using a real-time NMR assay, Song et al. characterize crosstalk between LSD1 and HDAC1 in the CoREST complex. Activation or inhibition of one enzyme strongly affects activity of the other. Electron microscopy studies of the complex reveal a bi-lobed structure with implications for the mode of interaction with nucleosomes.

Trophoblast are extra-embryonic cells that are essential to maintain pregnancy. Human trophoblasts arise from the morula as trophectoderm (TE), which, after implantation, differentiates into cytotrophoblast (CT), syncytiotrophoblast (ST), and extravillous trophoblast (EVT) composing the placenta. Here we show that naïve, but not primed, human pluripotent stem cells (PSCs) recapitulate trophoblast development. Naïve PSC-derived TE and CT (nCT) recreated the human and monkey TE-to-CT transition. nCT self-renewed as CT stem cells and had the characteristics of proliferating villous CT and CT in the cell column of the first trimester. Notably, although primed PSCs differentiated into trophoblast-like cells (pBAP), pBAP were distinct from nCT and human placenta-derived CT stem cells, exhibiting properties consistent of the amnion. Our findings establish an authentic paradigm for human trophoblast development, demonstrating the invaluable properties of naïve human PSCs. Our system will provide a platform to study the molecular mechanisms underlying trophoblast development and related diseases.

Trophoblasts are extraembryonic cells that are essential for maintaining pregnancy. Human trophoblasts arise from the morula as trophectoderm (TE), which, after implantation, differentiates into cytotrophoblasts (CTs), syncytiotrophoblasts (STs), and extravillous trophoblasts (EVTs), composing the placenta. Here we show that naive, but not primed, human pluripotent stem cells (PSCs) recapitulate trophoblast development. Naive PSC-derived TE and CTs (nCTs) recreated human and monkey TE-to-CT transition. nCTs self-renewed as CT stem cells and had the characteristics of proliferating villous CTs and CTs in the cell column of the first trimester. Notably, although primed PSCs differentiated into trophoblast-like cells (BMP4, A83-01, and PD173074 [BAP]-treated primed PSCs [pBAPs]), pBAPs were distinct from nCTs and human placenta-derived CT stem cells, exhibiting properties consistent with the amnion. Our findings establish an authentic paradigm for human trophoblast development, demonstrating the invaluable properties of naive human PSCs. Our system provides a platform to study the molecular mechanisms underlying trophoblast development and related diseases.

Unroofing, which is the mechanical shearing of a cell to expose the cytoplasmic surface of the cell membrane, is a unique preparation method that allows membrane cytoskeletons to be observed by cryo-electron microscopy, atomic force microscopy, freeze-etching electron microscopy and other methods. Ultrasound and adhesion have been known to mechanically unroof cells. In this study, unroofing using these two means was denoted sonication unroofing and adhesion unroofing, respectively. We clarified the mechanisms by which cell membranes are removed in these unroofing procedures and established efficient protocols for each based on the mechanisms. In sonication unroofing, fine bubbles generated by sonication adhered electrostatically to apical cell surfaces and then removed the apical (dorsal) cell membrane with the assistance of buoyancy and water flow. The cytoplasmic surface of the ventral cell membrane remaining on the grids became observable by this method. In adhesion unroofing, grids charged positively by coating with Alcian blue were pressed onto the cells, thereby tightly adsorbing the dorsal cell membrane. Subsequently, a part of the cell membrane strongly adhered to the grids was peeled from the cells and transferred onto the grids when the grids were lifted. This method thus allowed the visualization of the cytoplasmic surface of the dorsal cell membrane. This paper describes robust, improved protocols for the two unroofing methods in detail. In addition, micro-unroofing (perforation) likely due to nanobubbles is introduced as a new method to make cells transparent to electron beams.

