Biology subjects

Tsao, K. K.

Publications and source records attributed to Tsao, K. K..

3 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↗

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↗