RT Journal Article
SR Electronic
T1 Live-Cell Imaging and Quantification of PolyQ Aggregates by Stimulated Raman Scattering of Selective Deuterium Labeling
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 820217
DO 10.1101/820217
A1 Kun Miao
A1 Lu Wei
YR 2019
UL http://biorxiv.org/content/early/2019/11/08/820217.abstract
AB Huntington’s disease, a major neurodegenerative disorder, involves deposition of aggregation-prone proteins with long polyglutamine (polyQ) expansions. The ability to non-perturbatively visualize the formation of aggregates could offer new molecular insight for their pathologic roles. Here, we propose stimulated Raman scattering imaging of deuterium-labeled glutamine to investigate native polyQ aggregates in live cells with subcellular resolution. Through the enrichment of deuterated glutamine in the polyQ sequence of mutant Huntingtin (mHtt) proteins, we first achieved sensitive and specific SRS imaging of carbon-deuterium bonds (C-D) from aggregates without GFP labeling. These aggregates become 1.8-fold denser compared to those with GFP. Second, we performed ratiometric quantification, which revealed a dependence of protein compositions on aggregation sizes. Moreover, we calculated the absolute concentrations for sequestered mHtt and non-mHtt proteins within the same aggregates. Our method may readily reveal new features of polyQ aggregates and could be suited for in vivo investigations on multicellular organisms.