Recording transcriptional histories of a cell would enable deeper understanding of cellular developmental trajectories and responses to external perturbations. Here we describe an engineered protein fiber that incorporates diverse fluorescent marks during its growth to store a ticker tape-like history. An embedded HaloTag reporter incorporates user-supplied dyes, leading to colored stripes that map the growth of each individual fiber to wall clock time. A co-expressed eGFP tag driven by a promoter of interest records a history of transcriptional activation. High-resolution multi-spectral imaging on fixed samples reads the cellular histories, and interpolation of eGFP marks relative to HaloTag timestamps provides accurate absolute timing. We demonstrate recordings of doxycycline-induced transcription in HEK cells and cFos promoter activation in cultured neurons, with a single-cell absolute accuracy of 30–40 minutes over a 12-hour recording. The protein-based ticker tape design we present here could be generalized to achieve massively parallel single-cell recordings of diverse physiological modalities.
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Data comprising images and time-lapse recordings of iPAK4 fibers, as well as patch-clamp recordings, are available from the corresponding authors upon reasonable request.
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We thank D. Kim, Y. Baskaran and E. Manser for helpful discussions. We thank B. Cui for the PAK4 plasmid and C. Hellriegel at the Harvard Center for Biological Imaging for assistance with microscopy. We thank S. Begum, A. Preecha and J. Koob for technical assistance. This work was supported by Schmidt Futures (A.E.C., X.L., E.M., P.P. and B.T.), a Vannevar Bush Faculty Fellowship (A.E.C.), the Howard Hughes Medical Institute (A.E.C., J.B.G., N.F., L.D.L., H.S. and D.B.), National Institutes of Health grant 1-R21-1EY033669 (A.E.C., D.L. and X.L.), a John S. LaDue Fellowship (B.Z.J.) and the Harvard Brain Science Initiative (D.L.).
A.E.C., D.L. and X.L. have filed a US patent application on protein-based ticker tapes for intracellular recordings. The remaining authors declare no competing interests.
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Supplementary Movie Captions 1–5, Supplementary Calculations 1 and 2 and Supplementary Figs. 1–16.
Supplementary Video 1
Time-lapse recording of iPAK4 fiber growth in HEK293T cells. HEK293T cells were co-infected with CMV::iPAK4 (90%), CMV::HT-iPAK4 (5%) and CMV::eGFP-iPAK4 (5%) lentiviruses and stained with JF669. Time-lapse video microscopy was acquired over 43 hours, starting 20 hours after lentiviral infection, with one frame every 10 minutes.
Supplementary Video 2
Tracking individual iPAK4 fibers in HEK293T cells with real-time growth profiles. Left: Time-lapse movies showing the tracking of single iPAK4 fibers. Right: Real-time growth profiles of the corresponding fibers.
Supplementary Video 3
Time-lapse recording of iPAK4 fiber growth with one dye switch in HEK293T cells. HEK293T cells co-infected with CMV::iPAK4 (95%) and CMV::HT-iPAK4 (5%) lentiviruses. The dye was switched (from JF552 to JF669) right before the imaging. Time-lapse video microscopy was acquired over 40 hours, starting 20 hours after transfection, with one frame every 5 minutes.
Supplementary Video 4
Time-lapse recording of iPAK4 fiber growth in neurons. Primary neurons co-infected with CMV::iPAK4 (95%) and CMV::HT-iPAK4 (5%) lentiviruses. Time-lapse video microscopy was acquired over 40 hours, starting 20 hours after transfection, with one frame every 5 minutes.
Supplementary Video 5
Tracking individual iPAK4 fibers in primary neurons with real-time growth profiles. Left: Time-lapse movies showing the tracking of single iPAK4 fibers. Right: Real-time growth profiles of the corresponding fibers.
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Lin, D., Li, X., Moult, E. et al. Time-tagged ticker tapes for intracellular recordings. Nat Biotechnol 41, 631–639 (2023). https://doi.org/10.1038/s41587-022-01524-7
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