Summary High-purity cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) are promising for drug development and myocardial regeneration. However, most hiPSC-derived CMs morphologically and functionally resemble immature rather than adult CMs, which could hamper their application. Here, we obtained high-quality cardiac tissue-like constructs (CTLCs) by cultivating hiPSC-CMs on low-thickness aligned nanofibers made of biodegradable poly(D,L-lactic-co-glycolic acid) polymer. We show that multilayered and elongated CMs could be organized at high density along aligned nanofibers in a simple one-step seeding process, resulting in upregulated cardiac biomarkers and enhanced cardiac functions. When used for drug assessment, CTLCs were much more robust than the 2D conventional control. We also demonstrated the potential of CTLCs for modeling engraftments in vitro and treating myocardial infarction in vivo. Thus, we established a handy framework for cardiac tissue engineering, which holds high potential for pharmaceutical and clinical applications., Graphical Abstract, Highlights • hiPSC-CMs are seeded on aligned nanofibers to obtain 3D cardiac tissue-like constructs • Drug assessment using CTLCs can be more robust than conventional cultures • CTLCs can be used to model in vitro cardiac tissue engraftment • CTLCs improve the function of rat hearts with myocardial infarction, Although high-purity cardiomyocytes can be achieved, they are randomly distributed and resemble immature rather than adult CMs. In this article, Liu Li and her colleagues show that 3D and aligned cardiac tissue-like constructs (CTLCs) can be obtained using hPSC-CMs and aligned nanofiber. They also demonstrate the robust drug responses and repair capability of CTLCs in a myocardial infarction model.

Significance Alteration of splicing is emerging as a relevant cause of human disease. The C-terminal region of p73 is subject to complex alternative splicing that can give rise to seven different isoforms. Here, using a newly generated mouse model, we determine the functional consequence of in vivo ectopic switch from the physiologically expressed and most abundant isoform p73α to the shorter p73β isoform. Expression of p73β leads to neurodevelopmental defects with functional and morphological abnormalities in the mouse hippocampus. The ectopic isoform switch results in depletion of Cajal–Retzius (CR) neurons in embryonic stages, leading to aberrant hippocampal architecture. Our study indicates that deregulation in p73 alternative splicing might underlie neurodevelopmental human conditions., 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, p73β. These mice (Trp73Δ13/Δ13) display severe neurodevelopmental defects with significant functional and morphological abnormalities. Replacement of p73α with p73β 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.

Lack of photobleaching and blinking of new silicon nanocrystals allows direct observation of single-molecule receptor internalization and large-scale mosaicism in the plasma membrane., Fluorescence microscopy is used extensively in cell-biological and biomedical research, but it is often plagued by three major problems with the presently available fluorescent probes: photobleaching, blinking, and large size. We have addressed these problems, with special attention to single-molecule imaging, by developing biocompatible, red-emitting silicon nanocrystals (SiNCs) with a 4.1-nm hydrodynamic diameter. Methods for producing SiNCs by simple chemical etching, for hydrophilically coating them, and for conjugating them to biomolecules precisely at a 1:1 ratio have been developed. Single SiNCs neither blinked nor photobleached during a 300-min overall period observed at video rate. Single receptor molecules in the plasma membrane of living cells (using transferrin receptor) were imaged for ≥10 times longer than with other probes, making it possible for the first time to observe the internalization process of receptor molecules at the single-molecule level. Spatial variations of molecular diffusivity in the scale of 1–2 µm, i.e., a higher level of domain mosaicism in the plasma membrane, were revealed.

Membrane fusion under mildly acidic pH occurs naturally during viral infection in cells and has been exploited in the field of nanoparticle-mediated drug delivery to circumvent endosomal entrapment of the cargo. Herein, we aimed to confer virus-like fusogenic activity to HDL in the form of a ca. 10-nm disc comprising a discoidal lipid bilayer and two copies of a lipid-binding protein at the edge. A series of HDL mutants were prepared with a mixture of three lipids and a cell-penetrating peptide (TAT, penetratin, or Arg8) fused to the protein. In a lipid-mixing assay with anionic liposomes at pH 5.5, one HDL mutant showed the fusogenic activity higher than known fusogenic liposomes. In live mammalian cells, this HDL mutant showed high plasma membrane-binding activity in the presence of serum independent of pH. In the absence of serum, a mildly acidic pH dependency for binding to the plasma membrane and the subsequent lipid mixing between them was observed for this mutant. We propose a novel strategy to develop HDL-based drug carriers by taking advantage of the HDL lipid/protein composite structure.

