Wang Yanru, Jerdeva Galina, Yarber Francie A, da Costa Silvia R, Xie Jiansong, Qian Limin, Rose Chadron M, Mazurek Constance, Kasahara Noriyuki, Mircheff Austin K, Hamm-Alvarez Sarah F
Department of Pharmaceutical Sciences, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90033, USA.
J Cell Sci. 2003 May 15;116(Pt 10):2051-65. doi: 10.1242/jcs.00398. Epub 2003 Apr 1.
A major function of the acinar cells of the lacrimal gland is the production and stimulated release of tear proteins into ocular surface fluid. We investigate the participation of cytoplasmic dynein in carbachol-stimulated traffic to the apical plasma membrane in primary rabbit lacrimal acinar epithelial cells. Confocal fluorescence microscopy revealed a major carbachol-induced, microtubule-dependent recruitment of cytoplasmic dynein and the dynactin complex into the subapical region. Colocalization studies, sorbitol density gradient/phase partitioning analysis and microtubule-affinity purification of membranes showed that some dynein and dynactin complex were associated with VAMP2-enriched membranes. Adenovirus-mediated overexpression of p50/dynamitin inhibited the recruitment and colocalization of dynein, the dynactin complex and VAMP2 in the subapical region. Nocodazole treatment and p50/dynamitin overexpression also depleted subapical stores of rab3D in resting acini, suggesting that dynein activity was also involved in maintenance of rab3D-enriched secretory vesicles. These data implicate cytoplasmic dynein in stimulated traffic to the apical plasma membrane in these secretory epithelial cells.
泪腺腺泡细胞的一个主要功能是产生泪液蛋白并将其刺激释放到眼表液体中。我们研究了细胞质动力蛋白在原代兔泪腺腺泡上皮细胞中，对卡巴胆碱刺激的向顶端质膜运输过程中的参与情况。共聚焦荧光显微镜显示，卡巴胆碱主要诱导细胞质动力蛋白和动力蛋白激活蛋白复合物在微管依赖下募集到顶端下区域。共定位研究、山梨醇密度梯度/相分离分析以及膜的微管亲和纯化表明，一些动力蛋白和动力蛋白激活蛋白复合物与富含VAMP2的膜相关联。腺病毒介导的p50/动力蛋白抑制素过表达抑制了动力蛋白、动力蛋白激活蛋白复合物和VAMP2在顶端下区域的募集和共定位。诺考达唑处理和p50/动力蛋白抑制素过表达也耗尽了静息腺泡中顶端下区域的rab3D储备，这表明动力蛋白活性也参与了富含rab3D的分泌小泡的维持。这些数据表明细胞质动力蛋白参与了这些分泌上皮细胞中向顶端质膜的刺激运输。
