Spatial Multiplexing of Fluorescent Reporters for Imaging Signaling Network Dynamics

Overview

Journal Cell

Publisher Cell Press

Specialty Cell Biology

Date 2020 Nov 24

PMID 33232692

Citations 27

Authors

Changyang Linghu

Shannon L Johnson

Pablo A Valdes

Or A Shemesh

Won Min Park

Demian Park

Kiryl D Piatkevich

Asmamaw T Wassie

Yixi Liu

Bobae An

Stephanie A Barnes

Orhan T Celiker

Chun-Chen Yao

Chih-Chieh Jay Yu

Ru Wang

Katarzyna P Adamala

Mark F Bear

Amy E Keating

Edward S Boyden

Affiliations

Soon will be listed here.

Abstract

In order to analyze how a signal transduction network converts cellular inputs into cellular outputs, ideally one would measure the dynamics of many signals within the network simultaneously. We found that, by fusing a fluorescent reporter to a pair of self-assembling peptides, it could be stably clustered within cells at random points, distant enough to be resolved by a microscope but close enough to spatially sample the relevant biology. Because such clusters, which we call signaling reporter islands (SiRIs), can be modularly designed, they permit a set of fluorescent reporters to be efficiently adapted for simultaneous measurement of multiple nodes of a signal transduction network within single cells. We created SiRIs for indicators of second messengers and kinases and used them, in hippocampal neurons in culture and intact brain slices, to discover relationships between the speed of calcium signaling, and the amplitude of PKA signaling, upon receiving a cAMP-driving stimulus.

Citing Articles

Genetically encoded protein oscillators for FM streaming of single-cell data.

Rajasekaran R, Galateo T, Xu Z, Bolshakov D, Weix E, Coyle S bioRxiv. 2025; .

PMID: 40060462 PMC: 11888400. DOI: 10.1101/2025.02.28.640587.

Innovations in aging biology: highlights from the ARDD emerging science & technologies workshop.

Unfried M, Schmauck-Medina T, Amin N, Boyden E, Fuellen G, Han J NPJ Aging. 2025; 11(1):8.

PMID: 39966395 PMC: 11836439. DOI: 10.1038/s41514-025-00193-5.

A history-dependent integrase recorder of plant gene expression with single-cell resolution.

Maranas C, George W, Scallon S, VanGilder S, Nemhauser J, Guiziou S Nat Commun. 2024; 15(1):9362.

PMID: 39472426 PMC: 11522408. DOI: 10.1038/s41467-024-53716-1.

Genetically encoded green fluorescent sensor for probing sulfate transport activity of solute carrier family 26 member a2 (Slc26a2) protein.

Lai C, Yang L, Pathiranage V, Wang R, Subach F, Walker A Commun Biol. 2024; 7(1):1375.

PMID: 39443638 PMC: 11499995. DOI: 10.1038/s42003-024-07020-9.

Neurotoxin-Derived Optical Probes for Elucidating Molecular and Developmental Biology of Neurons and Synaptic Connections : Toxin-Derived Optical Probes for Neuroimaging.

Bijjam R, Shorter S, Bratt A, OLeary V, Ntziachristos V, Ovsepian S Mol Imaging Biol. 2024; 26(6):912-925.

PMID: 39348040 PMC: 11634926. DOI: 10.1007/s11307-024-01954-6.

References

Mao L, Yang L, Arora A, Choe E, Zhang G, Liu Z . Role of protein phosphatase 2A in mGluR5-regulated MEK/ERK phosphorylation in neurons. J Biol Chem. 2005; 280(13):12602-10. DOI: 10.1074/jbc.M411709200. View

Tripet B, Yu L, Bautista D, Wong W, Irvin R, Hodges R . Engineering a de novo-designed coiled-coil heterodimerization domain off the rapid detection, purification and characterization of recombinantly expressed peptides and proteins. Protein Eng. 1996; 9(11):1029-42. DOI: 10.1093/protein/9.11.1029. View

Otsuguro K, Gautam S, Ito S, Habara Y, Saito T . Characterization of forskolin-induced Ca2+ signals in rat olfactory receptor neurons. J Pharmacol Sci. 2005; 97(4):510-8. DOI: 10.1254/jphs.fp0040883. View