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., Author summary One way neurons communicate with each other and with other tissues, such as muscle, is by releasing chemical compounds known as neurotransmitters at sites of interaction known as synapses. This synaptic transmission can be finely regulated by both the releasing neuron and the receiving neuron or muscle cell. Many signaling molecules and pathways are involved in neurotransmitter release. In this study, we have investigated one of such pathways and its role in modulating neurotransmitter release at the neuromuscular synapse of the larva of the fruit fly Drosophila melanogaster. This regulation involves nitric oxide, a freely diffusible reactive molecule that can be generated in response to activity in the motor neuron. Several neuronal proteins can be modified by nitric oxide, and our study identified a specific target molecule that regulates neurotransmitter release. This protein, called complexin, undergoes a posttranslational modification in response to increased levels of nitric oxide, changing its localization and function at the synapse and modulating neurotransmission. Our findings can explain how neurons may modulate communication in an activity-dependent manner utilizing nitric oxide signaling.

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-β 42 (Aβ42)/Aβ40 and Aβ43/Aβ40. 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.

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., 光を使って神経細胞の「痛み」感知を制御する手法を開発 -新しい鎮痛療法の可能性-. 京都大学プレスリリース. 2015-08-14.

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.

Caveolae are abundant flask-shaped invaginations of plasma membranes that buffer membrane tension through their deformation. Few quantitative studies on the deformation of caveolae have been reported. Each caveola contains approximately 150 caveolin-1 proteins. In this study, we estimated the extent of caveolar deformation by measuring the density of caveolin-1 projected onto a two-dimensional (2D) plane. The caveolin-1 in a flattened caveola is assumed to have approximately one-quarter of the density of the caveolin-1 in a flask-shaped caveola. The proportion of one-quarter-density caveolin-1 increased after increasing the tension of the plasma membrane through hypo-osmotic treatment. The one-quarter-density caveolin-1 was soluble in detergent and formed a continuous population with the caveolin-1 in the caveolae of cells under isotonic culture. The distinct, dispersed lower-density caveolin-1 was soluble in detergent and increased after the application of tension, suggesting that the hypo-osmotic tension induced the dispersion of caveolin-1 from the caveolae, possibly through flattened caveolar intermediates.

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.

A hydrophobic gadolinium bis(naphthalocyanine) sandwich complex (GdSand) possessing several absorbances across visible and infrared wavelengths (up to 2500 nm) was solubilized in aqueous solution by uptake into a nascent mutant high-density lipoprotein (HDL) nanocarrier. The HDL nanocarrier was additionally functionalized with a trans-activator of transcription peptide sequence to promote efficient cell penetration of the drug delivery system (cpHDL). The dye-loaded nanocarrier (GdSand@cpHDL) exhibited photothermal heat generation properties upon irradiation with near-infrared (NIR) laser light, with controllable heat generation abilities as a function of the incident laser light power. Comparison of the photothermal behavior of the dyes GdSand and the well-explored molecular photothermal agent indocyanine green (ICG) in the cpHDL nanocarrier (i.e., ICG@cpHDL) revealed two significant advantages of GdSand@cpHDL: (1) the ability to maintain elevated temperatures upon light absorption for extended periods of time, with a reduced degree of self-destruction of the dye, and (2) exclusive photothermal heat generation with no detectable singlet oxygen production leading to improved integrity of the cpHDL nanocarrier after irradiation. Finally, GdSand@cpHDL was successfully subjected to an in vitro study against NCI-H460 human lung cancer cells, demonstrating the proof-of-principle utility of lanthanide sandwich complexes in photothermal therapeutic applications.

