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Pooled tagging and hydrophobic targeting of endogenous proteins for unbiased mapping of unfolded protein responses
Pooled tagging and hydrophobic targeting of endogenous proteins for unbiased mapping of unfolded protein responses
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Length:
20 minutes
Released:
Jul 14, 2023
Format:
Podcast episode
Description
Link to bioRxiv paper:
http://biorxiv.org/cgi/content/short/2023.07.13.548611v1?rss=1
Authors: Sansbury, S. E., Serebrenik, Y. V., Lapidot, T., Burslem, G. M., Shalem, O.
Abstract:
System-level understanding of proteome organization and function requires methods for direct visualization and manipulation of proteins at scale. We developed an approach enabled by high-throughput gene tagging for the generation and analysis of complex cell pools with endogenously tagged proteins. Proteins are tagged with HaloTag to enable visualization or direct perturbation. Fluorescent labeling followed by in situ sequencing and deep learning-based image analysis identifies the localization pattern of each tag, providing a birds-eye-view of cellular organization. Next, we use a hydrophobic HaloTag ligand to unfold tagged proteins, inducing spatially restricted proteotoxic stress that is read out by single cell RNA sequencing. By integrating optical and perturbation data, we map compartment-specific responses to protein misfolding, revealing inter-compartment organization and direct cross-talk, and assigning proteostasis functions to uncharacterized genes. Altogether, we present a powerful and efficient method for large-scale studies of proteome dynamics, function, and homeostasis.
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Podcast created by Paper Player, LLC
http://biorxiv.org/cgi/content/short/2023.07.13.548611v1?rss=1
Authors: Sansbury, S. E., Serebrenik, Y. V., Lapidot, T., Burslem, G. M., Shalem, O.
Abstract:
System-level understanding of proteome organization and function requires methods for direct visualization and manipulation of proteins at scale. We developed an approach enabled by high-throughput gene tagging for the generation and analysis of complex cell pools with endogenously tagged proteins. Proteins are tagged with HaloTag to enable visualization or direct perturbation. Fluorescent labeling followed by in situ sequencing and deep learning-based image analysis identifies the localization pattern of each tag, providing a birds-eye-view of cellular organization. Next, we use a hydrophobic HaloTag ligand to unfold tagged proteins, inducing spatially restricted proteotoxic stress that is read out by single cell RNA sequencing. By integrating optical and perturbation data, we map compartment-specific responses to protein misfolding, revealing inter-compartment organization and direct cross-talk, and assigning proteostasis functions to uncharacterized genes. Altogether, we present a powerful and efficient method for large-scale studies of proteome dynamics, function, and homeostasis.
Copy rights belong to original authors. Visit the link for more info
Podcast created by Paper Player, LLC
Released:
Jul 14, 2023
Format:
Podcast episode
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