Biology subjects

Hiroshima, M.

Publications and source records attributed to Hiroshima, M..

6 recordsLinked to original sources

bioRxiv2024

Bilateral regulation of EGFR activity and local PI dynamics observed with superresolution microscopy

Anionic lipid molecules, including phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), are implicated in the regulation of epidermal growth factor receptor (EGFR). However, the role of the spatiotemporal dynamics of PI(4,5)P2 in the regulation of EGFR activity in living cells is not fully understood, as it is difficult to visualize the local lipid domains around EGFR. In this study, both EGFR and PI(4,5)P2 nanodomains in the plasma membrane were visualized using super-resolution single-molecule microscopy. The EGFR and PI(4,5)P2 nanodomains aggregated before stimulation with epidermal growth factor (EGF) through transient visits of EGFR to the PI(4,5)P2 nanodomains. The degree of coaggregation decreased after EGF stimulation and depended on phospholipase C{gamma}, the EGFR effector hydrolyzing PI(4,5)P2. Artificial reduction in the PI(4,5)P2 content of the plasma membrane reduced both the dimerization and autophosphorylation of EGFR after stimulation with EGF. Inhibition of PI(4,5)P2 hydrolysis after EGF stimulation decreased phosphorylation of EGFR-Thr654. Thus, EGFR kinase activity and the density of PI(4,5)P2 around EGFR molecules were found to be mutually regulated.

cell biology↗

bioRxiv2023

Single-molecule tracking-based drug screening

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 screened for epidermal growth factor receptor (EGFR) from 1,134 of FDA approved drugs. The 18 hit compounds included all EGFR-targeted tyrosine kinase inhibitors (TKIs) in the library that suppressed 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 did not inhibit EGF-induced EGFR phosphorylation. These non-TKI compounds affected the mobility and/or clustering of EGFR without EGF and induced EGFR internalization, to impede EGFR-dependent cell growth. Thus, single-molecule tracking provides a new modality for discovering novel therapeutics on various receptor functions with previously untargeted mechanisms.

pharmacology and toxicology↗

bioRxiv2021

Membrane cholesterol interferes with tyrosine phosphorylation but facilitates the clustering and signal transduction of EGFR

Epidermal growth factor receptor (EGFR) activates major cell signaling pathways that regulate various cell responses. Its dimerization and clustering coupled with its lateral mobility are critical for EGFR function, but the contribution of the plasma membrane environment to EGFR function is unknown. Here we show, using single-molecule analysis, that EGFR mobility and clustering are altered by the depletion of cholesterol or sphingomyelin, major lipids of membrane subdomains, causing significant changes in EGFR signaling. When cholesterol was depleted, the subdomain boundary in EGFR diffusion disappeared, the fraction of EGFR pre-dimers was increased, and the ligand-induced phosphorylation of EGFR was enhanced. In addition, the depletion of either lipid prevented the formation of immobile clusters after EGF association and decreased the phosphorylation of downstream proteins. Our results revealed that cholesterol plays dichotomous roles in the signaling pathway of EGFR and that clustering in the membrane subdomains is critical for EGFR signal transduction.

biophysics↗

bioRxiv2020

Origin of diverse phosphorylation patterns in the ERBB system

Intercellular signals induce various cellular responses, including growth, proliferation, and differentiation, via the dynamic processes of signal transduction pathways. For cell-fate decisions, ligand-binding induces the phosphorylation of ERBB receptors, which in turn activate downstream molecules. The ERBB family includes four subtypes, which diverged through two gene duplications from a common ancestor, of different kinetic properties. Differences in the expression patterns of the subtypes have been reported between different organs in the human body. However, how these different kinetic and expression properties influence the diverse phosphorylation dynamics of ERBB proteins is not well understood. Here we study the phosphorylation dynamics by experimental and mathematical analyses. The experimental measurements clarified that the phosphorylation dynamics heavily depend on the ERBB expression profiles. We developed a mathematical model consisting of the four subtypes as monomers, homodimers and heterodimers and estimated the rate constants governing the phosphorylation dynamics from the experimental data. To understand the origin of the diversity of the phosphorylation responses of the ERBB system, we analyzed the expression profiles and reaction rates of the ERBB subtypes. The difference in phosphorylation rates between ERBB subtypes showed a much larger contribution to the diversity of the phosphorylation responses than did the dimerization rates. This result implies that divergent evolution in phosphorylation reactions rather than in dimerization reactions after whole genome duplications was essential for increasing the diversity of the phosphorylation responses. Statement of SignificanceIt is known that the expression patterns of a protein family are different between different organs in the human body. This difference is considered essential for the functional diversity between organs. However, the dynamical processes that translate expression patterns into function are not well understood. Here we study the origin of the diversity in ERBB phosphorylation patterns by combining experimental and mathematical methods. We confirmed experimentally that phosphorylation patterns depend on the ERBB expression profiles. Our mathematical model found that differences in phosphorylation rates made the largest contribution to the diversity of phosphorylation patterns between ERBB subtypes.

