Expression, localization, and functional role for synaptotagmins in pancreatic acinar cells

Abstract

induced changes in intracellular Ca 2ϩ play a pivotal role in secretion in pancreatic acini yet the molecules that respond to Ca 2ϩ are uncertain. Zymogen granule (ZG) exocytosis is regulated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. In nerve and endocrine cells, Ca 2ϩ -stimulated exocytosis is regulated by the SNARE-associated family of proteins termed synaptotagmins. This study examined a potential role for synaptotagmins in acinar secretion. RT-PCR revealed that synaptotagmin isoforms 1, 3, 6, and 7 are present in isolated acini. Immunoblotting and immunofluorescence using three different antibodies demonstrated synaptotagmin 1 immunoreactivity in apical cytoplasm and ZG fractions of acini, where it colocalized with vesicle-associated membrane protein 2. Synaptotagmin 3 immunoreactivity was detected in membrane fractions and colocalized with an endolysosomal marker. A potential functional role for synaptotagmin 1 in secretion was indicated by results that introduction of synaptotagmin 1 C2AB domain into permeabilized acini inhibited Ca 2ϩ -dependent exocytosis by 35%. In contrast, constructs of synaptotagmin 3 had no effect. Confirmation of these findings was achieved by incubating intact acini with an antibody specific to the intraluminal domain of synaptotagmin 1, which is externalized following exocytosis. Externalized synaptotagmin 1 was detected exclusively along the apical membrane. Treatment with CCK-8 (100 pM, 5 min) enhanced immunoreactivity by fourfold, demonstrating that synaptotagmin is inserted into the apical membrane during ZG fusion. Collectively, these data indicate that acini express synaptotagmin 1 and support that it plays a functional role in secretion whereas synaptotagmin 3 has an alternative role in endolysosomal membrane trafficking. zymogen granules; exocytosis PANCREATIC ACINAR CELLS ARE a prototypical model of exocrine secretory cells that undergo stimulated exocytosis of digestive enzymes, which are necessary for the assimilation of the diet. Multiple intracellular signals including diacylglycerol, cAMP and inositol triphosphate (IP 3 )-mediated release of Ca 2ϩ have been shown to stimulate acinar exocytosis. However, the pivotal role of Ca 2ϩ in the overall secretory response is underscored by findings that double knockout of the type 2 and 3 IP 3 receptors fully abolished secretagogue-induced changes in cellular Ca 2ϩ and resulted in a complete lack of salivary and pancreatic acinar secretion (11). Although the critical importance of Ca 2ϩ signaling in acinar exocytosis is well established, the exocytic regulatory proteins that respond to elevated Ca 2ϩ in the cytoplasm are largely uncertain.Synaptotagmins are a family of membrane proteins composed of 15 known isoforms distributed widely in neuronal and nonneuronal tissues (for comprehensive reviews see Refs. 4,39,45). Family members have the same basic structure consisting of a short NH 2 -terminal intravesicular domain followed by a transmembrane domain and a large COOH-termina...

Vesicle Associated Membrane Protein 8 (VAMP8)-mediated Zymogen Granule Exocytosis Is Dependent on Endosomal Trafficking via the Constitutive-Like Secretory Pathway

“…We previously identified a potential role for the Ca 2ϩ -sensing regulatory protein syt1 in VAMP2-mediated exocytosis (43). Analysis of VAMP2 in whole cell lysates revealed a 4-fold increase in VAMP8 Ϫ/Ϫ acini consistent with the increased numbers of ZGs (Fig.…”

Tumor protein D52 controls trafficking of an apical endolysosomal secretory pathway in pancreatic acinar cells

“…Following 2-min CCK-8 stimulation, D52 was present along the cytoplasmic edge of actin filaments, but absent from the apical membrane. Previous studies using various antibodies have demonstrated that VAMP2-positive ZGs are present within the most apical aspects of the acinar cytoplasm (7,19,20,73) and rapidly accumulate within apical actin filaments and on apical plasma membranes following CCK-8 stimulation (73). In stimulated cells, D52 showed only minimal colocalization with VAMP2 in areas confined to the cytoplasmic side of actin filaments (Fig.…”

“…We previously demonstrated that ZG exocytosis can be analyzed in acinar cell whole preparations by surface labeling with an antibody that recognizes the ZG luminal domain of syt1 (see Fig. 5D), which is externalized upon acinar stimulation and ZG exocytosis (20). Analysis of CCK-8-stimulated acini viewed in a reconstructed 3D format shows incomplete overlap of the syt1-and LAMP1-associated fluorophores in apical regions of acini (Fig.…”

Multiple roles for the actin cytoskeleton during regulated exocytosis

“…Furthermore, there is evidence that this mechanism may be universally conserved, since 4 isoforms have been identified for complexin and sytI belongs to a family of 16 members that are expressed in several secretory tissues [5]. Indeed, SytI has been shown to play a role in the exocytosis of zymogen granules in the pancreas and dense core vesicles in the adrenal medulla [143], whereas sytII, V, VII and IX have been reported to regulate the exocytosis of Glut 4 vesicles [144, 145] and secretory granules in mast and NK cells [146, 147]. …”