Strategy for correlating and integrating structures over scales. Multi-resolution workflow
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The overall outreaching goal of my research is structure-function integration in space and time, aiming at formulating molecular models of key migration organelles. My primary biological interest is deciphering the molecular details underlying the assembly and regulation of the actin cytoskeleton at the leading edge of motile cells. High-resolution structure analysis of these cytoskelatal assemblies is highly challenging due to the large number and size of components, diversity of assembly types and variety of adhesion-mediated functions. Furthermore, the inherent transient nature of some assemblies, which can form and mature over a long time periods and then can also disassemble, produces a continuum of states that results in heterogeneity of functional states and structural conformations. When this heterogeneity is combined with an incomplete characterization of what cellular outputs are generated by which adhesions, the result is incomplete and leads to potentially misleading structure-function relationships.
Our primary approach in structural determination of these dynamic, large multiprotein complexes is Electron cryomicroscopy (cryo-EM). Cryo-EM has grown to be a powerful technique applicable to almost any kind of specimen, is parsimonious in its material requirements, and allows imaging the specimen close to their physiological environment. My group studies the structures of these large actin cytoskeleton assemblies by developing and combining various electron cryo-microscopies, image analysis and bioinformatics techniques using a two-pronged approach. The first involves characterization at high resolution (1-3nm) of reconstituted assemblies in vitro. The combination of the three-dimensional reconstructions of multiprotein machines that we derive by combining electron cryo-microscopy with atomic resolution structures of their components allows us to extract high-resolution structural information of these large dynamic assemblies in their fully hydrated state. The resulting 3D molecular models serve as a library of motifs for our next level of complexity, observing these assemblies in situ in eukaruotic cells.
Although high-resolution structural approaches provide critical information about individual molecules and complexes, a barrier to progress remains their structural and functional integration at the cellular level. Towards this end, we develop and apply techniques and protocols that allow us to image whole cells, in their fully hydrated state, and to use bioinformatics tools, to correlate between the high-resolution structural information motives with the in situ characterization obtained from living cells (correlative light and electron cryo-tomography). Some of the areas I am vested in exploring include the use of force transmission from the substrate to cells and to develop a model for mechanotransduction at matrix adhesions that integrates adhesion ultrastructure, biochemical interactions, temporal and spatial dynamics of multiprotein assemblies and signaling networks.