Fig 1.
Rab27A deficiency caused increased expression of Rab27B, but did not affect its activity.
(A and B) Examples of total lysates of isolated pancreatic acinar cells from wild-type C3H/HeSnJ or ashen mice were analyzed by western blot. Each lane represents samples from one mouse. (B) Densitometry analysis on the western blot results from all samples run as in (A). The results are mean ± SE from five mice of each genotype. *P < 0.05. (C) Active form of Rab27B and Rab3D at basal level in isolated acini was examined by GST-SHD and GST-Rim pulldown, respectively. Pulldown fractions were analyzed by western blot. This experiment was repeated three times with similar results. (D) The expression of major digestive enzymes (amylase, chymotrypsin, lipase, and elastase) and other Rab proteins (Rab6 and Rab11) was also not changed in western blots on lysates from isolated ashen mouse acinar cells. (E) The expression of major digestive enzymes (amylase, chymotrypsin, elastase, carboxypeptidase A and ribonuclease A) was also not changed in western blots of purified ashen mouse zymogen granules.
Fig 2.
Rab27A deficiency did not affect the morphology of pancreas.
(A) Pancreas tissues freshly obtained from WT or ashen mice were fixed, embedded with paraffin, sectioned and stained with hematoxylin and eosin. Images were taken using a 20X (upper panel) and 40X (lower panel) objective lens. (B) Pancreas tissues from WT and ashen mice were processed for electron microscopy as described in Material and Methods. Images were obtained at the magnification of 3400X.
Fig 3.
Rab27A deficiency did not affect the localization of zymogen granules, Rab27B and Rab3D.
Pancreas tissues were cryosectioned at 10 μm, stained with phalloidin to label actin (green), DAPI to label nuclei (blue) and anti-amylase (A), anti-Rab27B (B) or anti-Rab3D (C) antibodies (red), respectively. Images were obtained using confocal microscopy. Each panel is paired with the Nomarski image for the same section. Scale bar = 25 μm.
Fig 4.
Rab27A showed partial co-localization with zymogen granules.
Expression of CFP-Rab27A was mediated by adenoviral infection at a titer of 5×106 pfu/mL to avoid excessive expression of exogenous protein. Therefore the acinar cells were partially positive for CFP-Rab27A. Anti-GFP antibody was used to enhance the CFP-Rab27A signal and doubled stained with anti-Rab3D (A) or anti-amylase antibody (B), respectively. Fluorescence images were obtained by confocal microscopy. Scale bars in the insets represent 5 μm.
Fig 5.
Rab27A exhibited different subcellular localization than Rab27B and Rab3D.
(A) Cell fractionation samples from ICR mouse pancreas were loaded at 10 μg/lane and separated by PAGE. Antibodies against small G-proteins and marker proteins of different subcellular compartments were used in western blots. (B) Densitometry analysis on the western blot results from purified ZG membrane, mitochondrial and microsomal fractions shown in (A). The results are mean ± SE from three separate cell fractionation experiments. Pancreas tissues from 5–6 ICR mice were used in each experiment. *P < 0.05.
Fig 6.
Rab27A deficiency caused decreased amylase release from isolated acini.
(A, B and C) Freshly prepared acini were incubated with indicated concentrations of CCK (A and B) or carbachol (CCh, C) for 30 min. (D) Fresh acini were incubated with 2 μM A23187 alone, 500 nM PMA alone, a combination of 2 μM A23187 with 500 nM PMA or 100 μM 8-pCPT-2’-O-Me-cAMP (pCPT-cAMP) alone for 30 min. Amylase release results were expressed as percentage of total acinar amylase content (A,C and D) or released amylase units per mg DNA content (B). All results are mean ± SE from five independent experiments, respectively. One WT and one ashen mouse were used in each experiment. Each value was compared to the WT group at each concentration point of the indicated secretagogue. *P < 0.05.
Fig 7.
Visualization of zymogen granule secretory pathway by apical lumen marking with Dextran-Texas Red.
(A and B) Freshly made acini were incubated with 1 mg/mL 3000 Da Dextran-Texas Red, stimulated with or without 3 μM carbachol (CCh), fixed and visualized as described in Material and Methods. Confocal microscopy images were obtained with a 60X objective lens. Representative maximum intensity projections of z-stacks images are shown. (C) Quantitative analysis on images obtained from 14 to 22 individual acini from three separate experiments. One WT and one ashen mouse were used in each experiment. Statistical results are mean ± SE. **P < 0.01. Scale bar = 10 μm.
Fig 8.
Visualization of minor-regulated secretory pathway by apical cell surface labeling with anti-LAMP1 staining.
(A and B) Freshly made acini were stimulated with 30 pM CCK and processed as described in Material and Methods. Confocal images of LAMP1 staining (red) and actin (phalloidin, green) were obtained. Representative maximum intensity projections of z-stacks images are shown. (C) Quantitative analysis on the images obtained from 14 to 24 individual acini from three separate experiments. One WT and one ashen mouse were used in each experiment. Statistical results are mean ± SE. *P < 0.05. Scale bar = 10 μm.