Naive pluripotent stem cells (PSCs) utilize both glycolysis and oxidative phosphorylation (OXPHOS) to satisfy their metabolic demands. However, it is unclear how somatic cells acquire this hybrid energy metabolism during reprogramming toward naive pluripotency. Here, we show that when transduced with Oct4, Sox2, and Klf4 (OSK) into murine fibroblasts, Zic3 and Esrrb synergistically enhance the reprogramming efficiency by regulating cellular metabolic pathways. These two transcription factors (TFs) cooperatively activate glycolytic metabolism independently of hypoxia inducible factors (HIFs). In contrast, the regulatory modes of the TFs on OXPHOS are antagonistic: Zic3 represses OXPHOS, whereas Esrrb activates it. Therefore, when introduced with Zic3, Esrrb restores OXPHOS activity, which is essential for efficient reprogramming. In addition, Esrrb-mediated OXPHOS activation is critical for the conversion of primed PSCs into the naive state. Our study suggests that the combinatorial function of TFs achieves an appropriate balance of metabolic pathways to induce naive PSCs.

Ultraspeed single-molecule tracking with, The mechanisms by which the diffusion rate in the plasma membrane (PM) is regulated remain unresolved, despite their importance in spatially regulating the reaction rates in the PM. Proposed models include entrapment in nanoscale noncontiguous domains found in PtK2 cells, slow diffusion due to crowding, and actin-induced compartmentalization. Here, by applying single-particle tracking at high time resolutions, mainly to the PtK2-cell PM, we found confined diffusion plus hop movements (termed “hop diffusion”) for both a nonraft phospholipid and a transmembrane protein, transferrin receptor, and equal compartment sizes for these two molecules in all five of the cell lines used here (actual sizes were cell dependent), even after treatment with actin-modulating drugs. The cross-section size and the cytoplasmic domain size both affected the hop frequency. Electron tomography identified the actin-based membrane skeleton (MSK) located within 8.8 nm from the PM cytoplasmic surface of PtK2 cells and demonstrated that the MSK mesh size was the same as the compartment size for PM molecular diffusion. The extracellular matrix and extracellular domains of membrane proteins were not involved in hop diffusion. These results support a model of anchored TM-protein pickets lining actin-based MSK as a major mechanism for regulating diffusion.

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 Ca2+ 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.

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-1β secretion. We also confirmed that the existing anti-inflammatory compounds inhibited the abnormal IL-1β 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.

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 (DWP) and SP (DSP). The result indicates that DWP was larger than DSP 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.

Newly generated neurons pass through a series of well-defined developmental stages, which allow them to integrate into existing neuronal circuits. After exit from the cell cycle, postmitotic neurons undergo neuronal migration, axonal elongation, axon pruning, dendrite morphogenesis and synaptic maturation and plasticity. Lack of a global metabolic analysis during early cortical neuronal development led us to explore the role of cellular metabolism and mitochondrial biology during ex vivo differentiation of primary cortical neurons. Unexpectedly, we observed a huge increase in mitochondrial biogenesis. Changes in mitochondrial mass, morphology and function were correlated with the upregulation of the master regulators of mitochondrial biogenesis, TFAM and PGC-1α. Concomitant with mitochondrial biogenesis, we observed an increase in glucose metabolism during neuronal differentiation, which was linked to an increase in glucose uptake and enhanced GLUT3 mRNA expression and platelet isoform of phosphofructokinase 1 (PFKp) protein expression. In addition, glutamate-glutamine metabolism was also increased during the differentiation of cortical neurons. We identified PI3K-Akt-mTOR signalling as a critical regulator role of energy metabolism in neurons. Selective pharmacological inhibition of these metabolic pathways indicate existence of metabolic checkpoint that need to be satisfied in order to allow neuronal differentiation.

Nanotechnologies to allow the nondisruptive introduction of carriers in vivo have wide potential for therapeutic delivery system. We have prepared functional nano-magnetic particles (d = 3 nm) by silanization with (3-aminopropyl) triethoxysilane. For the purpose of functionalizing the surface of the nanoparticles with amino groups for subsequent cross-linking with pharmaceuticals and biomolecules. The extremely small particles were successfully introduced into living cells without any further modification to enhance endocytic internalization, such as the use of a cationic help. The cells containing the internalized particles continued to thrive, indicating that the particles have no inhibition effect for mitosis. In addition, the particles could be incorporated into the subcutaneous tissue of mouse's ear from ear skin and were able to be localized upon application of an external magnetic field. The functionalized nano-magnetic particles are expected to be useful as a new delivery tool.

