Gi regulation of secretory vesicle swelling examined by atomic force microscopy

Abstract

In the last decade, several monomeric and heterotrimeric guanine nucleotide binding proteins have been identified to associate with secretory vesicles and to be implicated in exocytosis. Vesicle volume also has been proposed to play a regulatory role in secretory vesicle fusion at the plasma membrane. However, the molecular mechanism of function of the guanine nucleotide binding proteins and of the regulation of secretory vesicle volume in the exocytotic process remains unclear. In this study, we report association of the secretory vesicle membrane with the alpha subunit of a heterotrimeric GTP binding protein G(alpha i3) and implicate its involvement in vesicle swelling. Using an atomic force microscope in combination with confocal microscopy, we were able to study the dynamics of isolated zymogen granules, the secretory vesicles in exocrine pancreas. Exposure of zymogen granules to GTP resulted in a 15-25% increase in vesicle height as measured by the atomic force microscope and a similar increase in vesicle diameter as determined by confocal microscopy. Mas7, an active mastoparan analog known to stimulate Gi proteins, was found to stimulate the GTPase activity of isolated zymogen granules and cause swelling. Increase in vesicle size in the presence of GTP, NaF, and Mas7 were irreversible and KCl-sensitive. Ca2+ had no effect on zymogen granule size. Taken together, the results indicate that G(alpha i3) protein localized in the secretory vesicle membrane mediates vesicle swelling, a potentially important prerequisite for vesicle fusion at the cell plasma membrane.

Figure 1

Isolated ZGs ranging in size from 0.2 to 1.2 μm obtained from rat pancreas as seen by electron and AFM. (a) An electron micrograph of the electron-dense ZGs. Note the purity of the ZG preparation. (Bar = 1 μm.) (b) A three-dimensional AFM image of isolated ZGs adhering to a Cell-Tak-coated mica sheet. Notice the size heterogeneity in the ZG population.

Figure 2

Immunolocalization and biochemical detection of a G_i protein with ZGs from rat exocrine pancreas. (a and b) Immunoblot analysis. Identification of G_αi3-immunoreactive antigen associated with ZGM. (a) Ten micrograms each of TH and ZGM was resolved using a single dimension 12.5% SDS gel electrophoresis before electrotransfer on to nitrocellulose and immunoblotting. A 44-kDa immunoreactive band was detected in both fractions but enriched in the ZGM. (b) Twenty five micrograms of ZGM protein was resolved on two-dimensional 16-BAC gel electrophoresis before electrotransfer and immunoblotting using the G_αi3-specific antibody. Note a single spot at ≈44 kDa, suggesting that only one G_αi3 isoform is present in the ZGM. (c) Detection of G_i-specific GTPase activity associated with ZG. GTPase activity in 25 μg of ZG incubated at 30°C for 5 min in the presence of varying concentrations of either Mas7 or Mas17 demonstrates a dose-dependent increase in activity in the presence of Mas7, which is absent in the presence of Mas17. Values are mean ± SE.

Figure 3

Increase in size of ZGs in the presence of GTP. (a–c) Two-dimensional AFM images of the same granules after exposure to 20 μM GTP at time 0 (a), 5 minutes (b), and 10 minutes (c). (d–f) The same granules are shown in three-dimensions: the three-dimensional image of the granules at time 0, 5 minutes, and 10 minutes, respectively, after exposure to GTP. (g-i) The GTP-induced increase in size of another group of ZGs observed by confocal microscopy. Confocal images of the same ZGs at time 0, 5 minutes, and 10 minutes after GTP exposure are shown. (Bar = 1 μm.) Values represent one of three representative experiments.

Figure 4

Change in ZG height and diameter measured by AFM and confocal microscopy, respectively. (a) Increase in the height of isolated ZGs were observed only in the presence of GTP. Analysis of height change in various population of ZGs (n = 14) exposed for 0 minutes and the same ZGs exposed for 10 minutes to 20 μM GTP, 100 μM Ca²⁺, or 200 μM EGTA is graphically depicted. A significant increase in ZG height was observed only in the GTP-exposed groups. Paired Student’s t tests, with P < 0.001, were used. (b) Similar increases in ZG diameter as measured by the confocal microscope were observed in ZGs exposed to GTP, Mas7, and NaF. Change in diameter of various population of ZGs (n = 10), exposed to 20 μM GTP, 20 μM Mas7, or 100 μM NaF for 10 minutes is shown. A significant (P < 0.001) increase in ZG diameter at the 10-minute time point over the 0-minute time point is seen. Twenty micromolars of Mas17 (control) had no effect on ZG size. Values represent one of three representative experiments.

Figure 5

Hypothetical model depicting the possible role of ZGM-associated G_αi3 in regulation of secretory vesicle swelling and fusion at the plasma membrane in pancreatic acinar cells. In the presence of GTP, the G_αi3 protein is activated by replacement of its GDP with GTP, resulting in the opening of ZGM-associated Cl⁻ and K⁺ channels. There is then a net increase in flow of Cl⁻ and K⁺ ions together with water molecules into the ZG lumen, resulting in vesicle swelling and subsequent fusion at the plasma membrane by an unknown mechanism.