Spagnol Stephen T, Dahl Kris Noel
Department of Chemical Engineering, Carnegie Mellon University, 5000 Forbes Ave., Pittsburgh, Pennsylvania, 15213, United States of America.
Department of Biomedical Engineering, Carnegie Mellon University, 5000 Forbes Ave., Pittsburgh, Pennsylvania, 15213, United States of America.
PLoS One. 2016 Jan 14;11(1):e0146244. doi: 10.1371/journal.pone.0146244. eCollection 2016.
The linear sequence of DNA encodes access to the complete set of proteins that carry out cellular functions. Yet, much of the functionality appropriate for each cell is nested within layers of dynamic regulation and organization, including a hierarchy of chromatin structural states and spatial arrangement within the nucleus. There remain limitations in our understanding of gene expression within the context of nuclear organization from an inability to characterize hierarchical chromatin organization in situ. Here we demonstrate the use of fluorescence lifetime imaging microscopy (FLIM) to quantify and spatially resolve chromatin condensation state using cell-permeable, DNA-binding dyes (Hoechst 33342 and PicoGreen). Through in vitro and in situ experiments we demonstrate the sensitivity of fluorescence lifetime to condensation state through the mechanical effects that accompany the structural changes and are reflected through altered viscosity. The establishment of FLIM for resolving and quantifying chromatin condensation state opens the door for single-measurement mechanical studies of the nucleus and for characterizing the role of genome structure and organization in nuclear processes that accompany physiological and pathological changes.
DNA的线性序列编码了对执行细胞功能的全套蛋白质的访问权限。然而，适合每个细胞的许多功能都嵌套在动态调控和组织的层次结构中，包括染色质结构状态的层次结构和细胞核内的空间排列。由于无法原位表征分层染色质组织，我们对核组织背景下基因表达的理解仍然存在局限性。在这里，我们展示了使用荧光寿命成像显微镜（FLIM），通过细胞可渗透的DNA结合染料（Hoechst 33342和PicoGreen）来量化和空间分辨染色质凝聚状态。通过体外和原位实验，我们证明了荧光寿命对凝聚状态的敏感性，这种敏感性通过结构变化伴随的机械效应体现，并通过粘度改变反映出来。用于解析和量化染色质凝聚状态的FLIM的建立，为细胞核的单测量力学研究以及表征基因组结构和组织在伴随生理和病理变化的核过程中的作用打开了大门。
