Electron tomographic analysis of Golgi trafficking in the scale-forming alga Scherffelia dubia

The Golgi of Scherffelia dubia has become a model for the study of intra-Golgi transport by cisternal maturation. Cisternal maturation is one of the many models that may explain the mechanism of intra-Golgi trafficking. This alga produces scales in its Golgi stacks that are only seen inside the Golgi cisternae and not in the surrounding vesicles. This observation is a cornerstone of the cisternal maturation hypothesis. To further test the structural predictions of this hypothesis, I have produced 3-D Golgi reconstructions of high-pressure frozen and freeze-substituted cells at ∼6 nm resolution using electron tomography. I have exploited the large stack size (5 cis-, 7-8 medial-, 7-8 trans-cisternae) and scales as a cargo marker to characterize the structural basis of this trafficking mode by electron tomography. This analysis has allowed a detailed characterization and quantification of the structures involved in intra-Golgi trafficking including: cis-cisterna assembly, vesicle budding from Golgi cisternae, and transformation of the trans-most cisterna into a compartment of the contractile vacuole.; The two large Golgi stacks in S. dubia are always found directly adjacent to transitional ER sites. Each transitional ER domain displays an average of nine ∼70 COPII-type budding profiles. ∼30% of the resulting vesicles form tethered clusters of 3--5 vesicles that, after attachment to the C2 cisterna, appear to fuse to create cisternal initials that grow into a new cisterna. Putative docking/fusion complexes appear to deform vesicles into a cone shape before they fuse with cisternal membranes. Cis- medial- and trans-types of Golgi cisternae can be distinguished based on (1) their position in the stack, (2) their luminal staining, (3) the degree of staining of the flagellar scales, and (4) the sub-types of COPI vesicles they bud. The cis-cisternae appear devoid of stained contents except for barely discernable immature scales.; The cis-to-medial Golgi cisternal transition is marked by the abrupt appearance of darkly stained luminal contents and marked changes in size and staining of the surface domains of the scales. Cis-cisternae give rise to COPIa-type vesicles, which differ from COPIb-type vesicles that originate from medial- and trans-cisternae by having a different coat and differently stained contents. Exposure of S. dubia cells to brefeldin A for 10 min results in a total loss of cis and a partial loss of medial- and trans-Golgi cisternae, an increase in COPII vesicles, a major loss of COPIa and COPIb vesicles, and a massive swelling of the contractile vacuole system.; In S. dubia, the trans-most Golgi cisterna is transformed into a contractile vacuole element that fuses with the contractile vacuole system. Proteoglycan scales are tethered to the contractile vacuole membrane and slide out to the cell surface after the contractile vacuole fuses with the plasma membrane at the flagellar groove. I have take the structural findings made in cisternal maturation model system, Scherffelia dubia , and compared them to the green alga Chlamydomonas reinhardtii and the land plant Dionea muscipula (Venus Flyrap) and found that they exhibit the same features. This suggests that they too traffic by cisternal maturation.