Rab3D is not required for exocrine exocytosis but for maintenance of normally sized secretory granules - PubMed (original) (raw)

Rab3D is not required for exocrine exocytosis but for maintenance of normally sized secretory granules

Dietmar Riedel et al. Mol Cell Biol. 2002 Sep.

Abstract

Rab3D, a member of the Rab3 subfamily of the Rab/ypt GTPases, is expressed on zymogen granules in the pancreas as well as on secretory vesicles in mast cells and in the parotid gland. To shed light on the function of Rab3D, we have generated Rab3D-deficient mice. These mice are viable and have no obvious phenotypic changes. Secretion of mast cells is normal as revealed by capacitance patch clamping. Furthermore, enzyme content and overall morphology are unchanged in pancreatic and parotid acinar cells of knockout mice. Both the exocrine pancreas and the parotid gland show normal release kinetics in response to secretagogue stimulation, suggesting that Rab3D is not involved in exocytosis. However, the size of secretory granules in both the exocrine pancreas and the parotid gland is significantly increased, with the volume being doubled. We conclude that Rab3D exerts its function during granule maturation, possibly by preventing homotypic fusion of secretory granules.

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Figures

FIG. 1.

FIG. 1.

(A) Gene targeting strategy for the generation of Rab3D-KO mice. Maps of the targeting vector and the resulting mutant gene are shown. Positions of exon 1 (black box with amino acids encoded by corresponding cDNA) and restriction enzyme sites are indicated. neo, neomycin resistance gene; TK, thymidine kinase gene. (B) Rab3D protein is lacking in homozygous Rab3D-KO mice. Synaptic vesicles from brain and zymogen granules from the pancreas were isolated from adult wild-type (+/+) and homozygous (−/−) mice and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting (20 μg/lane) with antibodies specific for Rab3A, Rab3B, and Rab3C and for SCAMP and synaptobrevin 2 (syb) as a marker for synaptic vesicle and zymogen granule membranes.

FIG. 1.

FIG. 1.

(A) Gene targeting strategy for the generation of Rab3D-KO mice. Maps of the targeting vector and the resulting mutant gene are shown. Positions of exon 1 (black box with amino acids encoded by corresponding cDNA) and restriction enzyme sites are indicated. neo, neomycin resistance gene; TK, thymidine kinase gene. (B) Rab3D protein is lacking in homozygous Rab3D-KO mice. Synaptic vesicles from brain and zymogen granules from the pancreas were isolated from adult wild-type (+/+) and homozygous (−/−) mice and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting (20 μg/lane) with antibodies specific for Rab3A, Rab3B, and Rab3C and for SCAMP and synaptobrevin 2 (syb) as a marker for synaptic vesicle and zymogen granule membranes.

FIG. 2.

FIG. 2.

Exocytosis and granule size of mast cell granules, measured as step increase of membrane capacitance, are unchanged in Rab3D-deficient mice. (A and B) Representative traces of vesicle fusion in mast cells isolated from wild-type and Rab3D-deficient mice, respectively. Stepwise increases of membrane capacitance typically identify exocytotic events of single vesicles. (C to F) Size distributions of secretory vesicles. Panels C and D show step size histograms of discrete and irreversible increases of membrane capacitance (wild-type [WT] cells [C], 21.3 ± 0.27 fF [mean ± standard error of the mean], n = 1,186; mutant cells [D], 23.15 ± 0.32 fF, n = 799). Panels E and F show the distribution of vesicle radii (wild-type cells [E], 0.40 ± 0.002 μm, n = 1,186; mutant cells [F], 0.42 ± 0.003 μm, n = 799). Data were obtained from seven cells derived from two wild-type mice and six cells from two mutant mice. (G) Initial plasma membrane capacitance in mast cells measured immediately before intracellular stimulation of exocytosis with GTPγS (wild-type cells, 6.22 ± 0.55 pF [mean ± standard error of the mean], from 14 cells derived from two mice; mutant cells, 6.17 ± 0.42 pF, from 17 cells derived from three mice). (H) Final cell membrane capacitance after stimulation of exocytosis with GTPγS (wild-type cells, 18.46 ± 2.04 pF, from 13 cells derived from two mice; mutant cells, 17.80 ± 1.12 pF, from 17 cells derived from three mice).

FIG. 3.

FIG. 3.

Amylase release is unchanged in the pancreas of Rab3D-deficient mice. Amylase release was measured with isolated pancreatic lobules prepared from wild-type and KO mice. Secretion is expressed as a percentage of amylase activity released into the medium with respect to total amylase content (sum of amylase discharged in the medium and amylase retained in the tissue). Each value represents the mean of four independent experiments; bars indicate the range. (A) Dose dependence of carbachol-induced amylase release. (B) Discharge kinetics of amylase unstimulated and in response to 0.1 μM and 0.5 μM carbachol.

FIG. 4.

FIG. 4.

Intracellular transport of proteins is not affected in the pancreas of Rab3D-deficient mice. Pancreatic lobules derived from wild-type and KO mice were incubated for 5 min in medium containing 3H-labeled amino acids. After chase to cold medium containing either no stimulants (0 μM) or 0.5 μM carbachol, aliquots of the medium (A) and the lobules (B) were removed as indicated, and trichloroacetic acid-precipitable radioactivity was determined. The values were normalized to the DNA content of the homogenates. Values are means of two different animals; bars indicate the range.

FIG. 5.

FIG. 5.

Electron micrographs obtained from ultrathin sections of parotid gland (A and B) or pancreas (C and D) derived from wild-type (A and C) and Rab3D-deficient (B and D) mice. Also shown are purified zymogen granules derived from a sucrose gradient from wild-type (E) and Rab3D-deficient (F) mice. Bars, 7 (A to D) and 1 (E and F) μm.

FIG. 6.

FIG. 6.

Secretory granules of the parotid gland and the pancreas are enlarged in Rab3D-deficient mice. (A and B) The radii of granules were measured in ultrathin sections from the parotid gland (A) and the pancreas (B) by using images obtained with a slow-scan charge-coupled device camera. Each data set represents a combination of values obtained from two mice. The values were normalized to the total number of granules. (C) Zymogen granules of Rab3D-deficient mice, purified by sucrose gradient centrifugation, retain the increase in the vesicle diameter after granule isolation.

FIG. 7.

FIG. 7.

The buoyant density of zymogen granules is unchanged in Rab3D-deficient mice. Fractions enriched in zymogen granules were subjected to isopycnic sucrose density gradient centrifugation (closed circles, wild-type mice; open circles, Rab3D-deficient mice). The gradient was fractionated in 300-μl aliquots, and the distribution of zymogen granules was measured by determining the amylase content of each fraction. Lines without circles indicate the sucrose concentration of each fraction, determined by refractometry. The values are means of two different experiments; the bars represent the range of the data points. Filled circles, Rab3D-deficient mice; open circles, wild-type controls.

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