van Weeren, L. | bioRxiv Science

bioRxiv2025

mScarlet3-H with low brightness and fluorescence lifetime has potential for cellular lifetime-unmixing and lifetime-based pH-sensing applications

An independent evaluation of the spectroscopic properties, cellular performance, merits and pitfalls of the redfluorescent protein mScarlet3-H as compared to mScarlet3 is reported. mScarlet3-H was generated from mScarlet3 by a single M163H mutation. Purified mScarlet3-H is characterized by a molar coeWicient of 79,040 M-1cm-1, afluorescence quantum yield of 17.8%, molecular brightness of 14.1 and a heterogeneous multiexponential decay with an averagefluorescence lifetime of 1 ns. Evaluation in living mammalian cells revealed a comparable maturation speed and eWiciency of mScarlet3 and mScarlet3-H, but the overall cellular brightness of mScarlet3-H was 5-fold lower than that of mScarlet3. Photobleaching analysis in live cells revealed identical photobleaching kinetics of mScarlet3-H and mScarlet-H. Thefluorescence intensity,fluorescence spectra andfluorescence lifetime of mScarlet3-H were found to be strongly pH-dependent between pH 4-8. Thefluorescence lifetime increased from 1 ns to 3 ns in lowering the pH from 8 to 4 with a pK of [~]6. The much lower lifetime of mScarlet3-H ([~]1 ns) as compared to mScarlet3 ([~]4 ns) allows dualfluorescence lifetime unmixing applications in single channel FLIM recordings in compartments with neutral to slightly alkaline pH. Furthermore, the strongly pH-dependentfluorescence lifetime of mScarlet3-H enablesfluorescence lifetime-based pH sensing in a pH region between pH 5 to 7. With this property autophagy of the cytoplasm can be visualized by the pH-dependentfluorescence lifetime with mScarlet3-H accumulation in lysosomes. Potential useful applications and pitfalls regarding the special properties of mScarlet3-H are discussed.

cell biology↗

bioRxiv2019

Seeing cells smell: Dynamic optical measurements of Ca2+ and cAMP signaling from Olfactory Receptors transiently expressed in HEK293TN cells

Olfactory receptors (ORs) constitute the largest family of G-protein coupled receptors. They are responsible for the perception of odor (olfaction) and also play important roles in other biological processes, including regulation of cell proliferation. Their increasing diagnostic and therapeutic potential, especially for cancer research, requests the ongoing development of methodologies that would allow their robust functional expression in non-olfactory cells, and dynamic analysis of their signaling pathways. To enable realtime detection of OR activity, we use single cell imaging with genetically encoded fluorescent biosensors, Yellow Cameleon or EPAC, which are routinely used for kinetic measurements of Ca2+ or cAMP signaling downstream of various G-protein coupled receptors. We demonstrate that the co-expression of Lucy-Rho tagged variants of ORs together with an accessory protein, RTP1s, in HEK293TN cells is sufficient to detect the activity of a panel of ORs. Using this methodology, we were able to detect both Ca2+ and cAMP signaling downstream of twelve ORs within 2 minutes from the application of odorant.

molecular biology↗