Project III
3-D Structure of the Golgi Complex
3-D, high-resolution (5nm) structure of the Golgi complex is being solved in order to accurately integrate the structure and function of this organelle. In order to record common and distinct features of the Golgi, various cell types are studied: NRK cells, _-cells within isolated mouse islets of Langerhans and mammary epithelial cells within secretory acini. Optimal preservation of the Golgi for this analysis is carried out by high pressure freezing/freeze-substitution of the cells/tissues. Models are prepared from electron tomographic data, generated by imaging 400 nm thick sections of plastic-embedded samples in the EM at 750 keV and collecting a tilt series at 1.5° intervals (±60°) about two orthogonal axes. Complementing this data, immuno-labeling is carried out on samples prepared in parallel. Taken together our studies have yielded several important observations about structures at the trans-side of the Golgi stack. Trans-Golgi cisternae can be differentiated from the preceeding medial- and cis-cisternae using three morphological and functional criteria. First, all trans-cisternae are similar in that they present numerous budding vesicles and tubules that extend into the region trans- of the Golgi stack, indicating that they are involved in exit processes. Second, a modified form of ER associates directly with all trans-cisternae in a manner analogous to the association of the Golgi cisternae themselves and is characterized by the presence of ribosomes only on the face non-adherent to cisternae. Third, at least two different types of trans-cisternae can be distinguished from one another by the observation that the trans-most cisterna in a stack produces exclusively clathrin-coated vesicles, while precerding trans-cisternae produce only non-clathrin-coated buds and tubules. Our working hypothesis developed from these studies is that cargo exits the Golgi directly from three distinct trans-cisternae, and that specialized ER plays a significant role in trans-Golgi function.
This project is carried out in collaboration with colleagues at the Laboratory for 3D Fine Structure on the Boulder campus.