Biology subjects

Yanagawa, M.

Publications and source records attributed to Yanagawa, M..

9 recordsLinked to original sources

bioRxiv2026

Membrane PI(4,5)P2 and ErbB2 abundance regulate ErbB receptor signaling through receptor oligomerization and activation

Because ErbB receptors play distinct roles in regulating diverse cellular functions, the mechanisms governing ErbB receptor activation are likely to be more diverse than previously recognized. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] positively regulates ErbB1 kinase activity, but the role of PI(4,5)P2 in regulating other ErbB family members in living cells remains poorly understood. We show that disruption of PI(4,5)P2 binding enhances ErbB4 oligomerization and kinase activity while reducing both processes in ErbB1. Analysis of chimeric receptors identified the juxtamembrane (JM) regions of ErbB1 and ErbB4 as key determinants of their distinct responses to PI(4,5)P2 during receptor oligomerization and kinase activation. Furthermore, the JM-kinase module of ErbB1 is more active in the presence of PI(4,5)P2, whereas that of ErbB4 is activated by the disruption of PI(4,5)P2 binding. In contrast, the JM-kinase module of ErbB2 exhibits weak dependence on PI(4,5)P2. ErbB2 preferentially promotes ErbB4 oligomerization over ErbB1 oligomerization, thereby enhancing ErbB4 activation. Collectively, these findings identify plasma membrane PI(4,5)P2 availability and ErbB2 abundance as two factors that jointly govern ErbB receptor oligomerization and activation.

cell biology↗

bioRxiv2026

Comprehensive dissection of GPCR signaling using a NanoBiT-based platform

G-protein-coupled receptors (GPCRs) signal through multiple heterotrimeric G proteins, {beta}-arrestins, GPCR kinases (GRKs), and downstream effectors, whose combinatorial interactions shape cellular responses. These events are typically measured with separate assay formats that each capture only part of the network, making comparison across signaling layers difficult. Here, we consolidate a broad set of previously reported GPCR signaling interactions and assay concepts, together with newly designed sensors, into a single NanoBiT split-luciferase framework, allowing multiple layers of signal transduction to be examined side by side in living cells. We show that rational sensor engineering, in particular the positioning of NanoBiT fragments and targeted modification of the tagged proteins, is essential for detecting transient protein-protein interactions. The framework implements assays for G-protein dissociation, {beta}-arrestin recruitment, conformational activation and trafficking, and GRK recruitment, and extends to G-protein-effector and inter-effector interactions across diverse G, G{beta}, G{gamma}, {beta}-arrestin, GRK, adenylyl cyclase, PLC{beta}, and RhoGEF subtypes. It also enables real-time monitoring of the difficult-to-access G12/13-RhoGEF-RhoA pathway. Together, these assays provide a unified NanoBiT readout for systematic, side-by-side dissection of GPCR signaling.

pharmacology and toxicology↗

bioRxiv2025

Single-molecule behavior and cell-growth regulation in human RTKs

Receptor tyrosine kinases (RTKs) are a major family of cell surface receptor proteins responsible for various cellular functions in animal cells, including fate decisions, metabolism, polarization and migration. Lateral mobility, dimerization, clustering, and oligomerization are crucial behaviors in the activation process of RTKs on the cell surface. However, relationships between these molecular behaviors and molecular function remain to be elucidated, except for a few RTK members. Here, using an automated live-cell single-molecule imaging and analysis system, we studied the behavior of 52 of the 58 human RTK species on living cells over time during stimulation with ligands. We extracted 72 single-molecule parameters for each RTK species to examine their relationship to function, structure, and evolution. We noticed that RTKs ability to inhibit or support cell growth, as observed in a large-scale loss-of-function experiment in the public domain, significantly relates to their behavior. Growth-inhibitory signaling was coupled with the immediate formation of immobile clusters, followed by the enlargement of immobile and slow-mobile domains. In contrast, growth-supportive signaling coupled with higher lateral diffusivity and delayed clustering of immobile molecules. The relationship between structure and function suggests that functional differences are related to partitioning into membrane rafts and changes in mobility associated with phosphatidylinositol turnover. In multiple linear regression models, molecular behavior explained half or more of the molecular function related to cell growth. This level of explainability is comparable to that of evolutionary grouping.

biophysics↗

bioRxiv2024

Four-color single-molecule imaging system for tracking GPCR dynamics with fluorescent HiBiT peptide

