Center Overview: Challenges and Approaches

Recent advances in high-throughput DNA sequencing have led to an explosion of the available genomic data from model organisms. Genomes of entire organisms can now be fully sequenced in a matter of weeks. There are well over one million gene entries in the NCBI database, encoding comparable numbers of proteins and their homologs and variants. These data provide us with a blueprint – describing a cell’s potential. What cells do however, is conferred, not by a static picture or blueprint, but by the dynamic interactions of the players defined by that blueprint. Fundamentally, this is the essence of biology that gives rise to the emergent properties of life. Current technologies are not suitable to reveal dynamic interactions between macromolecules at sufficient scale, with sufficient reliability or with sufficient sensitivity to keep pace with the genomic revolution brought about by sequencing technologies. At current pace, it will take us thousands of years to annotate the human proteome with respect to function and dynamic interactions. Thus, there is a desparate need for technologies that can reveal the cellular interactome, not simply to annotate function, or reveal possible (high affinity) interactions, but to illuminate the actual dynamic interplay of the macromolecular characters in cells that define life. We therefore propose innovative and dramatically new approaches to the isolation, detection and analysis of macromolecular complexes that will enable scientists to realize the full potential of the revolution brought about by genomics, interdisciplinary research, and proteomics technologies.

The approach is summarized in the figure above and can be broken into three stages:

1. PRODUCTION of intact macromolecular complexes. We begin by visualizing the tags in vivo allowing us to follow their dynamic behavior and synchronize cell growth. We are developing a series of protocols and devices that harvest cells at the precise moment the tagged complex is localized (e.g., in a particular stage of the cell cycle, subcellular transport, regulated expression, chromatin silencing). These approaches include:rapid isolation,immobilization with crosslinkers, and isolation of large assemblages.

2. ANALYSIS of the composition and dynamics of each complex. We are developing proteomic and genomic analytical techniques to rapidly and comprehensively identify macromolecular complex components (proteins, DNA, and RNA), their amounts, their modifications, how the components interact with each other in the complex, and the time-dependent and dynamic alterations of the complex under defined growth conditions.

3. INTEGRATION of data into a holistic view of the complex in the cell. A computational system for enumerating structures and trajectories of macromolecular complexes is being built, integrating all our data into dynamic structural pictures of macromolecular complexes, in the context of their neighboring complexes and the living cell.