Opening Windows into the Cell: Focused Ion Beam Micromachining of Eukaryotic Cells for Cryo-Electron Tomography

Abstract

Cryo-electron tomography (cryo-ET) provides unprecedented insights into the 3-D macromolecular organization of cells in their native state. However, the thickness of most cells makes them inaccessible to cryo-ET. Focused ion beam (FIB) milling can be used to prepare 200-500 nm lamellae from intact cells, opening large windows into the cell's interior, exposing their landscapes at molecular resolution. Advanced computational analysis makes the identification of macromolecular complexes possible, yielding visual proteomes of cells. Cryo-ET and FIB milling were used to study the structural dynamics of the nuclear pore complex, one of the largest macromolecular machines in the cell. It is composed of hundreds of proteins, selectively controlling all traffic between the nucleus and the cytoplasm. The architecture of the NPC is central to understanding nuclear transport. However, due to its sheer size, its local environment and its dynamic nature, determining its structure at molecular resolution remains a challenge for conventional techniques. Combining FIB milling, cryo-ET, and image processing enables the study of the NPC in its native environment, free of the distortions caused by purification. This approach has not only revealed the NPC architecture at unparalleled resolution, but also captured different conformational states in action. Other uses of cryo-FIB/ET to study diverse cellular environments at molecular detail will be presented, including actin networks, the architecture of cell division, and the distribution of macromolecular complexes within organelles such as mitochondria.