Biology subjects

Takahashi-Yamashiro, K.

Publications and source records attributed to Takahashi-Yamashiro, K..

8 recordsLinked to original sources

bioRxiv2026

Ratiometric Fluorescent Protein Biosensors Reveal Citrate Dynamics and Cellular Heterogeneity

Citrate is a central intermediate metabolite linking the tricarboxylic acid cycle and lipid biosynthesis. Tools for monitoring of spatiotemporal citrate dynamics are critical for getting a better understanding of cellular metabolism. Here, we develope genetically encoded excitation ratiometric biosensors for citrate, based on our previous intensiometric green fluorescence protein-based citrate biosensor, Citron1. We find that a single mutation in the Citron1 chromophore-forming tripeptide provided an excitation ratiometric response. Further rounds of directed evolution yield highly responsive variants, exhibiting citrate-dependent fluorescence changes between two excitation peaks. When expressed in mammalian cells, these biosensors enable citrate dynamics to be monitored in both the cytosol and mitochondria. Comparative analysis across multiple human breast cancer cell lines uncovers cell line-specific differences in citrate levels and their heterogeneity, which could be linked to their malignancy. Furthermore, flow cytometry-based measurements in mouse embryonic stem cells demonstrate the proteomics signatures underlying the population-level variability in citrate concentrations and citrate rewiring during stem cell differentiation. Together, these results show that these excitation ratiometric citrate biosensors enable quantitative, compartment-resolved, and population-scale analysis of cellular metabolism.

molecular biology↗

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

An ultrasensitive and modular platform to detect Siglec ligands and control immune cell function

Siglecs are immunomodulatory receptors that regulate immune cell function. A fundamental challenge in studying Siglec-ligand interactions is the low affinity of Siglecs for their ligands. Inspired by how nature uses multivalency, we developed Siglec-liposomes as a highly multivalent and versatile platform for detecting Siglec glycan ligands in which recombinant Siglecs were conjugated to liposomes using the SpyCatcher-SpyTag system. Siglec-liposomes offer tunable multivalency and a modular assembly, enabling presentation of different Siglecs on the same liposome. Using Siglec-liposomes, we profiled Siglec ligands on human leukocytes, revealing new insights into Siglec ligands. Moreover, Siglec-liposomes are in vivo compatible, where we demonstrated that Siglec-7-liposomes bind to the brain vasculature in a mucin-dependent manner. Given the abundance of Siglec ligands on T cells, we investigated whether Siglec-liposomes modulate T cell function and find that Siglec-7-liposomes increase T cell proliferation in a ST3Gal1-dependent and CD43-independent manner. Taken together, Siglec-liposomes are a versatile and sensitive tool for detecting Siglec ligands and immunomodulation.

biochemistry↗

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↗

bioRxiv2022

High performance genetically-encoded green fluorescent biosensors for intracellular L-lactate

L-Lactate is a monocarboxylate produced during the process of cellular glycolysis and has long been generally considered a waste product. However, studies in recent decades have provided new perspectives on the physiological roles of L-lactate as a major energy substrate and a signaling molecule. To enable further investigations of the physiological roles of L-lactate, we have developed a series of high-performance ({Delta}F/F = 15 to 30 in vitro), intensiometric, genetically-encoded green fluorescent protein (GFP)-based intracellular L-lactate biosensors with a range of affinities. We evaluated the performance of these biosensors by in vitro and live-cell characterization and demonstrated the utility with imaging applications in several cell lines.

bioengineering↗

bioRxiv2022

Biosensor optimization using a FRET pair based on mScarlet red fluorescent protein and an mScarlet-derived green fluorescent protein

Genetically encoded biosensors based on Forster resonance energy transfer (FRET) are indispensable tools for monitoring biochemical changes in cells. Green and red fluorescent protein-based FRET pairs offer advantages over the classically employed cyan and yellow fluorescent protein pairs, such as better spectral separation, lower phototoxicity, and less autofluorescence. Here, we describe the development of an mScarlet-derived green fluorescent protein (designated as mWatermelon) and its use as a FRET donor to the red fluorescent protein mScarlet-I as a FRET acceptor. We tested the functionality of this FRET pair by engineering biosensors for the detection of protease activity, Ca2+, and K+. Furthermore, we described a strategy to enhance the FRET efficiency of these biosensors by modulating the intramolecular association between mWatermelon and mScarlet-I.

bioengineering↗