Characterization of Local Conformational Structure and Mechanical Properties of Chromatin using Fluorescence Lifetime Imaging Microscopy and Phasor Analysis
Chromatin is a complex of macromolecules that plays a very important role in packaging the long strand of DNA inside the eukaryotic nucleus. Chromatin enables and regulates many DNA functions and interactions with other molecules including DNA transcription, replication, and repair. During the cell division, chromatin ensures proper division and transfer of the genetic material to daughter cells. To fulfill these roles, chromatin must constantly undergo reconfiguration and reorganization of its structure. Therefore, local conformational structure and mechanical properties of chromatin play a crucial role in creating the highly dynamic structure of chromatin. Furthermore, recent studies link mechanobiology of the nucleus, specifically nuclear stiffness and deformability, to cancer metastasis. Due to the fact that chromatin structure is highly compact and dynamic, visualizing and studying its structure, remodeling and mechanical properties is very difficult. In this research, we showed that Fluorescence Lifetime Imaging Microscopy (FLIM) and phasor analysis can be used as a powerful technique to study local conformational structure and mechanical properties of chromatin during mitotic cell division and in metastatic tumor cells. More specifically, the findings from the lifetime values revealed information about the size, stiffness, deformability, and accessibility of the minor grooves in chromatin. The findings from this study confirmed significant structural differences between metaphase and interphase chromatin, but they also revealed significant variations within chromatin at any stage. We postulated that these variations are largely sequence dependent. Two types of regions with distinct conformational and mechanical properties were identified in chromatin. One of them had a significantly longer lifetime which was indicative of more rigid and larger binding area. These regions were attributed to GC-rich minor grooves. The other regions with significantly shorter lifetime, indicating softer and smaller binding sites, were associated with AT-rich minor grooves. Moreover, the lifetime of metaphase and interphase chromatin differed more significantly in GC-associated grooves. Therefore, we concluded that there were far more AT-rich minor grooves accessible for binding in both metaphase and interphase chromatin to the extent that the effect of chromatin condensation during metaphase did not cause very significant differences in the lifetime values. Finally, the fluorescence lifetime analysis revealed that chromatin in highly aggressive metastatic tumors like MB231 and MFC7 was significantly softer and more deformable than non-tumorigenic cells like MCF10A. There was a correlation between softness and deformability of chromatin and the metastatic aggressiveness of the tumors.