systems biology↗

bioRxiv2019

Cell-to-cell diversification in ERBB-RAS-MAPK signal transduction that produces cell-type specific growth factor responses

Growth factors regulate cell fates, including their proliferation, differentiation, survival, and death, according to the cell type. Even when the response to a specific growth factor is deterministic for collective cell behavior, significant levels of fluctuation are often observed between single cells. Statistical analyses of single-cell responses provide insights into the mechanism of cell fate decisions but very little is known about the distributions of the internal states of cells responding to growth factors. Using multi-color immunofluorescent staining, we have here detected the phosphorylation of seven elements in the early response of the ERBB-RAS-MAPK system to two growth factors. Among these seven elements, five were analyzed simultaneously in distinct combinations in the same single cells. Although principle component analysis suggested cell-type and input specific phosphorylation patterns, cell-to-cell fluctuation was large. Mutual information analysis suggested that cells use multitrack (bush-like) signal transduction pathways under conditions in which clear cell fate changes have been reported. The clustering of single-cell response patterns indicated that the fate change in a cell population correlates with the large entropy of the response, suggesting a bet-hedging strategy is used in decision making. A comparison of true and randomized datasets further indicated that this large variation is not produced by simple reaction noise, but is defined by the properties of the signal-processing network. Author SummaryHow extracellular signals, such as growth factors (GFs), induce fate changes in biological cells is still not fully understood. Some GFs induce cell proliferation and others induce differentiation by stimulating a common reaction network. Although the response to each GF is reproducible for a cell population, not all single cells respond similarly. The question that arises is whether a certain GF conducts all the responding cells in the same direction during a fate change, or if it initially stimulates a variety of behaviors among single cells, from which the cells that move in the appropriate direction are later selected. Our current statistical analysis of single-cell responses suggests that the latter process, which is called a bet-hedging mechanism is plausible. The complex pathways of signal transmission seem to be responsible for this bet-hedging.

systems biology↗

bioRxiv2017

Single-molecule diffusion-based estimation of GPCR activity

G protein-coupled receptors (GPCRs) are major drug targets and have high potential for drug discovery. The development of a method for measuring the activities of GPCRs is essential for pharmacology and drug screening. However, it is difficult to measure the effects of a drug by monitoring the receptor on the cell surface, and changes in the concentrations of downstream signaling molecules, which depend on signaling pathway selectivity of the receptor, are used as an index of the receptor activity. Here, we show that single-molecule imaging analysis provides an alternative method for assessing ligand effects on GPCR. We monitored the dynamics of the diffusion of metabotropic glutamate receptor 3 (mGluR3), a class C GPCR, under various ligand conditions by using total internal reflection fluorescence microscopy (TIRFM). The single-molecule tracking analysis demonstrates that changes in the average diffusion coefficient of mGluR3 quantitatively reflect the ligand-dependent activity. Then, we reveal that the diffusion of receptor molecules is altered by the common physiological events associated with GPCRs, including G protein binding or accumulation in clathrin-coated pits, by inhibition experiments and dual-color single-molecule imaging analysis. We also confirm the generality of agonist-induced diffusion change in class A and B GPCRs, demonstrating that the diffusion coefficient is a good index for estimating the ligand effects on many GPCRs regardless of the phylogenetic groups, chemical properties of the ligands, and G protein-coupling selectivity.\n\nOne Sentence Summary: Single-molecule imaging for evaluating ligand effects on GPCRs by monitoring the diffusion dynamics on the cell surface.

biophysics↗