Develop novel single-molecule imaging tools to study chromatin modifications in cellular senescence

2013 new Scholar Award in aging

The Cang lab is interested in developing novel biophotonics tools to understand chromatin modifications in cellular senescence. The human genome is organized into chromatin, a complex of DNA and associated proteins, to fit inside a micrometer-sized nucleus. The structure of chromatin determines the accessibility of various proteins to genes and therefore plays an important role in genomic regulation. Aging occurs at various levels in animals. At the cellular level, aging is manifested as the lost of ability to divide (proliferative arrest). This phenomenon is termed as cellular senescence, which is associated with chromatin modifications. The most striking chromatin modification is the formation of heterochromatic foci, termed as senescence associated heterochromatic foci (SAHF), often induced by the activation of oncogenes such as Ras. The DNA is densely packed in these foci, and the genes that reside inside are silenced. The molecular mechanisms underpinning SAHF remain elusive. Due to the lack of a powerful tool to directly visualize SAHF with sufficient resolution, even their structures remain unknown. Under a normal fluorescence microscope, these heterochromatic foci appear as featureless bright dots. We propose to develop a novel microscopy with super-resolution capability to “zoom into” these dots. This microcopy is a marriage of two recently developed imaging techniques, namely, light-sheet microscopy and single-molecule Bayesian microscopy. At the Salk Institute, we have developed a prototype system that can image chromatin with ~30nm level resolution, well beyond the resolution of conventional microscopes. Our preliminary studies have revealed nanometer-scale fine features within heterochromatic foci for the first time. The prototype system offers only a two-dimensional super-resolution capability. In this proposal, we will further develop this microscopy to achieve 3D, multicolor, and live-cell, super-resolution imaging capabilities, so as to record a 3D time-lapsed movie of the formation of SAHF during the process of cellular senescence. This task is impossible to achieve with any existing techniques. This study will help understand the mechanism of chromatin alternation in cellular senescence. Furthermore, the imaging tool that we develop in this study will complement existing genetics and biochemistry methods to study epigenetics in aging.

Hu Cang Ph.D.
Salk Institute for Biological Studies