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

doi:10.1016/j.cell.2020.10.035

Spatial Multiplexing of Fluorescent Reporters for Imaging Signaling Network Dynamics

Cell. 2020 Dec 10;183(6):1682-1698.e24. doi: 10.1016/j.cell.2020.10.035. Epub 2020 Nov 23.

Authors

Changyang Linghu¹, Shannon L Johnson², Pablo A Valdes³, Or A Shemesh⁴, Won Min Park⁵, Demian Park², Kiryl D Piatkevich⁶, Asmamaw T Wassie⁷, Yixi Liu⁸, Bobae An⁹, Stephanie A Barnes¹⁰, Orhan T Celiker¹, Chun-Chen Yao¹¹, Chih-Chieh Jay Yu⁷, Ru Wang², Katarzyna P Adamala¹², Mark F Bear¹⁰, Amy E Keating¹³, Edward S Boyden¹⁴

Affiliations

¹ Department of Media Arts and Sciences, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA; McGovern Institute for Brain Research, MIT, Cambridge, MA 02139, USA; Center for Neurobiological Engineering, MIT, Cambridge, MA 02139, USA; Department of Electrical Engineering and Computer Science, MIT, Cambridge, MA 02139, USA.
² Department of Media Arts and Sciences, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA; McGovern Institute for Brain Research, MIT, Cambridge, MA 02139, USA; Center for Neurobiological Engineering, MIT, Cambridge, MA 02139, USA.
³ Department of Media Arts and Sciences, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA; Department of Neurosurgery, Harvard Medical School, Brigham and Women's Hospital, Boston, MA 02115, USA.
⁴ Department of Media Arts and Sciences, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA; McGovern Institute for Brain Research, MIT, Cambridge, MA 02139, USA; Center for Neurobiological Engineering, MIT, Cambridge, MA 02139, USA; Department of Biological Engineering, MIT, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, MIT, Cambridge, MA 02139, USA; Department of Neurobiology and Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.
⁵ Tim Taylor Department of Chemical Engineering, Kansas State University, Manhattan, KS 66506, USA.
⁶ Department of Media Arts and Sciences, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA; McGovern Institute for Brain Research, MIT, Cambridge, MA 02139, USA; Center for Neurobiological Engineering, MIT, Cambridge, MA 02139, USA; Department of Biological Engineering, MIT, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, MIT, Cambridge, MA 02139, USA; School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310024, China.
⁷ Department of Media Arts and Sciences, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA; McGovern Institute for Brain Research, MIT, Cambridge, MA 02139, USA; Center for Neurobiological Engineering, MIT, Cambridge, MA 02139, USA; Department of Biological Engineering, MIT, Cambridge, MA 02139, USA.
⁸ Department of Media Arts and Sciences, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA; McGovern Institute for Brain Research, MIT, Cambridge, MA 02139, USA; Department of Electrical Engineering and Computer Science, MIT, Cambridge, MA 02139, USA; Center for Neurobiological Engineering, MIT, Cambridge, MA 02139, USA.
⁹ Department of Media Arts and Sciences, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA; McGovern Institute for Brain Research, MIT, Cambridge, MA 02139, USA; Center for Neurobiological Engineering, MIT, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, MIT, Cambridge, MA 02139, USA.
¹⁰ Department of Brain and Cognitive Sciences, MIT, Cambridge, MA 02139, USA; Picower Institute for Learning and Memory, MIT, Cambridge, MA 02139, USA.
¹¹ Department of Brain and Cognitive Sciences, MIT, Cambridge, MA 02139, USA.
¹² Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA.
¹³ Department of Biological Engineering, MIT, Cambridge, MA 02139, USA; Department of Biology, MIT, Cambridge, MA 02139, USA; Koch Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
¹⁴ Department of Media Arts and Sciences, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA; McGovern Institute for Brain Research, MIT, Cambridge, MA 02139, USA; Center for Neurobiological Engineering, MIT, Cambridge, MA 02139, USA; Department of Biological Engineering, MIT, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, MIT, Cambridge, MA 02139, USA; Koch Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Cambridge, MA 02139, USA. Electronic address: edboyden@mit.edu.

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.

Keywords: cAMP; calcium imaging; fluorescent reporters; live-cell imaging; protein kinase; protein scaffold; signal transduction; signaling pathway; signaling reporter islands; spatial multiplexing.

Publication types

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

MeSH terms

Animals
Calcium / metabolism
Cyclic AMP / metabolism
Cyclic AMP-Dependent Protein Kinases / metabolism
Female
Fluorescent Dyes / metabolism*
Genes, Reporter*
Green Fluorescent Proteins / metabolism
HeLa Cells
Hippocampus / metabolism
Humans
Mice
Neurons / metabolism
Optical Imaging*
Peptides / metabolism
Proteins / metabolism
Pyramidal Cells / metabolism
Signal Transduction*

Substances

Fluorescent Dyes
Peptides
Proteins
Green Fluorescent Proteins
Cyclic AMP
Cyclic AMP-Dependent Protein Kinases
Calcium

Abstract

Publication types

MeSH terms

Substances

Grants and funding