Src family tyrosine kinases are essential for neural development, but their in vivo functions remain elusive because of functional compensation among family members. To elucidate the roles of individual Src family members in vivo, we generated transgenic mice expressing the neuronal form of c-Src (n-Src), Fyn, and their constitutively active forms in cerebellar Purkinje cells using the L7 promoter. The expression of the constitutively active n-Src retarded the postnatal development of Purkinje cells and disrupted dendritic morphogenesis, whereas the wild-type n-Src had only moderate effects. Neither wild-type nor constitutively active Fyn over-expression significantly affected Purkinje-cell morphology. The aberrant Purkinje cells in n-Src transgenic mice retained multiple dendritic shafts extending in non-polarized directions and were located heterotopically in the molecular layer. Ultrastructural observation of the dendritic shafts revealed that the microtubules of n-Src transgenic mice were more densely and irregularly arranged, and had structural deformities. In primary culture, Purkinje cells from n-Src transgenic mice developed abnormally thick dendritic shafts and large growth-cone-like structures with poorly extended dendrites, which could be rescued by treatment with a selective inhibitor of Src family kinases, PP2. These results suggest that n-Src activity regulates the dendritic morphogenesis of Purkinje cells through affecting microtubule organization.

Fusogenic liposomes have a wide-range of applications as DDS and gene/protein delivery into living cells. A variety of surface modifications of drug carriers, to enable fusion with cells, have been proposed, however, the mechanism of fusion has still not been determined. To further improve the efficiency of drug carriers, a simple and easily examinable model of a living cell surface is needed. In this study, the time-course of a fusion phenomena was made by measuring the surface pressure increase of a phospholipid monolayer spread at the air/water interface due to the fusion of liposomes carrying PEO-lipid (dialkyl-terminated polyethylene oxide) reconstituted on their outer surface. The kinetics of the surface pressure change appeared to be bimodal, indicating the coexistence of different fusion pathways. It was found that the presence of the PEO-lipid on the liposome surface led to a faster lipid transfer compared to non-modified DMPC liposomes. This indicated that the reconstitution of PEO-lipid provided an alternative transfer pathway to that for non-fusogenic liposomes that show only a slow lipid transfer to phospholipid monolayers. The relation between the rate of fusion and the surface pressure of the host membrane is discussed.

Intracellular drug delivery by nanoparticles is often hampered by their endosomal entrapment followed by their degradation in the lysosomal compartment and/or exocytosis. Here, we show that internalization and endosomal escape of cargoes in a cationized natural nanocarrier, high-density lipoprotein (HDL), can be controlled in a pH-dependent manner through stable complexation with a membranolytic anionic block polymer. A genetically and chemically cationized form of HDL (catHDL) is prepared for the first time by both genetic fusion with YGRKKRRQRRR peptide and incorporation of 1,2-dioleoyloxy-3-(trimethylammonium)propane. Upon addition of poly(ethylene glycol)-block-poly(propyl methacrylate-co-methacrylic acid) (PA), catHDL yields inhibition of internalization at neutral pH and its subsequent recovery at mildly acidic pH. catHDL forms a stable discoidal-shape complex with PA (catHDL/PA) (ca. 50 nm in diameter), even in the presence of serum. Significant enhancement of endosomal escape of a catHDL component is observed after a 1-h treatment of human cancer cells with catHDL/PA. Doxorubicin and curcumin, fluorescent anti-cancer drugs, encapsulated into catHDL/PA are also translocated outside of endosomes, compared with that into catHDL, and their cytotoxicities are enhanced inside the cells. These data suggest that catHDL/PA may have a potential benefit to improve the cellular delivery and endosomal escape of therapeutics under mildly acidic conditions such as in tumor tissues.

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 ∼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.

SummaryThe serine/threonine kinase Akt (also called protein kinase B) is well known as an important regulator of cell survival and growth and has also been shown to be required for cell migration in different organisms. However, the mechanism by which Akt functions to promote cell migration is not understood. Here, we identify an Akt substrate, designated Girdin/APE (Akt-phosphorylation enhancer), which is an actin binding protein. Girdin expresses ubiquitously and plays a crucial role in the formation of stress fibers and lamellipodia. Akt phosphorylates serine at position 1416 in Girdin, and phosphorylated Girdin accumulates at the leading edge of migrating cells. Cells expressing mutant Girdin, in which serine 1416 was replaced with alanine, formed abnormal elongated shapes and exhibited limited migration and lamellipodia formation. These findings suggest that Girdin is essential for the integrity of the actin cytoskeleton and cell migration and provide a direct link between Akt and cell motility.

