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Phosphatidylinositol 3,4,5-trisphosphate (PIP3) and PIP3 phosphatase (PTEN) are enriched mutually exclusively on the anterior and posterior membranes of eukaryotic motile cells. However, the mechanism that causes this spatial separation between the two molecules is unknown. Here we develop a method to manipulate PIP3 levels in living cells and used it to show PIP3 suppresses the membrane localization of PTEN. Single-molecule measurements of membrane-association and -dissociation kinetics and of lateral diffusion reveal that PIP3 suppresses the PTEN binding site required for stable PTEN membrane binding. Mutual inhibition between PIP3 and PTEN provides a mechanistic basis for bistability that creates a PIP3-enriched/PTEN-excluded state and a PTEN-enriched/PIP3-excluded state underlying the strict spatial separation between PIP3 and PTEN. The PTEN binding site also mediates the suppression of PTEN membrane localization in chemotactic signaling. These results illustrate that the PIP3-PTEN bistable system underlies a cell’s decision-making for directional movement irrespective of the environment. 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.

Signaling-biased ligands acting on G-protein-coupled receptors (GPCRs) differentially activate heterotrimeric G proteins and β-arrestins. Although a wealth of structural knowledge about signaling bias at the GPCR level exists (preferential engagement of a specific transducer), little is known about the bias at the transducer level (different functions mediated by a single transducer), partly due to a poor understanding of GPCR kinase (GRK)-mediated GPCR phosphorylation. Here, we reveal a unique role of the Gq heterotrimer as a determinant for GRK-subtype selectivity that regulates subsequent β-arrestin conformation and function. Using the angiotensin II (Ang II) type-1 receptor (AT1R), we show that β-arrestin recruitment depends on both GRK2/3 and GRK5/6 upon binding of Ang II, but solely on GRK5/6 upon binding of the β-arrestin-biased ligand TRV027. With pharmacological inhibition or genetic loss of Gq, GRK-subtype selectivity and β-arrestin functionality by Ang II is shifted to those of TRV027. Single-molecule imaging identifies relocation of AT1R and GRK5, but not GRK2, to an immobile phase under the Gq-inactive, AT1R-stimulated conditions. These findings uncover a previously unappreciated Gq-regulated mechanism that encodes GRK-subtype selectivity and imparts distinct phosphorylation-barcodes directing downstream β-arrestin functions. 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.

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. EGFR is a primary target molecule in drug exploration for cancer therapeutics. Here, the authors show a demonstration of single-molecule tracking-based drug screening for EGFR, proving the selectivity for tyrosine kinase inhibitors and potential to find drugs with previously untargeted mechanisms.

Excitable systems of eukaryotic chemotaxis can generate asymmetric signals of Ras-GTP-enriched domains spontaneously to drive random cell migration without guidance cues. However, the molecules responsible for the spontaneous signal generation remain elusive. Here, we characterized RasGEFs encoded in Dictyostelium discoideum by live-cell imaging of the spatiotemporal dynamics of Ras-GTP and hierarchical clustering, finding that RasGEFX is primarily required for the spontaneous generation of Ras-GTP-enriched domains and is essential for random migration in combination with RasGEFB/M/U in starved cells, and they are dispensable for chemotaxis to chemoattractant cAMP. RasGEFX and RasGEFB that co-localize with Ras-GTP regulate the temporal periods and spatial sizes of the oscillatory Ras-GTP waves propagating along the membrane, respectively, and thus control the protrusions of motile cells differently, while RasGEFU and RasGEFM regulate adhesion and migration speed, respectively. Remarkably, RasGEFX is also important for Ras/PIP3-driven macropinocytosis in proliferating cells, but RasGEFB/M/U are not. These findings illustrate a specific and coordinated control of the cytoskeletal dynamics by multiple RasGEFs for spontaneous motility and macropinocytosis. Live-cell-imaging-based screens identify RasGEFX as an essential component of the pathway that triggers spontaneous Ras excitation for basal cell migration in combination with RasGEFB/M/U and for macropinocytic cup formation and fluid uptake.

