Scientists at Germany’s Max Planck Institute for Medical Research have developed protein recorders that can capture and analyze biological activities over multiple periods. The method is scalable and suitable for recording various biological activities, as demonstrated by recording protein-protein interactions, G protein-coupled receptor activation, and increases in intracellular calcium in vivo.

The research article “Recording physiological history of cells with chemical labeling” was published today in Science.

Direct, continuous, and sequential

Documenting transient cellular events is essential for elucidating biological mechanisms. At the moment, most recordings of cells across a tissue are made by linking an event to the transcription of a reporter gene or using a CRISPR/Cas-based tool. However, the coupling between cellular activity and the recording is only indirect.

The ideal approach to recording fleeting cellular events would be to directly access huge populations of cells simultaneously across whole tissues and to do it with excellent spatiotemporal resolution. In addition, for a large-scale parallel analysis of transient cellular events, it is crucial to separate the recording from its analysis; doing so would involve turning transient events into permanent marks for future analysis. However, there is a dearth of approaches that match these requirements.

Direct recording of activities for in situ and post hoc analyses is possible with optical and optogenetic approaches, which control recording through illumination of live specimens. These methods offer an outstanding spatiotemporal resolution. One example is the biosensor CaMPARI, which is used in different model organisms to record neuronal activity. When illuminated in high Ca2+ levels, it undergoes an irreversible color change.

An alternative to using light is activity-based labeling of cells with chemical probes. The addition and washout of an ideal, highly permeable, and harmless probe determine the recording window for this method, which can directly record the activity of interest. Therefore, it could be used in conjunction with the aforementioned approaches. However, the current methods of activity-based chemical labeling are neither well-suited for continuous recordings nor permit the recording of numerous consecutive events. 

Biological activity in technicolor

Co-authors Magnus-Carsten Huppertz and Jonas Wilhelm, as well as colleagues, developed Split-HaloTag—proteins that become labeled in the presence of both a specific cellular activity and a fluorescent substrate. The combination of a fluorescent substrate and a transient cellular activity, such as an interaction between proteins or an increase in intracellular Ca2+ levels, can be used to rationally design recorders to capture fleeting events for analysis at a later time.

The addition and washout of the fluorescent substrate determine the recording period. Any activities before or after these steps do not affect the recorder’s labeling. Since various colored fluorescent substrates are readily available, recording distinct time intervals within the same sample or organism is possible. Split-HaloTag recorders allow for the spatial resolution recording of cellular activities because the activity of interest activates them and can be directed to specific cellular locations.

Because the fluorescent signal is detectable for days and resistant to fixation strategies, this method can be easily scaled to simultaneously analyze large populations of cells or organisms. Furthermore, the fluorescent signal’s persistence enables cell sorting through recorder labeling and subsequent transcriptomic analysis.

All of these characteristics are demonstrated in the experiments using a split-HaloTag recorder designed for Ca2+-dependent protein labeling (Caprola). Huppertz and Wilhelm recorded neuronal activity in zebrafish larvae and adult flies using Caprola. They also sorted and analyzed the transcriptomes of diverse cell populations based on their Ca2+ levels. Given the widespread use of Ca2+ signaling and the success of HaloTag labeling in numerous model systems, Caprola could potentially be used in different branches of biology.

According to the authors, it should be possible to design recorders for additional cellular activities by utilizing the design principles of other protein-based fluorescent biosensors. This is because the design principle for biological recording links a cellular activity to the increased proximity of the two components of split-HaloTag. The split-HaloTag system opens the door to new recorders that could connect cellular physiology to phenotypes in the biological realm.

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