Rho-kinase and myosin phosphatase cooperatively regulate the phosphorylation level of myosin light chain and are involved in the formation of stress fibres and smooth muscle contraction. Rho-kinase has been known to be localized at stress fibres, but little is known about the mechanism of its localization. Here we identified non-muscle myosin heavy chain IIA and IIB as the pleckstrin homology domain-interacting molecules by affinity column chromatography. The pleckstrin homology domain of Rho-kinase binds to myosin II directly in in vitro cosedimentation assay. The C-terminal region of the pleckstrin homology domain was important for this interaction, and the point mutations in the pleckstrin homology domain mutant (W1170A, W1340L) resulted in a decrease in the binding. We also found that the pleckstrin homology domain, but not the pleckstrin homology domain mutant (W1170A, W1340L), was localized at stress fibres in fibroblasts. These results indicate that Rho-kinase is localized at stress fibres through binding of the pleckstrin homology domain to myosin II.

Surface engineering of mesoscopic metal nanoparticles to increase biocompatibility and cell interaction is important for improvement of their therapeutic properties. Here, we describe a strategy to stabilize mesoscopic metal nanoparticles and to enhance their cell interaction by stepwise addition of (Z)-9-octadecenoate (oleate) and a cell-penetrating peptide-fused high-density lipoprotein (cpHDL). Oleate replaces a cytotoxic dispersant on the surface of gold nanorods (AuNRs), which enables subsequent cpHDL binding without causing aggregation. Notably, these two lipidic dispersants are probably intercalated on the surface. This procedure was also used to stabilize 20 nm spherical gold nanoparticles and 40 nm aggregates of 10 nm magnetite nanoparticles. cpHDL-bound AuNRs were internalized greater than 80 times more efficiently than poly(ethylene glycol)-conjugated AuNRs and were able to elicit cancer cell photoablation.

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.

Modification of egg phosphatidylcholine (eggPC) and dimyristoylphosphatidylcholine liposomes with poly(ethylene oxide)-substituted lipids [PEO-lipid(m,n); m and n mean, respectively, the chain length of alkyl group and the number of PEO segments]. Morphologies of these supermolecules were investigated by means of laser dynamic light scattering and transmission electron microscopy. Reconstitution of the PEO-lipid to eggPC liposome was possible below 40–50 mol% of total lipids for PEO-lipid(12,5), and 30–40 mol% for PEO-lipid(12,13) and PEO-lipid(12,31). Effective encapsulation of FITC-dextran (1.96 kDa) into the liposome was obtained using the eggPC liposome modified with PEO-lipid. This PEO-modified liposome showed sufficient barrier function to water-soluble polymers such as dextran. Through various studies, it was found that PEO-lipid(12,13) is relatively compatible for modifying conventional eggPC liposome.

To better understand the dynamics of PEO-lipid-modified phosphatidylcholine liposomes (“fusogenic liposomes”), the mobility of the PEO segment on the liposomal surface and the fluidity of the bilayer were investigated by measuring the spin-lattice relaxation time (T1by 13C-NMR spectroscopy and by fluorescence depolarization. 13C-NMR studies revealed that the PEO-lipids in this work exhibit similar behavior on liposomal surfaces irrespective of the PEO chain length. Furthermore, with regard to the relationship between morphology and dynamics of the PEO-lipid and phosphatidylcholine mixture, it was found that the mobility of PEO segments are altered by the morphology; liposome < aqueous micelle < monomeric dispersion in organic solution. Fluorescence depolarization studies revealed that liposomal membranes were tighter with the addition of PEO-lipid(12,a31) than with the addition of PEO-lipid(12,a13). In contrast, the reconstitution of PEO-lipid(12,a5) into liposomes increased membrane fluidity.