The complex of DNA, protein, and RNA known as chromatin is the substrate for essential cellular processes such as gene transcription, regulation, replication, and repair. Unravelling its structure and dynamics is therefore essential f we are to understand the mechanics of these processes and their effects in development and disease. Chromatin is, however, a difficult target to study: it is found in a crowded environment within the nucleus, is structurally organized on multiple length scales, is variable within and between nuclei, and is highly dynamic. Capturing this information requires instrumentation which can (i) measure on multiple length scales, from the whole cell down to tens of nm, (ii) follow chromatin dynamics in living cells, and (iii) acquire and quantify thousands of images in a manageable time frame to overcome the intrinsic variability and provide a statistical description of chromatin behavior. Such an instrument does not yet exist, with existing instrumentation being limited in resolution, dynamic speed, and throughput. We propose an innovative multi-disciplinary approach that combines developments in optics, data processing and modeling to realize an integrated system for automated high-throughput super- resolution imaging and dense single-molecule tracking in the cell nucleus.
Our Specific Aims are: 1) Develop an automated multicolor 3D single-molecule switching (SMS) nanoscope for dynamic imaging and particle-tracking in the nucleus, 2) Develop data processing tools for high-throughput 3D- SMS nanoscopy of 100-1,000 cells/h, 3) Develop chromatin modeling tools that take advantage of the unprecedented level of detail and statistical depth of the 4D data provided by high-throughput particle- tracking and SMS nanoscopy, and 4) test the performance and refine our technical developments by applying them to a diverse set of representative and important questions in the field of chromatin architecture, including the mobility and dynamics of transcription factors and nucleosomes, and the processes of synaptonemal assembly and telomere recombination. The proposal represents a fundamental departure from the traditional view of the nanoscopy image generation procedure as a hands-on process heavily involving an expert user to an automated, high- throughput method with focus on quantification and efficiency. By making nanoscopy studies of tens of thousands of cells feasible, we anticipate that our instrument will enable, for the first time, the spatiotemporal dynamics of the nucleome to be quantitatively investigated down to the single nucleosome level.
Unraveling how chromatin, the complex of DNA, proteins, and RNA which is responsible for organizing the genetic information within a cell, is structured and functions is relevant to public health if we are to understand diseases, such as cancer and developmental disorders. In this project we will develop a new microscope platform which will permit the nanoscale analysis of chromatin in a much more systematic and large-scale manner than possible so far. Thus, this research is relevant to the part of NIH's mission that pertains to develop fundamental knowledge that will help to enhance health.
