Versatile nanogold tagging system applied to the identification of heterochromatin through cryo-FIB-ET.

Abstract

Cryo-Focused Ion Beam-Electron Tomography (cryo-FIB-ET) is swiftly becoming a powerful tool for investigating structure-function relationships in native environments, enabling the discovery of new biology. However, features which can be identified are those which are sufficiently large that they can be unambiguously identified visually (whole organelles, ribosomes, the nuclear pore complex, proteasomes), or through the matching of high resolution signatures (2D template matching) of sufficient molecular weight. The need for additional tagging methods is paramount, in order to identify macromolecules and expand our scientific inquiries. We have developed a delivery and tagging system utilizing a gold nanoparticle that specifically targets heterochromatin to be visualized by cryo-FIB-ET and resolved on a nucleosome basis by subtomogram analysis. There has been enormous progress in determining chromatin chain topology in vitro and in situ through the use of electron microscopy on a variety of conditions: frozen-hydrated samples, resin embedded samples, and cryo-conditions. As such, a useful tool for researchers would be the ability to distinguish between heterochromatin and euchromatin in native contexts, such as areas of active vs inactive gene expression. In general, the labeling, delivery, and identification strategy presented here would facilitate future progress for distinguishing and identifying various macromolecules endogenously, with minimal perturbations, imaged within the cell at high spatial resolution.