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PreprintCited by 21Lipid Membrane Structure and BehaviorCellular transport and secretionEndoplasmic Reticulum Stress and Disease

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Pooled tagging and hydrophobic targeting of endogenous proteins for unbiased mapping of unfolded protein responses

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Sansbury, Stephanie E., Serebrenik, Yevgeniy V., Lapidot, Tomer, Burslem, George M., Shalem, Ophir·2023·Molecular Cell
DOI 10.1101/2023.07.13.548611PMC PMC12115883

Preprint — published as 10.1016/j.molcel.2025.04.002

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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 bird’s-eye-view of cellular organization. Next, we use a hydrophobic HaloTag ligand to misfold 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 crosstalk, 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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Metadata

Authors
Sansbury, S. E., Serebrenik, Y. V., Lapidot, T., Burslem, G. M., & Shalem, O.
Affiliations
  • Children's Hospital of Philadelphia (US)
  • University of Pennsylvania (US)
Corresponding author
  • Serebrenik, Yevgeniy V.
  • Shalem, Ophir
ORCID
Venue
Molecular Cell
Publisher
openRxiv
Published
2023-07-14
Volume
85
Issue
9
Pages
1868-1886.e12
ISSN
1097-2765 (ISSN-L)
DOI
10.1101/2023.07.13.548611
PMC
PMC12115883
OpenAlex
W4409730592
Cited by
21
References
1
Citations by year
2026: 7 · 2025: 6 · 2024: 7 · 2023: 1
FWCI
1.87
Type
journal article
Language
en
Open access
Locations
  • Molecular Cell (acceptedVersion)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • SSRN Electronic Journal (acceptedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed Central (submittedVersion)
Funders
  • Pennsylvania Department of Health
  • Center for Strategic Scientific Initiatives, National Cancer Institute (F32CA239499)
  • National Institutes of Health
  • National Institute on Aging (K99AG075256, R00AG075256)
  • National Human Genome Research Institute (F31HG011185)
  • National Cancer Institute (F32CA239499)
  • National Institute of General Medical Sciences (4100083086, T32GM008216, DP2GM137416, R35GM142505)
  • National Institute of Neurological Disorders and Stroke (R03NS111447)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Lipid Membrane Structure and Behavior
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Endoplasmic Reticulum Stress and Disease
Keywords
membrane tubulation, glycocalyx, glycopolymers, spontaneous curvature, line tension, polymer physics, Golgi apparatus, endoplasmic reticulum

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