Biology subjects

Terai, T.

Publications and source records attributed to Terai, T..

7 recordsLinked to original sources

bioRxiv2026

Development of a photostable pH biosensor based on mStayGold

pH-sensitive fluorescent proteins (FPs) play a crucial role in investigating pH-related cellular processes, such as endocytosis and exocytosis. Existing pH-sensitive FPs generated from Aequorea victoria green fluorescent protein (GFP), such as superecliptic pHluorin (SEP) and Lime, have been widely employed to study these processes, but suffer from low photostability. Here, we report the development and characteristics of serapH, a genetically encodable pH biosensor with improved photostability compared to GFP analogues, which we generated using mStayGold as a scaffold. To aid in the development of serapH, we developed a method for screening pH-sensitive FP variants by directly evaluating both brightness and pH sensitivity in bacterial colonies on agar. This significantly increased the number of colonies that could be screened per round and reduced the time needed per round. The photostability of serapH should improve spatiotemporal resolution by increasing tolerance to higher excitation intensities and longer imaging durations, thereby expanding the range of applications of pH-sensitive FPs.

biochemistry↗

bioRxiv2026

A StayGold-based calcium ion indicator

Genetically encoded calcium ion (Ca2+) indicators (GECIs) enable visualization of Ca2+ dynamics in living systems but often suffer from limited photostability during prolonged imaging. The recent discovery of StayGold, a green fluorescent protein (FP) with exceptional brightness and photostability, opened the possibility of addressing this longstanding challenge. Here, we sought to establish whether a monomeric variant of StayGold (mStayGold) could be converted into a single FP-based GECI. Through extensive protein engineering, we generated a functional mStayGold-based GECI, HiCaRI (Highly intensiometric Ca2+ Responsive Indicator) by fusing Calmodulin (CaM) and the ckkap binding peptide from K-GECO1 into mStayGold(J). HiCaRI exhibits a large Ca2+-dependent inverse fluorescence response ({Delta}F/Fmin = -15) while retaining high brightness and improved photostability relative to previously reported GFP-based GECIs. Although the current variant represents a first-generation prototype with shortcomings in terms of Ca2+ affinity and photostability (relative to StayGold and mStayGold(J)), this work demonstrates the feasibility of constructing single FP-based GECIs from a highly photostable fluorescent protein.

bioengineering↗

bioRxiv2025

A sensitive orange fluorescent calcium ion indicator for imaging neural activity

Genetically encoded calcium indicators (GECIs) are vital tools for fluorescence-based visualization of neuronal activity with high spatial and temporal resolution. However, current highest-performance GECIs are predominantly green or red fluorescent, limiting multiplexing options and efficient excitation with fixed-wavelength femtosecond lasers operating at 1030 nm. Here, we introduce OCaMP (also known as O-GECO2), an orange fluorescent GECI engineered from O-GECO1 through targeted substitutions to improve calcium affinity while retaining the favorable photophysical properties of mOrange2. OCaMP exhibits improved two-photon cross-section, responsiveness, photostability, and calcium affinity relative to O-GECO1. In cultured neurons, zebrafish, and mouse cortex, OCaMP outperforms the red GECIs jRCaMP1a and jRGECO1a in sensitivity, kinetics, and signal-to-noise ratio. These properties establish OCaMP as a robust tool for high-fidelity neural imaging optimized for 1030 nm excitation and a compromise-free option within the spectral gap between existing green and red GECIs.

neuroscience↗

bioRxiv2025

High-performance genetically-encoded green and red fluorescent biosensors for pyruvate

Pyruvate is the end-product of glycolysis and a central metabolite involved in many biochemical pathways. However, a lack of high-performance (i.e., {Delta}F/F0 > 10) single fluorescent protein (FP)-based biosensors has hindered efforts to investigate the physiological role of pyruvate. Here, we present the GreenPy1 and ApplePy1 series, which are green FP (GFP)-based and red FP (RFP)-based pyruvate biosensors, respectively. Both series exhibit large fluorescence intensity change ({Delta}F/F0 [~] 20 to >40) and a range of affinities (10s of M to several mM). We demonstrate the utility of these pyruvate biosensors for multicolor imaging of metabolite concentration changes in mammalian cells.

