Biology subjects

Togashi, Y.

Publications and source records attributed to Togashi, Y..

3 recordsLinked to original sources

bioRxiv2018

HERC2 promotes BLM and WRN to suppress G-quadruplex DNA

BLM and WRN are RecQ DNA helicases essential for genomic stability. Here we demonstrate that HERC2, a HECT E3 ligase, is critical for their functions to suppress G-quadruplex (G4) DNA. HERC2 interacts with BLM, WRN, and replication protein A (RPA) complexes during S-phase of the cell cycle. Depletion of HERC2 dissociates RPA from BLM and WRN complexes and significantly increases G4 formation. Triple depletion revealed that HERC2 has an epistatic relationship with BLM and WRN in their G4- suppressing function. In vitro, HERC2 releases RPA onto single-stranded DNA (ssDNA), rather than anchoring onto RPA-coated ssDNA. CRISPR/Cas9-mediated deletion of the catalytic ubiquitin-binding site of HERC2 causes RPA accumulation in the helicase complexes and increases G4, indicating an essential role for E3 activity in G4 suppression. Both HERC2 depletion and E3 inactivation sensitize cells to the G4-interacting compounds, telomestatin and pyridostatin. Overall, HERC2 is a master regulator of G4 suppression and affects the sensitivity of cells to G4 stabilizers.

molecular 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↗

bioRxiv2017

Bridging the dynamics and organization of chromatin domains by mathematical modeling

The genome is three-dimensionally organized in the cell, and the mammalian genome DNA is partitioned into submegabase-sized chromatin domains. Genome functions are regulated within and across the domains according to their organization, whereas the chromatin itself is highly dynamic. However, the details of such dynamic organization of chromatin domains in living cells remain unclear. To unify chromatin dynamics and organization, we recently demonstrated that structural information of chromatin domains in living human cells can be extracted from analyses of the subdiffusive nucleosome movement using mathematical modeling. Our mathematical analysis suggested that as the chromatin domain becomes smaller and more compact, nucleosome movement becomes increasingly restricted. Here, we show the implication of these results for bridging the gap between chromatin dynamics and organization, and provide physical insight into chromatin domains as efficient units to conduct genome functions in the thermal noisy environment of the cell.\n\nExtra View toShinkai S, Nozaki T, Maeshima K, Togashi Y. Dynamic Nucleosome Movement Provides Structural Information of Topological Chromatin Domains in Living Human Cells. PLoS Comput Biol. 2016;12(10):e1005136. doi: 10.1371/journal.pcbi.1005136. PubMed PMID: 27764097; PubMed Central PMCID: PMCPMC5072619.\n\nFundingThis work was supported by Platform Project for Supporting in Drug Discovery and Life Science Research (Platform for Dynamic Approaches to Living System) from MEXT and AMED; KAKENHI under Grant JP16H01408, JP23115007, JP23115005, JP16H04746; CREST grant (JPMJCR15G2) from JST; and Research Fellowship for Young Scientists under Grant JP13J04821, JP16J07205.\n\nDisclosureNo potential conflicts of interest were disclosed.\n\nAcknowledgementsWe would like to thank Dr. Takashi Toda for comments regarding the manuscript.

biophysics↗