(2/10) We believe this is the beginning of a journey to record longitudinal biological events in as many cells as possible across the whole organ of interest, which is heretofore unachievable.

(3/10) The “ticker tape” is engineered from iPAK4 protein (Nat. Commun. 6, 8681 (2015)) that can assemble intracellularly into fibers. The crystal structure exhibits spacious 1D channels to accommodate N-terminus fusions without disrupting its crystallization process.

(4/10) We then engineered HaloTag-fused building blocks for crystal formation. Sequential staining using Janelia Fluor dyes with HaloTag Ligand (generous support from @rhodamine110) affords colored stripes in fibers that map their growth to wall-clock time.

(5/10) The expression of a tagged iPAK4 can be made dependent on a designated biological process. For example, an eGFP-iPAK4 fusion driven by activity-dependent IEG promoters (cFos) can report the neural activation events as green stripes on the fibers.

(6/10) Surprisingly, the in-cellulo fibers do not affect the cell viability or significantly alter the electrophysiological properties of neurons, permitting the utilization of iPAK4 as the intracellular “ticker tape”.

(7/10)To test its growth kinetics, we successively washed the HT-iPAK4 expressing cells with various colors of HT-ligand dyes at designated intervals. Steady growth rate and sharp boundaries were clearly identified, permitting the reservation of temporal order in fiber growth.

(8/10) As the fibers grow steadily, the onset of a recorded signal can be precisely determined by linear interpolation from the fiducial timestamps. Our “ticker tapes” were proven to report artificial intracellular events at a temporal resolution of 20-50min within a 10h window.

(9/10) At the end, cFos::eGFP-iPAK4 was coexpressed to encode the chemically (PMA) induced neural activation in the in-cellulo fibers. Similarly, the onsets of neural activation can be determined with high precision.