High-Resolution Imaging of Human Cancer Proteins Using Microprocessor Materials.

Mutations in tumor suppressor genes, such as Tumor Protein 53 (TP53), are heavily implicated in aggressive cancers giving rise to gain- and loss-of-function phenotypes. While individual domains of the p53 protein have been studied extensively, structural information for full-length p53 remains incomplete. Functionalized microprocessor chips (microchips) with properties amenable to electron microscopy permitted us to visualize complete p53 assemblies for the first time. The new structures revealed p53 in an inactive dimeric state independent of DNA binding. Residues located at the protein-protein interface corresponded with modification sites in cancer-related hot spots. Changes in these regions may amplify the toxic effects of clinical mutations. Taken together, these results contribute advances in technology and imaging approaches to decode native protein models in different states of activation.

Cell-matrix tension contributes to hypoxia in astrocyte-seeded viscoelastic hydrogels composed of collagen and hyaluronan.

Astrocyte activation is crucial for wound contraction and glial scar formation following central nervous system injury, but the mechanism by which activation leads to astrocyte contractility and matrix reorganization in the central nervous system (CNS) is unknown. Current means to measure cell traction forces within three-dimensional scaffolds are limited to analyzing individual or small groups of cells, within extracellular matrices, whereas gap junctions and other cell-cell adhesions connect astrocytes to form a functional syncytium within the glial scar. Here, we measure the viscoelastic properties of cell-seeded hydrogels to yield insight into the collective contractility of astrocytes as they exert tension on the surrounding matrix and change its bulk mechanical properties. Our results indicate that incorporation of the CNS matrix component hyaluronan into a collagen hydrogel increases expression of the intermediate filament protein GFAP and results in a higher shear storage modulus of the cell/matrix composite, establishing the correlation between astrocyte activation and increased cell contractility. The effects of thrombin and blebbistatin, known mediators of actomyosin-mediated contraction, verify that cell-matrix tension dictates the hydrogel mechanical properties. Viability assays indicate that increased cell traction exacerbates cell death at the center of the scaffold, and message level analysis reveals that cells in the hyaluronan-containing matrix have a ~ 3-fold increase in HIF-1α gene expression. Overall, these findings suggest that astrocyte activation not only increases cell traction, but may also contribute to hypoxia near sites of central nervous system injury.