molecular biology↗

bioRxiv2025

GPR182 is a lipoprotein receptor for dietary fat absorption

The lymphatic system plays a central role in lipid absorption, which transports chylomicrons from the small intestine to the circulation. However, the molecular mechanism by which chylomicrons get into the intestinal lymphatics is unknown. Here we demonstrated that GPR182, a receptor in lymphatic endothelial cells (LECs), mediates dietary fat absorption. GPR182 knockout mice are resistant to dietary-induced obesity. GPR182 ablation in mice leads to poor lipid absorption and thereby a delay in growth during development. GPR182 binds and endocytoses lipoproteins broadly. Mechanistically, loss of GPR182 prevents chylomicrons from entering the lacteal lumen of the small intestine. GPR182 blockage with a monoclonal antibody (mAb) protects mice from dietary induced obesity. Together, our study identifies GPR182 as a lipoprotein receptor that mediates dietary fat absorption.

physiology↗

bioRxiv2024

PinkyCaMP a mScarlet-based calcium sensor with exceptional brightness, photostability, and multiplexing capabilities

Genetically encoded calcium (Ca2+) indicators (GECIs) are widely used for imaging neuronal activity, yet current limitations of existing red fluorescent GECIs have constrained their applicability. The inherently dim fluorescence and low signal-to-noise ratio of red-shifted GECIs have posed significant challenges. More critically, several red-fluorescent GECIs exhibit photoswitching when exposed to blue light, thereby limiting their applicability in all-optical experimental approaches. Here, we present the development of PinkyCaMP, the first mScarlet-based Ca2+ sensor that outperforms current red fluorescent sensors in brightness, photostability, signal-to-noise ratio, and compatibility with optogenetics and neurotransmitter imaging. PinkyCaMP is well-tolerated by neurons, showing no toxicity or aggregation, both in vitro and in vivo. All imaging approaches, including single-photon excitation methods such as fiber photometry, widefield imaging, miniscope imaging, as well as two-photon imaging in awake mice, are fully compatible with PinkyCaMP.

neuroscience↗

bioRxiv2024

Synthesis and application of a photocaged L-lactate

O_SCPLOWLC_SCPLOW-Lactate, once considered a metabolic waste product of glycolysis, is now recognized as a vitally important metabolite and signaling molecule in multiple biological pathways. However, exploring O_SCPLOWLC_SCPLOW-lactates emerging intra- and extra-cellular roles is hindered by a lack of tools to locally perturb O_SCPLOWLC_SCPLOW-lactate concentration intracellularly and extracellularly. Photocaged compounds are a powerful way to introduce bioactive molecules with spatial and temporal precision using illumination. Here, we report the development of a photocaged derivative of O_SCPLOWLC_SCPLOW-lactate, 4-methoxy-7-nitroindolinyl O_SCPLOWLC_SCPLOW-lactate (MNI-O_SCPLOWLC_SCPLOW-lac), that releases O_SCPLOWLC_SCPLOW-lactate upon UV illumination. We validated MNI-O_SCPLOWLC_SCPLOW-lac in cell culture by demonstrating that the photorelease of O_SCPLOWLC_SCPLOW-lactate elicits a response from genetically encoded extra- and intracellular O_SCPLOWLC_SCPLOW-lactate biosensors. These results indicate that MNI-O_SCPLOWLC_SCPLOW-lac may be useful for perturbing the concentration of endogenous O_SCPLOWLC_SCPLOW-lactate in order to investigate O_SCPLOWLC_SCPLOW-lactates roles in metabolism and signaling pathways.

biochemistry↗