Rising Stars Seminar Series: Spatial Multiplexing of Fluorescent Reporters for Imaging Signaling Network Dynamics

Biological cells are composed of a variety of dynamic, interacting molecular and biophysical signaling building blocks. These building blocks collectively form signal transduction networks that process and store biological information, allowing cells to achieve complex physiological functions. 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 human-created 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, even if the fluorescent spectra of the reporters overlap or are identical. We created SiRIs for indicators of second messengers and kinase activities and used them, in hippocampal neurons in culture and intact brain slices, to discover relationships between the speed of calcium signaling, and the amplitude and duration of protein kinase A (PKA) signaling, upon receiving a cAMP-driving stimulus. We further showed that it is possible to image four, and even five, signals at once by expressing SiRIs containing fluorescent reporters of Ca2+, cAMP, PKA, protein kinase C (PKC), and extracellular signal-regulated kinase (ERK) within single cells. We are freely distributing SiRI reporters at the nonprofit plasmid repository, Addgene, and we envision the spatial multiplexing concept, and the SiRI protein architecture, to enable simultaneous measurements of many more biological dynamics and to provide a broad utility in biological science.

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