Genetically Encoded Protein Oscillators for FM Streaming of Single-cell Data

Overview

Journal bioRxiv

Date 2025 Mar 10

PMID 40060462

Authors

Rohith Rajasekaran

Thomas M Galateo

Zhejing Xu

Dennis T Bolshakov

Elliott W Z Weix

Scott M Coyle

Affiliations

Soon will be listed here.

Abstract

Radios and cellphones use frequency modulation (FM) of an oscillating carrier signal to reliably transmit multiplexed data while rejecting noise. Here, we establish a biochemical analogue of this paradigm using genetically encoded protein oscillators (GEOs) as carrier signals in circuits that enable continuous, real-time FM streaming of single-cell data. GEOs are constructed from evolutionarily diverse MinDE-family ATPase and activator modules that generate fast synthetic protein oscillations when co-expressed in human cells. These oscillations serve as a single-cell carrier signal, with frequency and amplitude controlled by GEO component levels and activity. We systematically characterize 169 ATPase/activator GEO pairs and engineer composite GEOs with multiple competing activators to develop a comprehensive platform for waveform programming. Using these principles, we design circuits that modulate GEO frequency in response to cellular activity and decode their responses using a calibrated machine-learning model to demonstrate sensitive, real-time FM streaming of transcription and proteasomal degradation dynamics in single cells. GEOs establish a dynamically controllable biochemical carrier signal, unlocking noise-resistant FM data-encoding paradigms that open new avenues for dynamic single-cell analysis.

References

Shaner N, Patterson G, Davidson M . Advances in fluorescent protein technology. J Cell Sci. 2007; 120(Pt 24):4247-60. DOI: 10.1242/jcs.005801. View

Fange D, Elf J . Noise-induced Min phenotypes in E. coli. PLoS Comput Biol. 2006; 2(6):e80. PMC: 1484588. DOI: 10.1371/journal.pcbi.0020080. View

Qian Y, Celiker O, Wang Z, Guner-Ataman B, Boyden E . Temporally multiplexed imaging of dynamic signaling networks in living cells. Cell. 2023; 186(25):5656-5672.e21. PMC: 10843875. DOI: 10.1016/j.cell.2023.11.010. View

Elowitz M, Leibler S . A synthetic oscillatory network of transcriptional regulators. Nature. 2000; 403(6767):335-8. DOI: 10.1038/35002125. View

Linghu C, Johnson S, Valdes P, Shemesh O, Park W, Park D . Spatial Multiplexing of Fluorescent Reporters for Imaging Signaling Network Dynamics. Cell. 2020; 183(6):1682-1698.e24. PMC: 7771521. DOI: 10.1016/j.cell.2020.10.035. View

Day R, Davidson M . The fluorescent protein palette: tools for cellular imaging. Chem Soc Rev. 2009; 38(10):2887-921. PMC: 2910338. DOI: 10.1039/b901966a. View

Vecchiarelli A, Li M, Mizuuchi M, Hwang L, Seol Y, Neuman K . Membrane-bound MinDE complex acts as a toggle switch that drives Min oscillation coupled to cytoplasmic depletion of MinD. Proc Natl Acad Sci U S A. 2016; 113(11):E1479-88. PMC: 4801307. DOI: 10.1073/pnas.1600644113. View

Higuchi-Sanabria R, Garcia E, Tomoiaga D, Munteanu E, Feinstein P, Pon L . Characterization of Fluorescent Proteins for Three- and Four-Color Live-Cell Imaging in S. cerevisiae. PLoS One. 2016; 11(1):e0146120. PMC: 4699809. DOI: 10.1371/journal.pone.0146120. View

Cusanovich D, Daza R, Adey A, Pliner H, Christiansen L, Gunderson K . Multiplex single cell profiling of chromatin accessibility by combinatorial cellular indexing. Science. 2015; 348(6237):910-4. PMC: 4836442. DOI: 10.1126/science.aab1601. View

10.

Rajasekaran R, Chang C, Weix E, Galateo T, Coyle S . A programmable reaction-diffusion system for spatiotemporal cell signaling circuit design. Cell. 2024; 187(2):345-359.e16. PMC: 10842744. DOI: 10.1016/j.cell.2023.12.007. View

11.

Regot S, Hughey J, Bajar B, Carrasco S, Covert M . High-sensitivity measurements of multiple kinase activities in live single cells. Cell. 2014; 157(7):1724-34. PMC: 4097317. DOI: 10.1016/j.cell.2014.04.039. View

12.

Pulianmackal L, Limcaoco J, Ravi K, Yang S, Zhang J, Tran M . Multiple ParA/MinD ATPases coordinate the positioning of disparate cargos in a bacterial cell. Nat Commun. 2023; 14(1):3255. PMC: 10241942. DOI: 10.1038/s41467-023-39019-x. View

13.

Shamir M, Bar-On Y, Phillips R, Milo R . SnapShot: Timescales in Cell Biology. Cell. 2016; 164(6):1302-1302.e1. DOI: 10.1016/j.cell.2016.02.058. View

14.

Loose M, Kruse K, Schwille P . Protein self-organization: lessons from the min system. Annu Rev Biophys. 2011; 40:315-36. DOI: 10.1146/annurev-biophys-042910-155332. View

15.

Chen Z . Protein circuit design using de novo proteins. Trends Biotechnol. 2023; 41(5):593-594. DOI: 10.1016/j.tibtech.2023.02.011. View

16.

Regev A, Teichmann S, Lander E, Amit I, Benoist C, Birney E . The Human Cell Atlas. Elife. 2017; 6. PMC: 5762154. DOI: 10.7554/eLife.27041. View

17.

Skylaki S, Hilsenbeck O, Schroeder T . Challenges in long-term imaging and quantification of single-cell dynamics. Nat Biotechnol. 2016; 34(11):1137-1144. DOI: 10.1038/nbt.3713. View

18.

Nandagopal N, Elowitz M . Synthetic biology: integrated gene circuits. Science. 2011; 333(6047):1244-8. PMC: 4117316. DOI: 10.1126/science.1207084. View

19.

Kelly R . Single-cell Proteomics: Progress and Prospects. Mol Cell Proteomics. 2020; 19(11):1739-1748. PMC: 7664119. DOI: 10.1074/mcp.R120.002234. View

20.

Iwamoto M, Bjorklund T, Lundberg C, Kirik D, Wandless T . A general chemical method to regulate protein stability in the mammalian central nervous system. Chem Biol. 2010; 17(9):981-8. PMC: 2943492. DOI: 10.1016/j.chembiol.2010.07.009. View