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Live-Cell Imaging and Quantification of PolyQ Aggregates by Stimulated Raman Scattering of Selective Deuterium Labeling

Miao, Kun and Wei, Lu (2020) Live-Cell Imaging and Quantification of PolyQ Aggregates by Stimulated Raman Scattering of Selective Deuterium Labeling. ACS Central Science, 6 (4). pp. 478-486. ISSN 2374-7943. PMCID PMC7181319. https://resolver.caltech.edu/CaltechAUTHORS:20191029-113111264

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Abstract

Polyglutamine (polyQ) diseases are a group of neurodegenerative disorders, involving the deposition of aggregation-prone proteins with long polyQ expansions. However, the cytotoxic roles of these aggregates remain highly controversial, largely due to a lack of proper tools for quantitative and nonperturbative interrogations. Common methods including in vitro biochemical, spectroscopic assays, and live-cell fluorescence imaging all suffer from certain limitations. Here, we propose coupling stimulated Raman scattering microscopy with deuterium-labeled glutamine for live-cell imaging, quantification, and spectral analysis of native polyQ aggregates with subcellular resolution. First, through the enrichment of deuterated glutamine in the polyQ sequence of mutant Huntingtin (mHtt) exon1 proteins for Huntington’s disease, we achieved sensitive and specific stimulated Raman scattering (SRS) imaging of carbon–deuterium bonds (C–D) from aggregates without GFP labeling, which is commonly employed in fluorescence microscopy. We revealed that these aggregates became 1.8-fold denser compared to those with GFP. Second, we performed ratiometric quantifications, which indicate a surprising dependence of protein compositions on aggregation sizes. Our further calculations, for the first time, reported the absolute concentrations for sequestered mHtt and non-mHtt proteins within the same aggregates. Third, we adopted hyperspectral SRS for Raman spectroscopic studies of aggregate structures. By inducing a cellular heat shock response, a potential therapeutic approach for inhibiting aggregate formation, we found a possible aggregate intermediate state with changed solvation microenvironments. Our method may hence readily unveil new features and mechanistic insight of polyQ aggregates and pave the way for comprehensive in vivo investigations.


Item Type:Article
Related URLs:
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https://doi.org/10.1021/acscentsci.9b01196DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/pmc7181319PubMed CentralArticle
https://doi.org/10.1101/820217DOIDiscussion Paper
https://resolver.caltech.edu/CaltechAUTHORS:20201221-095807659Related ItemConference Abstract
ORCID:
AuthorORCID
Miao, Kun0000-0001-6567-3650
Wei, Lu0000-0001-9170-2283
Additional Information:© 2020 American Chemical Society. This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Received: November 19, 2019; Published: March 6, 2020. We would like to thank Dr. C. Qian, D. Lee, J. Du, and Dr. L. Voong for helpful discussions. We are grateful for the plasmid (mHtt-97Q-GFP) shared by Prof. R. Kopito and Prof. F.-U. Hartl. We thank Prof. Z. Liu for sharing the stable embryonic stem cell-lines. L.W. acknowledges the support of start-up funds from California Institute of Technology. The authors declare no competing financial interest.
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CaltechUNSPECIFIED
Issue or Number:4
PubMed Central ID:PMC7181319
Record Number:CaltechAUTHORS:20191029-113111264
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20191029-113111264
Official Citation:Live-Cell Imaging and Quantification of PolyQ Aggregates by Stimulated Raman Scattering of Selective Deuterium Labeling. Kun Miao and Lu Wei. ACS Central Science 2020 6 (4), 478-486; DOI: 10.1021/acscentsci.9b01196
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:99535
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:29 Oct 2019 19:23
Last Modified:21 Dec 2020 17:59

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