Changes in kinesin distribution and phosphorylation occur during regulated secretion in pancreatic acinar cells.
In secretory cells, microtubule- (Mt-) based motor enzymes are thought to support transport of secretory vesicles to the cell surface for subsequent release. At present, the role of Mts and kinesin in secretory vesicle transport in exocrine epithelial cells has not been defined. Furthermore, it is unclear whether an agonist-induced secretory event modifies kinesin function and distribution, thus altering vesicle transport. To this end, we utilized isolated rat pancreatic acini and cultured rat pancreatic acinar cells to examine the role of Mts and kinesin in regulated secretion. Exposure of cells to cytoskeletal antagonistic drugs demonstrated that the observed movements of apically clustered zymogen granules (ZGs) are supported by Mts, but not actin. Morphological studies of Mt organization in polarized acini show that Mt plus ends extend outward from the apical membrane toward the cell center. Immunofluorescence microscopy in both cell models revealed a clear association of kinesin with apical ZGs, while quantitative immunoblot analysis of pancreatic subcellular fractions confirmed kinesin enrichment on ZG membranes. In addition, microinjection of kinesin antibodies into cultured acinar cells inhibited ZG movements. Indirect immunofluorescence staining of isolated cells and quantitative Western blotting of isolated ZGs revealed that kinesin association with granule membranes increased up to 3-fold in response to a secretory stimulus. Autoradiographic studies of 32P-labeled acini showed up to a 6-fold increase in kinesin heavy chain (KHC) phosphorylation during stimulated secretion. These studies provide the first direct evidence that Mts and kinesin support ZG movements and that physiological agonists induce a marked phosphorylation of KHC while increasing the association of kinesin with ZG membranes. These changes during agonist stimulation suggest that the participation of kinesin in zymogen secretion is regulated.
Marlowe
K J
KJ
Center for Basic Research in Digestive Diseases, Mayo Clinic and Foundation, Rochester, MN 55905, USA.
Farshori
P
P
Torgerson
R R
RR
Anderson
K L
KL
Miller
L J
LJ
McNiven
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D003594:Cytoplasmic Granules; D004792:Enzyme Precursors / Q000378:metabolism; D016547:Kinesins / Q000378:metabolism*; D008870:Microtubules / Q000502:physiology; D008969:Molecular Sequence Data; D010179:Pancreas / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D012633:Secretin / Q000494:pharmacology; D012844:Sincalide / Q000494:pharmacology
D004792:Enzyme Precursors; D012633:Secretin; D016547:Kinesins; D012844:Sincalide