Milieu-induced, selective aggregation of regulated secretory proteins in the trans-Golgi network

Control and hormone-treated (48 h) GH4C1 cells grown on polylysinecoated coverslips were subjected to indirect immunofluorescence using published protocols (Ross et al., 1989) with minor modifications. Both the antiserum against rat SgII (Ross et al ., 1985) and the rhodamine-conjugated sheep anti-rabbit IgG antibodies (Cappel Laboratories, Cockranville, PA) were used at a 1 :200 dilution . Control and hormone-treated cells were photographed at the same exposure and negatives were printed under identical conditions . EM Based on previous work CTooze et al., 1991) , a fraction enriched in immature secretary granules was prepared from PC12 cells as follows . A postnuclear supernatant was prepared from PC12 cells as described (Tboze and Huttner, 1990 ) except that the cells were homogenized in 0.25 M sucrose containing 1 mM EDTA and 1 mM Tris-HCI, pH 7.4 . The postnuclear supernatant (1-ml aliquots) was subjected to differential centrifugation at 4°C . First, most (>95%) of TGN membranes, mitochondria, and mature secretory granules were pelleted by centrifugation for 35 min at 10,000 rpm . This and all subsequent centrifugations were carried out using polycarbonate tubes and a TLS-55 rotor in a Beckman TT-100 ultracentrifuge with the brake off. The supernatants were subjected to a second centrifugation for 22 min at 25,000 rpm to sediment immature secretary granules. The pellets were resuspended in 50#1 of 0.25 M sucrose and mixed with either 50 pl of 0.25 M sucrose (control) or with 50 Al of 0.25 M sucrose containing 1 mg/ml saponin and two times concentrated nonaggregative or aggregative milieu. After 5 min of incubation at 0°C, samples were either mixed with paraformaldehyde (1% final concentration, added from a 16% stock), fixed for 20 min at 0°C and centrifuged for 30 min at 55,000 rpm, or centrifuged without prior fixation. All pellets were then overlaid with 8 % paraformaldehyde and kept overnight at 4°C. Samples were then washed in 0.1 M sodium cacodylate buffer, pH 7.2, and postfixed in cacodylate buffer containing 1% OsO4 and 1 .5 % magnesium ferrocyanide . After washes in cacodylate buffer and H2O, pellets were incubated for 30 min in 1.5 % magnesium uranyl acetate in water. They were then dehydrated in ethanol, incubated in propylene oxide, and embedded in Epon. Thin sections were contrasted with lead citrate and examined in a Philips 400 microscope. Pictures were taken at random from the bottom of the pellets and dense cores were counted in 10 fields representing a total area of 150 pmt .