Our efforts focus on developing imaging technology platform that quantitatively link macroscopic cellular outputs to molecular-resolution structural changes in the crowded cellular environment. Our central biological interest is the structures of actin cytoskeleton macromolecular assemblies; the girders and cables that control the shapes and movements of all living cells. The anchoring sites of the girders, between the cell and the extracellular matrix or other cells, are mechano-sensitive multi-protein assemblies that transmit force across the cell membrane and regulate biochemical signals in response to changes in the mechanical environment. The combined functions in force transduction, signaling and mechanosensing are crucial for cell and tissue behaviors in development, homeostasis and disease.
The heterogeneity and dynamic nature of the multi-component complexes responsible for the transmission of force between the actin cytoskeleton and integrin receptors in migrating and mechano-active cells have hindered structural studies aimed at unrevealing the underlying the molecular mechanisms of their assembly and disassembly. A large body of work carried out over the last two decades has identified key components that directly link the actomyosin system to the extracellular domain (via integrin). Here I will describe how a hybrid approach that involves a combination of correlative light and electron microscopy, cellular cryo-electron tomography, image analysis, computational docking, and biophysical tools allows us to “ “directly view” the 3D the molecular architecture of these nanomachinery in situ.
Recently, we acquired cellular cryo tomograms of whole mammalian cells using intermediate voltage TEMs equipped with various imaging devices / energy filter configurations and employed drift movie correction in conjunction with feature extraction to extend these studies and view single membrane receptors at the cell membrane extracellular matrix interface. here I will describe these results.
These studies are supported by the National Institute of General Sciences (NIGMS) Grant Number P01 GM098412 (DH and NV).