Biology subjects

Yoshii, T.

Publications and source records attributed to Yoshii, T..

4 recordsLinked to original sources

bioRxiv2021

A chemogenetic platform for controlling plasma membrane signaling and synthetic signal oscillation

Chemogenetic methods that enable the rapid translocation of specific signaling proteins in living cells using small molecules are powerful tools for manipulating and interrogating intracellular signaling networks. However, existing techniques rely on chemically induced dimerization of two protein components and have certain limitations, such as a lack of reversibility, bioorthogonality, and usability. Here, by expanding our self-localizing ligand-induced protein translocation (SLIPT) approach, we have developed a versatile chemogenetic system for plasma membrane (PM)-targeted protein translocation. In this system, a novel engineered Escherichia coli dihydrofolate reductase in which a hexalysine (K6) sequence is inserted in a loop region (iK6DHFR) is used as a universal protein tag for PM-targeted SLIPT. Proteins of interest that are fused to the iK6DHFR tag can be specifically recruited from the cytoplasm to the PM within minutes by addition of a myristoyl-O_SCPLOWDC_SCPLOW-Cys-tethered trimethoprim ligand (mDcTMP). We demonstrated the broad applicability and robustness of this engineered protein-synthetic ligand pair as a tool for the conditional activation of various types of signaling molecules, including protein and lipid kinases, small GTPases, heterotrimeric G proteins, and second messengers. In combination with a competitor ligand and a culture-medium flow chamber, we further demonstrated the application of the system for chemically manipulating protein localization in a reversible and repeatable manner to generate synthetic signal oscillations in living cells. The present bioorthogonal iK6DHFR/mDcTMP-based SLIPT system affords rapid, reversible, and repeatable control of the PM recruitment of target proteins, offering a versatile and easy-to-use chemogenetic platform for chemical and synthetic biology applications.

synthetic biology↗

bioRxiv2020

Synthetic protein condensates that recruit and release protein activity in living cells

Compartmentation of proteins into biomolecular condensates or membraneless organelles formed by phase separation is an emerging principle for the regulation of cellular processes. Creating synthetic condensates that accommodate specific intracellular proteins on demand would have various applications in chemical biology, cell engineering and synthetic biology. Here, we report the construction of synthetic protein condensates capable of recruiting and/or releasing proteins of interest in living mammalian cells in response to a small molecule or light. We first present chemogenetic protein-recruiting and -releasing condensates, which rapidly inhibited and activated signaling proteins, respectively. An optogenetic condensate system was successfully constructed that enables reversible release and sequestration of protein activity using light. This proof-of-principle work provides a new platform for chemogenetic and optogenetic control of protein activity in mammalian cells and represents a step towards tailor-made engineering of synthetic protein condensates with various functionalities.

synthetic biology↗

bioRxiv2020

Amplitude of circadian rhythms becomes weaker in the north, but there is no cline in the period of rhythm in a beetle

Many species show rhythmicity in activity, from the timing of flowering in plants to that of foraging behaviour in animals. The free-running periods and amplitude (sometimes called strength or power) of circadian rhythms are often used as indicators of biological clocks. Many reports have shown that these traits highly geographically variable, and interestingly, they often show latitudinal or altitudinal clines. In many cases, the higher the latitude is, the longer the free-running circadian period (i.e., period of rhythm) in insects and plants. However, reports of positive correlations between latitude or longitude and circadian rhythm traits, including free-running periods, the power of the rhythm and locomotor activity, are limited to certain taxonomic groups. Therefore, we collected a cosmopolitan stored-product pest species, the red flour beetle Tribolium castaneum, in various parts of Japan and examined its rhythm traits, including the power of the rhythm and period of the rhythm, which were calculated from locomotor activity. The analysis revealed that power was significantly lower for beetles collected in northern areas compared with southern areas in Japan. However, it is worth noting that the period of circadian rhythm did not show any clines; specifically, it did not vary among the sampling sites, despite the very large sample size (n = 1585). We discuss why these cline trends were observed in T. castaneum.

animal behavior and cognition↗

bioRxiv2020

Improved synthetic lipidation-based protein translocation system for SNAP-tag fusion proteins

The ability to artificially attach lipids to specific intracellular protein targets would be a valuable approach for controlling protein localization and function in cells. We recently devised a chemogenetic method in which a SNAP-tag fusion protein can be translocated from the cytoplasm to the plasma membrane by post-translationally and covalently conjugating a synthetic lipopeptide in cells. However, the first-generation system lacked general applicability. Herein, we present an improved synthetic lipidation system that enables efficient plasma membrane translocation of SNAP-tag fusion proteins in cells. This second-generation system is now applicable to the control of various cell-signaling molecules, offering a new and useful research tool in chemical biology and synthetic biology.

biochemistry↗