This video shows the 3D visualization of a Golgi apparatus from the green alga Chlamydomonas reinhardtii. The unicellular alga was flash-frozen without any artificial stains or fixatives. Prior to imaging with cryo-electron tomography (cryo-ET), a thin cryo-lamella sample was prepared from the vitrified cell by cryo-focused ion beam milling (cryo-FIB). The Golgi apparatus is an organelle located near the nucleus, serving as the major sorting center for the cell. It is made up of stacks of membranous sacs called cisternae, which are not connected to each other. The complex network of membranes and associated vesicles is known as the trans-Golgi network (TGN). Within the Golgi, proteins are modified, sorted and packaged; they undergo posttranslational modification through the process of glycosylation. The TGN consists of a cis face at which the proteins enter and a trans face from which the proteins exit. Upon exiting the Golgi apparatus, proteins are usually sent to either lysosomes, secretory granules or the plasma membrane, depending on their encoded signal. Vesicular transport within the Golgi complex is mediated by the coatomer protein I (COPI) complex. The COPI protein coat mediates trafficking of proteins from the cis end of the Golgi complex back to the rough endoplasmic reticulum and between Golgi cisternae.
For the first time, cryo-ET has made it possible to determine the in situ structure of the COPI coat within vitrified Chlamydomonas reinhardtii cells. While the in situ structure supports the existing in vitro structural model (based on purified, reconstituted coatomer components), it also revealed bound cargo. Tomographic analysis of the native Golgi not only showed that the coat machinery remains constant during vesicle trafficking, but that vesicles change morphology and cargo content during their progression from the cis to the trans face. These findings were made possible by applying and combining several different methods within one workflow: cryo-FIB, cryo-ET and subtomogram averaging. Sample thinning by cryo-FIB is key for such an approach since it allows the production of sufficiently thin samples for cryo-ET in the first place. The Thermo Scientific Aquilos Cryo-FIB is a dedicated cryo-DualBeam microscope that facilitates the preparation of thin, electron-transparent lamellae from a target region within a vitrified cell.
Data segmentation and visualization by Thermo Scientific Amira Software. Data courtesy of Dr. Benjamin Engel, Department of Molecular Structural Biology, Max Planck Institute for Biochemistry, Martinsried, Germany.
