TY - JOUR
T1 - Label-free imaging of the native, living cellular nanoarchitecture using partial-wave spectroscopic microscopy
AU - Almassalha, Luay M.
AU - Bauer, Greta M.
AU - Chandler, John E.
AU - Gladstein, Scott
AU - Cherkezyan, Lusik
AU - Stypula-Cyrus, Yolanda
AU - Weinberg, Samuel
AU - Zhang, Di
AU - Ruhoff, Peder Thusgaard
AU - Roy, Hemant K.
AU - Subramanian, Hariharan
AU - Chandel, Navdeep S.
AU - Szleifer, Igal
AU - Backman, Vadim
N1 - Funding Information:
MDA-MB-231 cells were provided by the O'Halloran Laboratory at Northwestern University Evanston, IL. TEM was performed at the Biological Imaging Facility at Northwestern University. Flow cytometry was performed by the Northwestern University Flow Cytometry Facility, which received support from National Cancer Institute Grant CA060553. This material was based on work supported by a National Science Foundation Graduate Research Fellowship under Grant DGE-082416; National Science Foundation Grants CBET-1249311 and CBET-1240416; National Institutes of Health (NIH) Grants U54CA193419, R01CA200064, R01CA155284, and R01CA165309; NIH T32 Training Grants T32GM008152 and T32HL076139; and Chicago Biomedical Consortium Lever Award L-006 for the Chicago Center for Physical Science Oncology Innovation and Translation.
Publisher Copyright:
© 2016, National Academy of Sciences. All rights reserved.
PY - 2016/10/18
Y1 - 2016/10/18
N2 - The organization of chromatin is a regulator of molecular processes including transcription, replication, and DNA repair. The structures within chromatin that regulate these processes span from the nucleosomal (10-nm) to the chromosomal (>200-nm) levels, with little known about the dynamics of chromatin structure between these scales due to a lack of quantitative imaging technique in live cells. Previous work using partial-wave spectroscopic (PWS) microscopy, a quantitative imaging technique with sensitivity to macromolecular organization between 20 and 200 nm, has shown that transformation of chromatin at these length scales is a fundamental event during carcinogenesis. As the dynamics of chromatin likely play a critical regulatory role in cellular function, it is critical to develop live-cell imaging techniques that can probe the real-time temporal behavior of the chromatin nanoarchitecture. Therefore, we developed a live-cell PWS technique that allows high-throughput, label-free study of the causal relationship between nanoscale organization and molecular function in real time. In this work, we use live-cell PWS to study the change in chromatin structure due to DNA damage and expand on the link between metabolic function and the structure of higher-order chromatin. In particular, we studied the temporal changes to chromatin during UV light exposure, show that live-cell DNA-binding dyes induce damage to chromatin within seconds, and demonstrate a direct link between higher-order chromatin structure and mitochondrial membrane potential. Because biological function is tightly paired with structure, live-cell PWS is a powerful tool to study the nanoscale structure-function relationship in live cells.
AB - The organization of chromatin is a regulator of molecular processes including transcription, replication, and DNA repair. The structures within chromatin that regulate these processes span from the nucleosomal (10-nm) to the chromosomal (>200-nm) levels, with little known about the dynamics of chromatin structure between these scales due to a lack of quantitative imaging technique in live cells. Previous work using partial-wave spectroscopic (PWS) microscopy, a quantitative imaging technique with sensitivity to macromolecular organization between 20 and 200 nm, has shown that transformation of chromatin at these length scales is a fundamental event during carcinogenesis. As the dynamics of chromatin likely play a critical regulatory role in cellular function, it is critical to develop live-cell imaging techniques that can probe the real-time temporal behavior of the chromatin nanoarchitecture. Therefore, we developed a live-cell PWS technique that allows high-throughput, label-free study of the causal relationship between nanoscale organization and molecular function in real time. In this work, we use live-cell PWS to study the change in chromatin structure due to DNA damage and expand on the link between metabolic function and the structure of higher-order chromatin. In particular, we studied the temporal changes to chromatin during UV light exposure, show that live-cell DNA-binding dyes induce damage to chromatin within seconds, and demonstrate a direct link between higher-order chromatin structure and mitochondrial membrane potential. Because biological function is tightly paired with structure, live-cell PWS is a powerful tool to study the nanoscale structure-function relationship in live cells.
KW - Cell dynamics
KW - Chromatin
KW - DNA damage
KW - Microscopy
KW - Mitochondrial metabolism
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U2 - 10.1073/pnas.1608198113
DO - 10.1073/pnas.1608198113
M3 - Article
C2 - 27702891
AN - SCOPUS:84991730814
VL - 113
SP - E6372-E6381
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 42
ER -