Marlowe K J, Farshori P, Torgerson R R, Anderson K L, Miller L J, McNiven M A
Center for Basic Research in Digestive Diseases, Mayo Clinic and Foundation, Rochester, MN 55905, USA.
Eur J Cell Biol. 1998 Feb;75(2):140-52. doi: 10.1016/S0171-9335(98)80056-3.
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.
在分泌细胞中，基于微管（Mt）的运动酶被认为支持分泌囊泡向细胞表面的运输以便随后释放。目前，微管和驱动蛋白在外分泌上皮细胞的分泌囊泡运输中的作用尚未明确。此外，尚不清楚激动剂诱导的分泌事件是否会改变驱动蛋白的功能和分布，从而改变囊泡运输。为此，我们利用分离的大鼠胰腺腺泡和培养的大鼠胰腺腺泡细胞来研究微管和驱动蛋白在调节性分泌中的作用。将细胞暴露于细胞骨架拮抗药物表明，观察到的顶端聚集的酶原颗粒（ZG）的运动由微管支持，而非肌动蛋白。对极化腺泡中微管组织的形态学研究表明，微管的正端从顶端膜向外延伸至细胞中心。两种细胞模型中的免疫荧光显微镜检查均显示驱动蛋白与顶端ZG有明显关联，而对胰腺亚细胞组分的定量免疫印迹分析证实驱动蛋白在ZG膜上富集。此外，将驱动蛋白抗体显微注射到培养的腺泡细胞中可抑制ZG运动。对分离细胞的间接免疫荧光染色和对分离的ZG的定量蛋白质印迹分析表明，响应分泌刺激，驱动蛋白与颗粒膜的结合增加了多达3倍。对32P标记的腺泡的放射自显影研究表明，在刺激分泌过程中，驱动蛋白重链（KHC）的磷酸化增加了多达6倍。这些研究提供了首个直接证据，即微管和驱动蛋白支持ZG运动，并且生理激动剂在增加驱动蛋白与ZG膜结合的同时诱导KHC发生显著磷酸化。激动剂刺激期间的这些变化表明，驱动蛋白在酶原分泌中的参与是受调控的。