Chen T, Wardill T, Sun Y, Pulver S, Renninger S, Baohan A . Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature. 2013; 499(7458):295-300. PMC: 3777791. DOI: 10.1038/nature12354. View

Berg J, Hung Y, Yellen G . A genetically encoded fluorescent reporter of ATP:ADP ratio. Nat Methods. 2009; 6(2):161-6. PMC: 2633436. DOI: 10.1038/nmeth.1288. View

Redmond L, Kashani A, Ghosh A . Calcium regulation of dendritic growth via CaM kinase IV and CREB-mediated transcription. Neuron. 2002; 34(6):999-1010. DOI: 10.1016/s0896-6273(02)00737-7. View

Mehta S, Zhang J . Reporting from the field: genetically encoded fluorescent reporters uncover signaling dynamics in living biological systems. Annu Rev Biochem. 2011; 80:375-401. PMC: 4384825. DOI: 10.1146/annurev-biochem-060409-093259. View

Tinevez J, Perry N, Schindelin J, Hoopes G, Reynolds G, Laplantine E . TrackMate: An open and extensible platform for single-particle tracking. Methods. 2016; 115:80-90. DOI: 10.1016/j.ymeth.2016.09.016. View

Huang P, Boyken S, Baker D . The coming of age of de novo protein design. Nature. 2016; 537(7620):320-7. DOI: 10.1038/nature19946. View

10.

Averaimo S, Nicol X . Intermingled cAMP, cGMP and calcium spatiotemporal dynamics in developing neuronal circuits. Front Cell Neurosci. 2014; 8:376. PMC: 4230202. DOI: 10.3389/fncel.2014.00376. View

11.

Zaccai N, Chi B, Thomson A, Boyle A, Bartlett G, Bruning M . A de novo peptide hexamer with a mutable channel. Nat Chem Biol. 2011; 7(12):935-41. PMC: 3223406. DOI: 10.1038/nchembio.692. View

12.

Bodenmiller B . Multiplexed Epitope-Based Tissue Imaging for Discovery and Healthcare Applications. Cell Syst. 2016; 2(4):225-38. DOI: 10.1016/j.cels.2016.03.008. View

13.

Hsia Y, Bale J, Gonen S, Shi D, Sheffler W, Fong K . Design of a hyperstable 60-subunit protein dodecahedron. [corrected]. Nature. 2016; 535(7610):136-9. PMC: 4945409. DOI: 10.1038/nature18010. View

14.

Bruce J, Straub S, Yule D . Crosstalk between cAMP and Ca2+ signaling in non-excitable cells. Cell Calcium. 2003; 34(6):431-44. DOI: 10.1016/s0143-4160(03)00150-7. View

15.

Micheva K, Smith S . Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits. Neuron. 2007; 55(1):25-36. PMC: 2080672. DOI: 10.1016/j.neuron.2007.06.014. View

16.

Lin J, Izar B, Wang S, Yapp C, Mei S, Shah P . Highly multiplexed immunofluorescence imaging of human tissues and tumors using t-CyCIF and conventional optical microscopes. Elife. 2018; 7. PMC: 6075866. DOI: 10.7554/eLife.31657. View

17.

Grigoryan G, Kim Y, Acharya R, Axelrod K, Jain R, Willis L . Computational design of virus-like protein assemblies on carbon nanotube surfaces. Science. 2011; 332(6033):1071-6. PMC: 3264056. DOI: 10.1126/science.1198841. View

18.

Lee H, Kameyama K, Huganir R, Bear M . NMDA induces long-term synaptic depression and dephosphorylation of the GluR1 subunit of AMPA receptors in hippocampus. Neuron. 1998; 21(5):1151-62. DOI: 10.1016/s0896-6273(00)80632-7. View

19.

Ohta Y, Furuta T, Nagai T, Horikawa K . Red fluorescent cAMP indicator with increased affinity and expanded dynamic range. Sci Rep. 2018; 8(1):1866. PMC: 5789972. DOI: 10.1038/s41598-018-20251-1. View

20.

Wang G, Jones S, Marra M, Sadar M . Identification of genes targeted by the androgen and PKA signaling pathways in prostate cancer cells. Oncogene. 2006; 25(55):7311-23. DOI: 10.1038/sj.onc.1209715. View