Imaging Complex Protein Metabolism in Live Organisms by Stimulated Raman Scattering Microscopy with Isotope Labeling

Protein metabolism, consisting of both synthesis and degradation, is highly complex, playing an indispensable regulatory role throughout physiological and pathological processes. Over recent decades, extensive efforts, using approaches such as autoradiography, mass spectrometry, and fluorescence mic...

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Bibliographic Details
Published in:ACS chemical biology 2015-03, Vol.10 (3), p.901-908
Main Authors: Wei, Lu, Shen, Yihui, Xu, Fang, Hu, Fanghao, Harrington, Jamie K, Targoff, Kimara L, Min, Wei
Format: Article
Language:English
Subjects:
Amino Acids - metabolism
Animals
Brain - metabolism
Brain - ultrastructure
Cells, Cultured
Deuterium - metabolism
Embryo, Nonmammalian
HeLa Cells
Humans
Isotope Labeling - methods
Mice
Microscopy - instrumentation
Microscopy - methods
Molecular Imaging - instrumentation
Molecular Imaging - methods
Neurons - metabolism
Neurons - ultrastructure
Protein Biosynthesis
Proteins - chemistry
Proteins - metabolism
Proteolysis
Spectrum Analysis, Raman - instrumentation
Spectrum Analysis, Raman - methods
Zebrafish
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Summary:Protein metabolism, consisting of both synthesis and degradation, is highly complex, playing an indispensable regulatory role throughout physiological and pathological processes. Over recent decades, extensive efforts, using approaches such as autoradiography, mass spectrometry, and fluorescence microscopy, have been devoted to the study of protein metabolism. However, noninvasive and global visualization of protein metabolism has proven to be highly challenging, especially in live systems. Recently, stimulated Raman scattering (SRS) microscopy coupled with metabolic labeling of deuterated amino acids (D-AAs) was demonstrated for use in imaging newly synthesized proteins in cultured cell lines. Herein, we significantly generalize this notion to develop a comprehensive labeling and imaging platform for live visualization of complex protein metabolism, including synthesis, degradation, and pulse–chase analysis of two temporally defined populations. First, the deuterium labeling efficiency was optimized, allowing time-lapse imaging of protein synthesis dynamics within individual live cells with high spatial–temporal resolution. Second, by tracking the methyl group (CH3) distribution attributed to pre-existing proteins, this platform also enables us to map protein degradation inside live cells. Third, using two subsets of structurally and spectroscopically distinct D-AAs, we achieved two-color pulse–chase imaging, as demonstrated by observing aggregate formation of mutant hungtingtin proteins. Finally, going beyond simple cell lines, we demonstrated the imaging ability of protein synthesis in brain tissues, zebrafish, and mice in vivo. Hence, the presented labeling and imaging platform would be a valuable tool to study complex protein metabolism with high sensitivity, resolution, and biocompatibility for a broad spectrum of systems ranging from cells to model animals and possibly to humans.
ISSN:1554-8929
1554-8937
1554-8937
DOI:10.1021/cb500787b