Spatial Multiplexing of Fluorescent Reporters for Imaging Signaling Network Dynamics

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

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish (US)
Pages (from-to)1682-1698.e24
Issue number6
StatePublished - Dec 10 2020

Bibliographical note

Funding Information:
We thank Daniel Goodwin for help with data analysis and Guangyu Xu and Jian-Ping Zhao for useful discussion. C.L. acknowledges the Friends of the McGovern Institute Graduate Fellowship and the J. Douglas Tan Postdoctoral Fellowship . S.L.J. acknowledges the MIT K. Lisa Yang and Hock E. Tan Center for Molecular Therapeutics in Neuroscience Fellowship and the MIT Media Lab . E.S.B. was supported by Lisa Yang , John Doerr , the Open Philanthropy Project , the HHMI-Simons Faculty Scholars Program and HHMI Investigatorship , Human Frontier Science Program RGP0015/2016 , U.S. Army Research Laboratory , and the U.S. Army Research Office under contract/grant number W911NF1510548 , the MIT Media Lab , the Picower Institute Innovation Fund , NIH R01MH122971 , NIH RF1NS113287 , NIH 1R24MH106075 , NIH 1R01NS102727 , NIH R44EB021054 , NIH 1R01DA045549 , NIH 1R01MH114031 , NIH 2R01DA029639 , NIH 1R01EB024261 , NIH Director’s Pioneer Award 1DP1NS087724 , NIH 1R01GM104948 , and NSF CBET 1344219 .

Publisher Copyright:
© 2020 The Author(s)


  • cAMP
  • calcium imaging
  • fluorescent reporters
  • live-cell imaging
  • protein kinase
  • protein scaffold
  • signal transduction
  • signaling pathway
  • signaling reporter islands
  • spatial multiplexing

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.


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