HVEM TOMOGRAPHY OF THE TRANS-GOLGI NETWORK - STRUCTURAL INSIGHTS AND IDENTIFICATION OF A LACE-LIKE VESICLE COAT

High voltage electron microscopy and computer axial tomography have be en used to study the 3-D structure of trans-Golgi cisternae and trans- Golgi networks (TGNs) in NRK cells. Both structures were specifically labeled by photoconversion of a fluorescent analogue of ceramide using a modification of the technique of Pagano et al. (J. Cell Biol. 1991. 113: 1267-1279). Regions of the Golgi ribbon in fixed, stained cells were cut in 250-nm sections and analyzed by tilt series microscopy aci d subsequent tomographic reconstruction. Resolution of the reconstruct ions ranged from 6 to 10 nm. The size and structure of the TGN varied considerably throughout the Golgi ribbon; all reconstructions were mad e from regions with pronounced TGN. Most regions analyzed contained mu ltiple (2-4) Golgi cisternae that stain with ceramide. These ''peel of f'' from the closely stacked cisternae and are continuous at their end s with tubules that contribute to the TGN. Most vesicular profiles vis ualized in the TGN are connected to TGN tubules. The budding of vesicl es appears to occur synchronously along the length of a TGN tubule. Tw o distinct coats were visualized on budding vesicles: clathrin cages a nd a novel, lace-like structure. Individual TGN tubules produce vesicl es of only one coat type. These observations lead to the following pre dictions: (a) sorting of molecules must occur prior to the formation o f TGN tubules; (b) vesicle formation takes place almost synchronously along a given TGN tubule; and (c) lace-like coats form on exocytic ves icles.