timer and tocky | Masahiro Ono

Fluorescent Timer Protein

As a BBSRC David Phillips Fellow, Dr. Ono developed a novel experimental method for analyzing the temporal changes in cellular activities and differentiation through flow cytometric analysis using a Fluorescent Timer protein, which was developed by the Verkhusha group (Subach et al., 2009). This Fluorescent Timer protein is an mCherry mutant (fastFT).

The chromophore of the Fluorescent Timer protein emits blue fluorescence upon translation, which then matures into red chromophore with a half-life of 4 hours. This maturation process was experimentally determined through flow cytometric analysis of T-cells expressing the Timer protein (Bending et al., 2018).

Left: Each Timer protein will emit either blue or red fluorescence (or be at the colorless stage). Right: figure from (Ono, 2024).

From Timer to Tocky: A New Perspective

With the Tocky reporter system, each cell expresses multiple Timer proteins. The expression dynamics are determined by the temporal dynamics of transcriptional activities for the Timer gene.

Thus, each cell will express multiple Timer proteins, each maturing from the blue form to the red one. This creates a continuous gradient of Timer fluorescence from blue to red (Bending et al., 2018).

As flow cytometry is a single-cell analysis method, the unique features of Timer fluorescence and Timer-expressing cells provide opportunities to dissect the temporal dynamics of cellular activities and differentiation.

This requires quantitative analysis. Enter the quantitative Timer fluorescence analysis method - Tocky.

Hence, the pivotal element that defines Tocky is the shift from protein-level analysis to single-cell level analysis.

References

2024

Unraveling T-cell dynamics using fluorescent timer: Insights from the Tocky system

Masahiro Ono

Biophysics and Physicobiology, 2024

Abstract Publication

Understanding the temporal dynamics of T-cell transcription is crucial for insights into immune cell function and development. In this study, we show the features of the Timer-of-Cell-Kinetics-and-Activity (Tocky) system, which enables analysis of temporal dynamics of cell activities and differentiation, leveraging Fluorescent Timer protein, which spontaneously changes its emission spectrum from blue to red fluorescence in known kinetics, as reporters. The current study examines the properties of the Tocky system, highlighting the Timer-Angle approach, which is a core algorithm of Tocky analysis and converts Timer Blue and Red fluorescence into Timer Angle and Intensity by trigonometric transformation. Importantly, Tocky analyzes time-related events within individual cells by the two phases of measurements, distinguishing between (1) the temporal sequence of cellular activities and differentiation within the time domain, and (2) the transcription frequency within the frequency domain. The transition from time measurement to frequency analysis, particularly at the Persistent locus that bridges these domains, highlights that system’s unique property in what is measured and analyzed by Tocky. Intriguingly, the sustained transcriptional activities observed in cells at the Persistent locus may have unique biological features as demonstrated in activated regulatory T-cells (Treg) and pathogenic T-cells, respectively, using Foxp3-Tocky and Nr4a3-Tocky models. In conclusion, the Tocky system can provide crucial data for advancing our understanding of T-cell dynamics and function.

2018

A temporally dynamic Foxp3 autoregulatory transcriptional circuit controls the effector Treg programme

David Bending , Alina Paduraru , Catherine B Ducker , Paz Prieto Martı́n , Tessa Crompton , and Masahiro Ono

The EMBO journal, 2018

The second Tocky paper from the Ono lab uncovers the temporally dynamic regulation of Foxp3 transcription, offering new insights into T cell regulation.

Abstract Publication

Regulatory T cells (Treg) are negative regulators of the immune response; however, it is poorly understood whether and how Foxp3 transcription is induced and regulated in the periphery during T‐cell responses. Using Foxp3‐Timer of cell kinetics and activity (Tocky) mice, which report real‐time Foxp3 expression, we show that the flux of new Foxp3 expressors and the rate of Foxp3 transcription are increased during inflammation. These persistent dynamics of Foxp3 transcription determine the effector Treg programme and are dependent on a Foxp3 autoregulatory transcriptional circuit. Persistent Foxp3 transcriptional activity controls the expression of coinhibitory molecules, including CTLA‐4 and effector Treg signature genes. Using RNA‐seq, we identify two groups of surface proteins based on their relationship to the temporal dynamics of Foxp3 transcription, and we show proof of principle for the manipulation of Foxp3 dynamics by immunotherapy: new Foxp3 flux is promoted by anti‐TNFRII antibody, and high‐frequency Foxp3 expressors are targeted by anti‐OX40 antibody. Collectively, our study dissects time‐dependent mechanisms behind Foxp3‐driven T‐cell regulation and establishes the Foxp3‐Tocky system as a tool to investigate the mechanisms behind T‐cell immunotherapies.
A timer for analyzing temporally dynamic changes in transcription during differentiation in vivo

David Bending , Paz Prieto Martı́n , Alina Paduraru , Catherine Ducker , Erik Marzaganov , Marie Laviron , Satsuki Kitano , Hitoshi Miyachi , Tessa Crompton , and Masahiro Ono

Journal of Cell Biology, 2018

The foundational publication introducing Tocky technology by the Ono lab, marking a breakthrough in T cell and B cell studies.

Abstract Publication

Understanding the mechanisms of cellular differentiation is challenging because differentiation is initiated by signaling pathways that drive temporally dynamic processes, which are difficult to analyze in vivo. We establish a new tool, Timer of cell kinetics and activity (Tocky; or toki [time in Japanese]). Tocky uses the fluorescent Timer protein, which spontaneously shifts its emission spectrum from blue to red, in combination with computer algorithms to reveal the dynamics of differentiation in vivo. Using a transcriptional target of T cell receptor (TCR) signaling, we establish Nr4a3-Tocky to follow downstream effects of TCR signaling. Nr4a3-Tocky reveals the temporal sequence of events during regulatory T cell (Treg) differentiation and shows that persistent TCR signals occur during Treg generation. Remarkably, antigen-specific T cells at the site of autoimmune inflammation also show persistent TCR signaling. In addition, by generating Foxp3-Tocky, we reveal the in vivo dynamics of demethylation of the Foxp3 gene. Thus, Tocky is a tool for cell biologists to address previously inaccessible questions by directly revealing dynamic processes in vivo.

Fluorescent Timer Protein

From Timer to Tocky: A New Perspective

References

2024

2018

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