Romagnoli P, Herzog V
Institut für Zellbiologie, University of Bonn, Federal Republic of Germany.
Exp Cell Res. 1991 Jun;194(2):202-9. doi: 10.1016/0014-4827(91)90355-x.
In order to analyze quantitatively the translocation of plasma membrane during endocytosis and transcytosis and the regulation of these processes in thyroid follicle cells, the apical cell surfaces of resting and TSH-stimulated inside-out follicles were labeled with cationized ferritin. Morphometric analyses showed that the rates of endocytosis and transcytosis are TSH-dependent. More interestingly, whereas the effect of TSH on endocytosis was transient (with a maximum at 16 min), the effect on transcytosis continued to increase until the end of the experiment (i.e, 70 min). During 1 h of endocytosis, the fraction of membrane involved in transcytosis increased by a factor 4 upon TSH stimulation, corresponding to about 12% of the internalized apical plasma membrane area. Cooling to 15 degrees C slowed down, but did not block endocytosis entirely, whereas transcytosis and transfer to lysosomes were totally inhibited In order to quantitate transcytosis of thyroglobulin (TG) and to ascertain whether this molecule undergoes cleavage during transcytosis, inside-out follicles were incubated in a medium containing 3H-labeled TG in the presence of TSH; upon washing and reopening of follicles, the luminal fluid containing TG after transcytosis was found to contain about 10% of the total radioactivity taken up by follicle cells. Transcytosed TG proved to be unmodified with respect to its electrophoretic mobility. We conclude that (i) the fraction of transcytosed TG corresponds approximately to the fraction of membrane involved in this process, (ii) TG does not undergo cleavage during transcytosis, (iii) endocytosis and transcytosis are regulated by TSH but differ in their kinetics after stimulation, and (iv) transcytosis is affected by temperature in a similar way as transfer to lysosomes, suggesting the existence of a common gating step for both pathways.
为了定量分析甲状腺滤泡细胞内吞作用和转胞吞作用过程中质膜的转运以及这些过程的调控，用阳离子铁蛋白标记静止和TSH刺激的外翻滤泡的顶端细胞表面。形态计量分析表明，内吞作用和转胞吞作用的速率依赖于TSH。更有趣的是，虽然TSH对内吞作用的影响是短暂的（在16分钟时达到最大值），但对转胞吞作用的影响持续增加直至实验结束（即70分钟）。在1小时的内吞作用过程中，TSH刺激后参与转胞吞作用的膜部分增加了4倍，相当于内化顶端质膜面积的约12%。冷却至15℃会减慢但不会完全阻断内吞作用，而转胞吞作用和向溶酶体的转运则完全被抑制。为了定量甲状腺球蛋白（TG）的转胞吞作用并确定该分子在转胞吞过程中是否发生裂解，将外翻滤泡在含有TSH的3H标记TG的培养基中孵育；冲洗并重新打开滤泡后，发现转胞吞作用后含有TG的腔液中含有滤泡细胞摄取的总放射性的约10%。经转胞吞作用的TG在电泳迁移率方面未被修饰。我们得出以下结论：（i）经转胞吞作用的TG部分大致对应于参与该过程的膜部分，（ii）TG在转胞吞过程中不发生裂解，（iii）内吞作用和转胞吞作用受TSH调节，但刺激后的动力学不同，（iv）转胞吞作用受温度影响的方式与向溶酶体的转运相似，表明这两种途径存在共同的门控步骤。