Single-molecule imaging provides information on diffusion dynamics, oligomerization, and protein-protein interactions in living cells. To simultaneously monitor different types of proteins at the single-molecule level, orthogonal fluorescent labeling methods with different photostable dyes are required. G-protein-coupled receptors (GPCRs), a major class of drug targets, are prototypical membrane receptors that have been studied using single-molecule imaging techniques. Here we developed a method for labeling cell-surface GPCRs inspired by the HiBiT system, which utilizes the high affinity complementation between LgBiT and HiBiT fragments of the NanoLuc luciferase. We synthesized four fluorescence-labeled HiBiT peptides (F-FiBiTs) with a different color dye (Setau-488, TMR, SaraFluor 650 and SaraFluor 720). We constructed a multicolor total internal reflection fluorescence microscopy system that allows us to track four color dyes simultaneously. As a proof-of-concept experiment, we labeled an N-terminally LgBiT-fused GPCR (Lg-GPCR) with a mixture of the four F-FiBiTs and successfully tracked each dye within a cell at the single molecule level. The F-FiBiT-labeled Lg-GPCRs showed agonist-dependent changes in the diffusion dynamics and accumulation into the clathrin-coated pits as observed with a conventional method using a C-terminally HaloTag-fused GPCR. Taking advantage of luciferase complementation by the F-FiBiT and Lg-GPCRs, the F-FiBiT was also applicable to bioluminescence plate-reader-based assays. By combining existing labeling methods such as HaloTag, SNAP-tag, and fluorescent proteins, the F-FiBiT method will be useful for multicolor single-molecule imaging and will enhance our understanding of GPCR signaling at the single-molecule level.

biophysics↗

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↗

bioRxiv2021

Threonine phosphorylation regulates the molecular assembly and signaling of EGFR in cooperation with membrane lipids

The cytoplasmic domain of the receptor tyrosine kinases (RTKs) plays roles as a phosphorylation enzyme and a protein scaffold but the allocation of these two functions is not fully understood. We here analyzed assembly of the transmembrane (TM)-juxtamembrane (JM) region of EGFR, one of the best studied species of RTKs, by combining single-pair FRET imaging and a nanodisc technique. The JM domain of EGFR contains a threonine residue (Thr654) that is phosphorylated after ligand association. We observed that the TM-JM peptides of EGFR form anionic lipid-induced dimers and cholesterol-induced oligomers. The two forms involve distinct molecular interactions, with a bias towards oligomer formation upon threonine phosphorylation. We further analyzed the functions and oligomerization of whole EGFR molecules, with or without a substitution of Thr654 to alanine, in living cells. The results suggested an autoregulatory mechanism in which Thr654 phosphorylation causes a switch of the major function of EGFR from kinase activation dimers to scaffolding oligomers.

biophysics↗

bioRxiv2020

Total workflows of the single-molecule imaging analysis in living cells: a tutorial guidance to the measurement of the drug effects on a GPCR

Single-molecule imaging (SMI) is a powerful method to measure the dynamics of membrane proteins on the cell membrane. The single-molecule tracking (SMT) analysis provides information about the diffusion dynamics, the oligomer size distribution, and the particle density change. The affinity and on/off-rate of a protein--protein interaction can be estimated from the dual-color SMI analysis. However, it is difficult for trainees to determine quantitative information from the SMI movies. The present protocol guides the detailed workflows to measure the drug-activated dynamics of a G protein-coupled receptor (GPCR) and metabotropic glutamate receptor 3 (mGluR3), by using the total internal reflection fluorescence microscopy (TIRFM). This tutorial guidance comprises an open-source software named smDynamicsAnalyzer, with which one can easily analyze the SMT dataset by just following the workflows after building a designated folder structure (https://github.com/masataka-yanagawa/IgorPro8-smDynamicsAnalyzer).

biophysics↗

bioRxiv2017

Lipid-protein interplay in dimerization of the juxtamembrane domains of epidermal growth factor receptor

Transmembrane (TM) helix and juxtamembrane (JM) domains (TM-JM) bridge the extracellular and intracellular domains of single-pass membrane proteins, including epidermal growth factor receptor (EGFR). TM-JM dimerization plays a crucial role in regulation of EGFR kinase activity at the cytoplasmic side. Although the interaction of JM with membrane lipids is thought to be important to turn on EGF signaling and phosphorylation of Thr654 on JM leads to desensitization, the underlying kinetic mechanisms remain unclear. Especially, how Thr654 phosphorylation regulates EGFR activity is largely unknown. Here, combining single-pair FRET imaging and nanodisc techniques, we showed that phosphatidylinositol 4,5-bis phosphate (PIP2) facilitated JM dimerization effectively. We also found that Thr654 phosphorylation dissociated JM dimers in the membranes containing acidic lipids, suggesting that Thr654 phosphorylation electrostatically prevented the interaction with basic residues in JM and acidic lipids. Based on the single-molecule experiment, we clarified the kinetic pathways of monomer (inactive state) - dimer (active state) transition of JM domains and alteration in the pathways depending on the membrane lipid species and Thr654 phosphorylation.

biophysics↗

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↗