An automated single-molecule imaging system developed for live-cell analyses based on artificial intelligence-assisted microscopy is presented. All significant procedures, i.e., searching for cells suitable for observation, detecting in-focus positions, and performing image acquisition and single-molecule tracking, are fully automated, and numerous highly accurate, efficient, and reproducible single-molecule imaging experiments in living cells can be performed. Here, the apparatus is applied for single-molecule imaging and analysis of epidermal growth factor receptors (EGFRs) in 1600 cells in a 96-well plate within 1 day. Changes in the lateral mobility of EGFRs on the plasma membrane in response to various ligands and drug concentrations are clearly detected in individual cells, and several dynamic and pharmacological parameters are determined, including the diffusion coefficient, oligomer size, and half-maximal effective concentration (EC 50 ). Automated single-molecule imaging for systematic cell signaling analyses is feasible and can be applied to single-molecule screening, thus extensively contributing to biological and pharmacological research. 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.

There have been many empirical studies regarding mergers in Japan’s paper industry from the standpoint of productivity and cost effectiveness. However, such studies only compared effectiveness based on data regarding existing companies. This article creates simulations of data on hypothetical mergers. A relative comparison will be made between such simulated data and the actual merger data regarding cost effectiveness using a data envelopment analysis (DEA). The results of the DEA efficiency analysis using such data show that, in mergers between large companies and midsized companies, in the long run, their efficiency tends to improve. A series of mergers in Japan’s paper industry, in general, can be positively evaluated since these mergers did not significantly reduce the cost-effective value of the surviving companies.

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.

Single-molecule imaging analysis of chemotactic response in eukaryotic cells has revealed a stochastic nature in the input signals and the signal transduction processes. This leads to a fundamental question about the signaling processes: how does the signaling system operate under stochastic fluctuations or noise? Here, we report a stochastic model of chemotactic signaling in which noise and signal propagation along the transmembrane signaling pathway by chemoattractant receptors can be analyzed quantitatively. The results obtained from this analysis reveal that the second-messenger-production reactions by the receptors generate noisy signals that contain intrinsic noise inherently generated at this reaction and extrinsic noise propagated from the ligand-receptor binding. Such intrinsic and extrinsic noise limits the directional sensing ability of chemotactic cells, which may explain the dependence of chemotactic accuracy on chemical gradients that has been observed experimentally. Our analysis also reveals regulatory mechanisms for signal improvement in the stochastically operating signaling system by analyzing how the SNR of chemotactic signals can be improved on or deteriorated by the stochastic properties of receptors and second-messenger molecules. Theoretical consideration of noisy signal transduction by chemotactic signaling systems can further be applied to signaling systems in general.

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. Different membrane proteins dynamically polarize to organize signal transduction, but the underlying mechanism is unclear. Here, the authors show that a differential diffusion mediated partitioning process is sufficient to drive such spatiotemporal patterning of membrane-associated signaling proteins.

Chemotactic eukaryote cells can sense chemical gradients over a wide range of concentrations via heterotrimeric G-protein signaling; however, the underlying wide-range sensing mechanisms are only partially understood. Here we report that a novel regulator of G proteins, G protein-interacting protein 1 (Gip1), is essential for extending the chemotactic range of Dictyostelium cells. Genetic disruption of Gip1 caused severe defects in gradient sensing and directed cell migration at high but not low concentrations of chemoattractant. Also, Gip1 was found to bind and sequester G proteins in cytosolic pools. Receptor activation induced G-protein translocation to the plasma membrane from the cytosol in a Gip1-dependent manner, causing a biased redistribution of G protein on the membrane along a chemoattractant gradient. These findings suggest that Gip1 regulates G-protein shuttling between the cytosol and the membrane to ensure the availability and biased redistribution of G protein on the membrane for receptor-mediated chemotactic signaling. This mechanism offers an explanation for the wide-range sensing seen in eukaryotic chemotaxis.