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Vibrational imaging of newly synthesized proteins in live cells by stimulated Raman scattering microscopy

Wei, Lu and Yu, Yong and Shen, Yihui and Wang, Meng C. and Min, Wei (2013) Vibrational imaging of newly synthesized proteins in live cells by stimulated Raman scattering microscopy. Proceedings of the National Academy of Sciences of the United States of America, 110 (28). pp. 11226-11231. ISSN 0027-8424. PMCID PMC3710790. https://resolver.caltech.edu/CaltechAUTHORS:20181119-102039803

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Abstract

Synthesis of new proteins, a key step in the central dogma of molecular biology, has been a major biological process by which cells respond rapidly to environmental cues in both physiological and pathological conditions. However, the selective visualization of a newly synthesized proteome in living systems with subcellular resolution has proven to be rather challenging, despite the extensive efforts along the lines of fluorescence staining, autoradiography, and mass spectrometry. Herein, we report an imaging technique to visualize nascent proteins by harnessing the emerging stimulated Raman scattering (SRS) microscopy coupled with metabolic incorporation of deuterium-labeled amino acids. As a first demonstration, we imaged newly synthesized proteins in live mammalian cells with high spatial–temporal resolution without fixation or staining. Subcellular compartments with fast protein turnover in HeLa and HEK293T cells, and newly grown neurites in differentiating neuron-like N2A cells, are clearly identified via this imaging technique. Technically, incorporation of deuterium-labeled amino acids is minimally perturbative to live cells, whereas SRS imaging of exogenous carbon–deuterium bonds (C–D) in the cell-silent Raman region is highly sensitive, specific, and compatible with living systems. Moreover, coupled with label-free SRS imaging of the total proteome, our method can readily generate spatial maps of the quantitative ratio between new and total proteomes. Thus, this technique of nonlinear vibrational imaging of stable isotope incorporation will be a valuable tool to advance our understanding of the complex spatial and temporal dynamics of newly synthesized proteome in vivo.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1073/pnas.1303768110DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3710790/PubMed CentralArticle
http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1303768110/-/DCSupplementalPublisherSupporting Information
ORCID:
AuthorORCID
Wei, Lu0000-0001-9170-2283
Min, Wei0000-0003-2570-3557
Additional Information:© 2013 National Academy of Sciences. Edited by David A. Tirrell, California Institute of Technology, Pasadena, CA, and approved May 31, 2013 (received for review February 27, 2013) We thank F. Hu, Z. Chen, V. W. Cornish, D. Peterka, and R. Yuste for helpful discussion. We are grateful to S. Buffington, M. Costa-Mattioli, and M. Sakamoto for providing hippocampal neurons, and Y. Li for his assistance on the spontaneous Raman microscope. We acknowledge support from Ellison Medical Foundation fellowships (to M.C.W.) and National Institutes of Health Director’s New Innovator Award (to W.M.). Author contributions: L.W., M.C.W., and W.M. designed research; L.W., Y.Y., and Y.S. performed research; L.W. analyzed data; and L.W., Y.Y., Y.S., M.C.W., and W.M. wrote the paper. Conflict of interest statement: Columbia University has filed a patent application based on this work. This article is a PNAS Direct Submission. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1303768110/-/DCSupplemental.
Funders:
Funding AgencyGrant Number
Ellison Medical FoundationUNSPECIFIED
NIHUNSPECIFIED
Subject Keywords:stable isotope labeling | stimulated Raman microscopy | protein synthesis
Issue or Number:28
PubMed Central ID:PMC3710790
Record Number:CaltechAUTHORS:20181119-102039803
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20181119-102039803
Official Citation:Vibrational imaging of newly synthesized proteins Lu Wei, Yong Yu, Yihui Shen, Meng C. Wang, Wei Min Proceedings of the National Academy of Sciences Jul 2013, 110 (28) 11226-11231; DOI: 10.1073/pnas.1303768110
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:91013
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:19 Nov 2018 19:14
Last Modified:23 Oct 2019 20:50

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