Visual barcodes for clonal-multiplexing of live microscopy-based assays

Abstract

While multiplexing samples using DNA barcoding revolutionized the pace of biomedical discovery, multiplexing of live imaging-based applications has been limited by the number of fluorescent proteins that can be deconvoluted using common microscopy equipment. To address this limitation, we develop visual barcodes that discriminate the clonal identity of single cells by different fluorescent proteins that are targeted to specific subcellular locations. We demonstrate that deconvolution of these barcodes is highly accurate and robust to many cellular perturbations. We then use visual barcodes to generate ‘Signalome’ cell-lines by mixing 12 clones of different live reporters into a single population, allowing simultaneous monitoring of the activity in 12 branches of signaling, at clonal resolution, over time. Using the ‘Signalome’ we identify two distinct clusters of signaling pathways that balance growth and proliferation, emphasizing the importance of growth homeostasis as a central organizing principle in cancer signaling. The ability to multiplex samples in live imaging applications, both in vitro and in vivo may allow better high-content characterization of complex biological systems.

Stepwise Stiffening/Softening of and Cell Recovery from Reversibly Formulated Hydrogel Double Networks

“…To that end, we designed a tdTomato hMSC reporter line based on the Signalome reporter system for YAP/TAZ, which gives a live output of YAP translocation, but does not introduce exogenous YAP that may disrupt signaling processes. [57] In this system, as the YAP/TAZ complex translocates to the nucleus and binds to the transcription factor TEAD, which then drives the expression of tdTomato (Supplementary Figure S7a-b). [58] We repeated the dynamic softening experiment with this modified cell line and live-imaged each gel on days 1, 3, 5, and 7; gels softened on days 3 and 5 were first imaged on their respective days of softening, and then treated with 4S9.…”

Pooled multicolour tagging for visualizing subcellular protein dynamics

“…We here show that multicolour tagging enables automated clone identification in cell pools. Rather than using specific tags solely for barcoding 43 , we directly use the combination of localization patterns and intensities of the tagged proteins as visual barcodes. We estimate that this method enables discriminating thousands of clones, with the option to further increase this number up to proteome-scale cell pools by expanding the set of BFP barcodes and modulating structural channel intensities.…”

pYtags enable spatiotemporal measurements of receptor tyrosine kinase signaling in living cells

“…Recent advances in live-cell biosensors have enabled the study of intracellular signaling at unprecedented spatiotemporal resolution. Signaling responses can be measured with a temporal precision on the order of seconds, and recent studies have described approaches to multiplex biosensors of multiple pathways in a single cell ( Regot et al, 2014 ) or to use barcoding strategies to monitor many biosensors within a population of cells ( Kaufman et al, 2022 ; Yang et al, 2021 ). Yet the development of biosensors for specific RTKs has been somewhat limited.…”