We are examining the three-dimensional (3-D) fine structure of the Golgi complex in rapidly-frozen pancreatic beta cells to provide new data on the sites of formation and sorting for secretory granules, constitutive secretory vesicles, and vesicles destined for the endo-lysosomal pathway. Insulinoma cell lines (HIT-T15 and MIN-6) and isolated rodent islets of Langerhans are being fixed by high-pressure freezing and freeze-substitution. 250-400 nm thick sections of plastic-embedded samples are imaged in a high-voltage electron microscope (HVEM) and serially tilted at 1.5x intervals (q60x) about two orthogonal axes. These digital images are used to generate high resolution (5-7 nm) tomographic reconstructions which can then be analyzed and modeled in 3-D. These reconstructions will be used to examine the relationship between trans-cisternae and the TGN, to resolve novel coat structures on nascent vesicles and granules, and to address hypotheses relating to cisternal progression-maturation and protein trafficking via membranous tubules. Our preliminary data show that small dense-core granules (average diameter 95 nm) form from cis-, medial- and trans-cisternae, not just from the trans-most cisterna or TGN. This suggests that secretory product condenses at many, if not all, levels of the Golgi complex. These dense- core granules appear to be connected to Golgi cisternae and to each other by tubular projections of uniform diameter and density, arranged like """"""""beads on a string"""""""". The result is a continuum of developing granules, connected with Golgi cisternae at one end and projecting away from the Golgi stack at the other. C3
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