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Abstract The single-molecule tracking of transmembrane receptors in living cells has provided significant insights into signaling mechanisms, such as mobility and clustering upon their activation/inactivation, making it a potential screening method for drug discovery. Here we show that single-molecule tracking-based screening can be used to explore compounds both detectable and undetectable by conventional methods for disease-related receptors. Using an automated system for a fast large-scale single-molecule analysis, we screen for epidermal growth factor receptor (EGFR) from 1134 of FDA approved drugs. The 18 hit compounds include all EGFR-targeted tyrosine kinase inhibitors (TKIs) in the library that suppress any phosphorylation-dependent mobility shift of EGFR, proving the concept of this approach. The remaining hit compounds are not reported as EGFR-targeted drugs and do not inhibit EGF-induced EGFR phosphorylation. These non-TKI compounds affect the mobility and/or clustering of EGFR without EGF and induce EGFR internalization, to impede EGFR-dependent cell growth. Thus, single-molecule tracking provides an alternative modality for discovering therapeutics on various receptor functions with previously untargeted mechanisms.

Single-molecule imaging in living cells is an effective tool for elucidating the mechanisms of cellular phenomena at the molecular level. However, the analysis was not designed for throughput and requires high expertise, preventing it from reaching large scale, which is necessary when searching for rare cells that induce singularity phenomena. To overcome this limitation, we have automated the imaging procedures by combining our own focusing device, artificial intelligence, and robotics. The apparatus, called automated in-cell single-molecule imaging system (AiSIS), achieves a throughput that is a hundred-fold higher than conventional manual imaging operations, enabling the analysis of molecular events by individual cells across a large population. Here, using AiSIS, we demonstrate the single-molecule imaging of molecular behaviors and reactions related to tau protein aggregation, which is considered a singularity phenomenon in neurological disorders. Changes in the dynamics and kinetics of molecular events were observed inside and on the basal membrane of cells after the induction of aggregation. Additionally, to detect rare cells based on the molecular behavior, we developed a method to identify the state of individual cells defined by the quantitative distribution of molecular mobility and clustering. Using this method, cellular variations in receptor behavior were shown to decrease following ligand stimulation. This cell state analysis based on large-scale single-molecule imaging by AiSIS will advance the study of molecular mechanisms causing singularity phenomena.

Abstract The plasma membrane is widely regarded as the hub of the numerous signal transduction activities. Yet, the fundamental biophysical mechanisms that spatiotemporally compartmentalize different classes of membrane proteins remain unclear. Using multimodal live-cell imaging, here we first show that several lipid-anchored membrane proteins are consistently depleted from the membrane regions where the Ras/PI3K/Akt/F-actin network is activated. The dynamic polarization of these proteins does not depend upon the F-actin-based cytoskeletal structures, recurring shuttling between membrane and cytosol, or directed vesicular trafficking. Photoconversion microscopy and single-molecule measurements demonstrate that these lipid-anchored molecules have substantially dissimilar diffusion profiles in different regions of the membrane which enable their selective segregation. When these diffusion coefficients are incorporated into an excitable network-based stochastic reaction-diffusion model, simulations reveal that the altered affinity mediated selective partitioning is sufficient to drive familiar propagating wave patterns. Furthermore, normally uniform integral and lipid-anchored membrane proteins partition successfully when membrane domain-specific peptides are optogenetically recruited to them. We propose “dynamic partitioning” as a new mechanism that can account for large-scale compartmentalization of a wide array of lipid-anchored and integral membrane proteins during various physiological processes where membrane polarizes.

In eukaryotic motile cells, the active Ras (Ras-GTP)-enriched domain is generated in an asymmetric manner on the cell membrane through the excitable dynamics of an intracellular signaling network. This asymmetric Ras signaling regulates pseudopod formation for both spontaneous random migration and chemoattractant-induced directional migration. While membrane lipids, such as sphingomyelin and phosphatidylserine, contribute to Ras signaling in various cell types, whether they are involved in the Ras excitability for cell motility is unknown. Here we report that functional Ras excitability requires the normal metabolism of sphingomyelin for efficient cell motility and chemotaxis. The pharmacological blockade of sphingomyelin metabolism by an acid-sphingomyelinase inhibitor, fendiline, and other inhibitors suppressed the excitable generation of the stable Ras-GTP-enriched domain. The suppressed excitability failed to invoke enough basal motility to achieve directed migration under shallow chemoattractant gradients. The fendiline-induced defects in Ras excitability, motility and stimulation-elicited directionality were due to an accumulation of sphingomyelin on the membrane, which could be recovered by exogenous sphingomyelinase or phosphatidylserine without changing the expression of Ras. These results indicate a novel regulatory mechanism of the excitable system by membrane lipids, in which sphingomyelin metabolism provides a membrane environment to ensure Ras excitation for efficient cellular motility and chemotaxis. Key words: cell polarity, cell migration, Ras, excitability, sphingomyelin

