Fluorescently Guided Optical Photothermal Infrared Microspectroscopy for Protein-Specific Bioimaging at Subcellular Level.

Infrared spectroscopic imaging is widely used for the visualization of biomolecule structures, and techniques such as optical photothermal infrared (OPTIR) microspectroscopy can achieve <500 nm spatial resolution. However, these approaches lack specificity for particular cell types and cell components and thus cannot be used as a stand-alone technique to assess their properties. Here, we have developed a novel tool, fluorescently guided optical photothermal infrared microspectroscopy, that simultaneously exploits epifluorescence imaging and OPTIR to perform fluorescently guided IR spectroscopic analysis. This novel approach exceeds the diffraction limit of infrared microscopy and allows structural analysis of specific proteins directly in tissue and single cells. Experiments described herein used epifluorescence to rapidly locate amyloid proteins in tissues or neuronal cultures, thus guiding OPTIR measurements to assess amyloid structures at the subcellular level. We believe that this new approach will be a valuable addition to infrared spectroscopy providing cellular specificity of measurements in complex systems for studies of structurally altered protein aggregates.

The vinculin C-terminal hairpin mediates F-actin bundle formation, focal adhesion, and cell mechanical properties.

Vinculin is an essential and highly conserved cell adhesion protein, found at both focal adhesions and adherens junctions, where it couples integrins or cadherins to the actin cytoskeleton. Vinculin is involved in controlling cell shape, motility, and cell survival, and has more recently been shown to play a role in force transduction. The tail domain of vinculin (Vt) contains determinants necessary for binding and bundling of actin filaments. Actin binding to Vt has been proposed to induce formation of a Vt dimer that is necessary for cross-linking actin filaments. Results from this study provide additional support for actin-induced Vt self-association. Moreover, the actin-induced Vt dimer appears distinct from the dimer formed in the absence of actin. To better characterize the role of the Vt strap and carboxyl terminus (CT) in actin binding, Vt self-association, and actin bundling, we employed smaller amino-terminal (NT) and CT deletions that do not perturb the structural integrity of Vt. Although both NT and CT deletions retain actin binding, removal of the CT hairpin (1061-1066) selectively impairs actin bundling in vitro. Moreover, expression of vinculin lacking the CT hairpin in vinculin knock-out murine embryonic fibroblasts affects the number of focal adhesions formed, cell spreading as well as cellular stiffening in response to mechanical force.