Spatial Multiplexing of Fluorescent Reporters for Imaging Signaling Network Dynamics

Linghu, Changyang; Johnson, Shannon; Valdés, Pablo; Shemesh, Or A.; Park, Won Min; Park, Demian; Piatkevich, Kiryl D.; Wassie, Asmamaw T.; Liu, Yixi; An, Bo; Barnes, Stephanie A.; Celiker, Orhan T.; Yao, Chun-Chen; Yu, Chih-Chieh Jay; Wang, Ru; Adamala, Katarzyna P.; Bear, Mark F.; Keating, Amy E.; Boyden, Edward S.

doi:10.1016/j.cell.2020.10.035

Cited by 43 publications

(31 citation statements)

References 98 publications

(100 reference statements)

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“…Another approach uses single-color probes for obsolete complex and laborious spectral unmixing and image processing. Detecting different biosensors of the same color permits synchronous detection of multiple parameters in aggregated protein clusters inside cells [ 18 ]. However, spatial and optical encoding of the various probes requires post hoc imaging experiments and image processing, such as immunostaining using specific epitopes fused to each biosensor.…”

Section: Introductionmentioning

confidence: 99%

A Co-Culture-Based Multiparametric Imaging Technique to Dissect Local H2O2 Signals with Targeted HyPer7

et al. 2021

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Multispectral live-cell imaging is an informative approach that permits detecting biological processes simultaneously in the spatial and temporal domain by exploiting spectrally distinct biosensors. However, the combination of fluorescent biosensors with distinct spectral properties such as different sensitivities, and dynamic ranges can undermine accurate co-imaging of the same analyte in different subcellular locales. We advanced a single-color multiparametric imaging method, which allows simultaneous detection of hydrogen peroxide (H2O2) in multiple cell locales (nucleus, cytosol, mitochondria) using the H2O2 biosensor HyPer7. Co-culturing of endothelial cells stably expressing differentially targeted HyPer7 biosensors paved the way for co-imaging compartmentalized H2O2 signals simultaneously in neighboring cells in a single experimental setup. We termed this approach COMPARE IT, which is an acronym for co-culture-based multiparametric imaging technique. Employing this approach, we detected lower H2O2 levels in mitochondria of endothelial cells compared to the cell nucleus and cytosol under basal conditions. Upon administering exogenous H2O2, the cytosolic and nuclear-targeted probes displayed similarly slow and moderate HyPer7 responses, whereas the mitochondria-targeted HyPer7 signal plateaued faster and reached higher amplitudes. Our results indicate striking differences in mitochondrial H2O2 accumulation of endothelial cells. Here, we present the method’s potential as a practicable and informative multiparametric live-cell imaging technique.

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Section: Introductionmentioning

confidence: 99%

A Co-Culture-Based Multiparametric Imaging Technique to Dissect Local H2O2 Signals with Targeted HyPer7

et al. 2021

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“…In this strategy, the multiplicity is limited to the number of sites that can be robustly distinguished in microscopic images. To our knowledge, the only generalizable approach for multiplexing biosensor imaging is the recently reported “signaling reporter islands” method, in which different biosensors are clustered at different spots in cells to allow for spatial separation of their signals ( Linghu et al, 2020 ). However, the method is technically demanding, requiring laborious post-imaging sample processing and analysis to identify the biosensor in each cluster, and is incompatible with translocation based biosensors.…”

Section: Discussionmentioning

confidence: 99%

Deciphering cell signaling networks with massively multiplexed biosensor barcoding

Yang

Chi

Liang

et al. 2021

Cell

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“…Ongoing activities to engineer promoters and expression systems that respond to calcium 39,40 and other physiological dynamics 25,41 , or stages of the cell cycle 42 , would enable XRI recordings of calcium activity or cell cycle. Another future direction will be to expand the XRI system for multiplexed recording of multiple kinds of biological information onto the same polymer chain, using a unique epitope tag for each kind of biological information, and multiplexed immunostaining methods 32 to read out each information. For example, one could use tags A and B to encode the gene expression history of genes 1 and 2, respectively, and use tag C to encode the calcium signal, by expressing all the components simultaneously, and then immunostaining all the tags after fixation.…”

Section: Discussionmentioning

confidence: 99%

Recording of cellular physiological histories along optically readable self-assembling protein chains

Linghu

Shpokayte

et al. 2021

Preprint

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Observing cellular physiological histories is key to understanding normal and disease-related processes, but longitudinal imaging is laborious and equipment-intensive. A tantalizing possibility is that cells could record such histories in the form of digital biological information within themselves, for later high-throughput readout. Here we show that this concept can be realized through information storage in the form of growing protein chains made out of multiple self-assembling subunits bearing different labels, each corresponding to a different cellular state or function, so that the physiological history of the cell can be visually read out along the chain of proteins. Conveniently, such protein chains are fully genetically encoded, and easily readable with simple, conventional optical microscopy techniques, compatible with visualization of cellular shape and molecular content. We use such expression recording islands (XRIs) to record gene expression timecourse downstream of pharmacological and physiological stimuli, in cultured neurons and in living mouse brain.

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Spatial Multiplexing of Fluorescent Reporters for Imaging Signaling Network Dynamics

Cited by 43 publications

References 98 publications

A Co-Culture-Based Multiparametric Imaging Technique to Dissect Local H2O2 Signals with Targeted HyPer7

A Co-Culture-Based Multiparametric Imaging Technique to Dissect Local H2O2 Signals with Targeted HyPer7

Deciphering cell signaling networks with massively multiplexed biosensor barcoding

Recording of cellular physiological histories along optically readable self-assembling protein chains

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