Eukaryotic cells exhibit random migration in the absence of extracellular directional cues. This random migration acts as basal motility for various migratory responses such as chemotaxis. The self-organization of random motility requires the internal signals that determine the anterior side of the migrating cell be generated spontaneously from the intrinsic activities of intracellular signaling networks. Recent studies have identified an excitable system as the mechanism of the spontaneous signal generation. Here, we discuss how the excitable system of Ras, a small G protein, regulates signaling networks in Dictyostelium discoideum as a model organism. The excitability produces a domain where an active form of Ras is enriched on the cell membrane without extracellular directional cues, such that Ras serves as the anterior signal. The typical spatiotemporal characteristics are mathematically explained by reaction-diffusion models. These models further enable a quantitative analysis of the dynamics that depends on the internal cellular states and surrounding environments. Downstream of the Ras excitable system, a phosphoinositide metabolic network composed of PI3K, PTEN, PI(3,4,5)P3 and PI(4,5)P2 exhibits bistability to discretize the anterior and posterior regions of the cell membrane. Upstream, a local excitation and global inhibition local excitation global inhibition network, which works for gradient sensing in the presence of chemoattractant gradients, spatiotemporally biases the excitability of Ras for chemotaxis. In parallel with the Ras excitable system, the cGMP signaling pathway constitutes another excitable system of its own periodicity to ensure flexible migratory dynamics. In addition to these intracellular signaling networks, an intercellular signaling network activated by secreted cAMP is coupled with the Ras excitable system for collective cell migration. Finally, we discuss how the excitable system of Ras operates as a platform of information integration by receiving multiple intrinsic and extrinsic signals to ensure spontaneous cellular activity and robust responses in eukaryotic cell migration under natural complex environments.

Abstract Glycosylation of proteins and lipids in viruses and their host cells is important for viral infection and is a target for antiviral therapy. Hepatitis B virus (HBV) is a major pathogen that causes acute and chronic hepatitis; it cannot be cured because of the persistence of its covalently closed circular DNA (cccDNA) in hepatocytes. Here we found that Pholiota squarrosa lectin (PhoSL), a lectin that specifically binds core fucose, bound to HBV particles and inhibited HBV infection of a modified human HepG2 cell line, HepG2-hNTCP-C4, that expresses an HBV receptor, sodium taurocholate cotransporting polypeptide. Knockout of fucosyltransferase 8, the enzyme responsible for core fucosylation and that aids receptor endocytosis, in HepG2-hNTCP-C4 cells reduced HBV infectivity, and PhoSL facilitated that reduction. PhoSL also blocked the activity of epidermal growth factor receptor, which usually enhances HBV infection. HBV particles bound to fluorescently labeled PhoSL internalized into HepG2-hNTCP-C4 cells, suggesting that PhoSL might inhibit HBV infection after internalization. As PhoSL reduced the formation of HBV cccDNA, a marker of chronic HBV infection, we suggest that PhoSL could impair processes from internalization to cccDNA formation. Our finding could lead to the development of new anti-HBV agents.

Abstract Calcium acts as a second messenger to regulate many cellular functions, including cell motility. In Dictyostelium discoideum, the cytosolic calcium level oscillates synchronously, and calcium waves propagate through the cell population during the early stages of development, including aggregation. In the unicellular phase, the calcium response through Piezo channels also functions in mechanosensing. However, calcium dynamics during multicellular morphogenesis are still unclear. Here, live imaging of cytosolic calcium revealed that calcium wave propagation, depending on cAMP relay, disappeared at the onset of multicellular body (slug) formation. Later, other forms of occasional calcium bursts and their propagation were observed in both anterior and posterior regions of migrating slugs. This calcium signaling also occurred in response to mechanical stimuli. Two pathways—calcium release from the endoplasmic reticulum via IP3 receptor and calcium influx from outside the cell—were involved in calcium signals induced by mechanical stimuli. These data suggest that calcium signaling is involved in mechanosensing in both the unicellular and multicellular phases of Dictyostelium development using different molecular mechanisms.

The applications of wear resistant material coatings or claddings by directed energy deposition (DED) are rapidly increasing in industrial engineering fields. DED allows us the use of non-expensive substrate materials and add functional materials such as Co-based superalloys on them. However, depending on the deposition parameters, cracks can easily occur on the workpiece. The determination of the appropriate deposition parameters is crucial because it is still difficult to predict and to control cracking. This paper discusses the effect of substrate preheating by an induction heater on the crack generation for DED of Co-based superalloys. An induction heater was adopted for its high efficiency, low waste heat and ability to quickly induce a high temperature in the workpiece directly. The material used in this study is Stellite® 1, which is widely used for die cut rolls or screw shafts. To investigate the effect of the preheating, experiments were conducted by changing the process parameters: preheating temperature and laser power. Sufficient hardness of 642 HV was obtained without cracking when the preheating temperature was 500 ℃ and the laser power was 1750 W. Although cracking did not occur when the preheating temperature or the laser power was higher, the hardness decreased. There is a trade-off between hardness and crack occurrence. This trade-off is considered to be influenced by thermal stress and the precipitation amount of chromium carbides, which are hard and brittle materials. The amount of chromium carbide precipitation is affected by the cooling rate of the deposited material. In addition, a higher cooling rate may result in greater thermal stress.

GPCR kinases (GRKs) phosphorylate active-form G-protein-coupled receptors (GPCRs). Here, the authors reveal that Gq heterotrimer coupled with the angiotensin II type-1 receptor (AT1R) determines the GRK subtypes recruited to the complex in a microdomain, thus defining subsequent AT1R phosphorylation patterns, β-arrestin conformation and functionality.

Chemotaxis describes directional motility along ambient chemical gradients and has important roles in human physiology and pathology. Typical chemotactic cells, such as neutrophils and Dictyostelium cells, can detect spatial differences in chemical gradients over a background concentration of a 105 scale. Studies of Dictyostelium cells have elucidated the molecular mechanisms of gradient sensing involving G protein coupled receptor (GPCR) signaling. GPCR transduces spatial information through its cognate heterotrimeric G protein as a guanine nucleotide change factor (GEF). More recently, studies have revealed unconventional regulation of heterotrimeric G protein in the gradient sensing. In this review, we explain how multiple mechanisms of GPCR signaling ensure the broad range sensing of chemical gradients in Dictyostelium cells as a model for eukaryotic chemotaxis.

Summary: We describe a protocol for identifying bacteria-derived lipid metabolites produced in the guts using antibiotic-treated mice, liquid chromatography tandem mass spectrometry-based lipidomics, and feature-based molecular spectrum networking (FBMN). Untargeted lipidomics using the MS-DIAL 4 program provides information on known and unknown complex lipid molecules. The FBMN technique clusters similar MS2 spectra, facilitating the identification of bacterial lipids. Targeted analysis was used as a complementary method to cover oxylipins. Here, we provide details for targeted and untargeted analyses.For complete details on the use and execution of this protocol, please refer to Yasuda et al. (2020).

This research has been conducted to develop computer aided manufacturing software, which can generate NC programs for a five-axis directed energy deposition (DED) type additive manufacturing system with following three focuses: 1. How to maintain consistent formation of a melt pool on the surface of the workpiece. 2. How to generate three-dimensional deposition path efficiently when a 3D part model is given. 3. How to control process parameters during additive operation. For focus 1, the work orientation has been controlled such that the surface to be deposited can remain perpendicular to the nozzle which is fixed to gravity direction and minimize distortion of the laser spot and powder distortion on the workpiece surface. For focus 2, a unique path generation method has been proposed based on the reverse play of a subtractive machining path, which removes the entire part to the null. For focus 3, preoperative process parameters control has been introduced to achieve the specified volumetric deposition rates along the additive tool path, which is equated with the material removal rates along the subtractive machining tool path. With methods mentioned above, the dedicated CAM system, CAMAM, has been prototyped and its feasibility has been verified experimentally by applying the NC program generation strategies to deposit several parts.

Gip1 sequesters heterotrimeric G proteins in the cytosolic pool which regulates G protein-coupled receptor signalling for eukaryotic chemotaxis. Here the authors provide the crystal structure of Gip1's G protein-binding region and show that mutations in this region lead to G protein sequestration and ultimately chemotaxis defects.

PIP3 and its phosphatase (PTEN) are enriched mutually exclusively on the anterior and posterior membranes of eukaryotic motile cells. Here authors manipulate PIP3 level and use single-molecule imaging to show that PIP3 suppresses the membrane localization of PTEN.

Large scale live cell screens often lack single-molecule resolution. Here the authors present an artificial intelligence-assisted TIRF microscope with automated cell searching and focusing, and use it for high-throughput single-molecule imaging of EGFR dynamics in response to various stimuli.

Lipid A is a characteristic molecule of Gram-negative bacteria that elicits an immune response in mammalian cells. The presence of structurally diverse lipid A types in the human gut bacteria has been suggested before, and this appears associated with the immune response. However, lipid A structures and their quantitative heterogeneity have not been well characterized. In this study, a method of analysis for lipid A using liquid chromatography–quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) was developed and applied to the analyses of Escherichia coli and Bacteroidetes strains. In general, phosphate compounds adsorb on stainless-steel piping and cause peak tailing, but the use of an ammonia-containing alkaline solvent produced sharp lipid A peaks with high sensitivity. The method was applied to E. coli strains, and revealed the accumulation of lipid A with abnormal acyl side chains in knockout strains as well as known diphosphoryl hexa-acylated lipid A in a wild-type strain. The analysis of nine representative strains of Bacteroidetes showed the presence of monophosphoryl penta-acylated lipid A characterized by a highly heterogeneous main acyl chain length. Comparison of the structures and amounts of lipid A among the strains suggested a relationship between lipid A profiles and the phylogenetic classification of the strains.