Mata L R, Christensen E I
Department of Cell Biology, Gulbenkian Institute of Science, Oeiras, Portugal.
Biol Cell. 1990;68(3):183-93. doi: 10.1016/0248-4900(90)90307-o.
This study was performed to clarify the fate of membrane constituents internalized from the apical domain in secretory cells, in particular their possible recycling and the compartments involved in it. Glycoproteins of the apical membrane of seminal vesicle secretory cells from guinea-pig were covalently labeled in vitro (0 degrees C, 20 min) with 3H-galactose and the epithelium incubated for 15 min (37 degrees C, first incubation) to allow endocytosis. The label which was not internalized was then exposed to enzymatic hydrolysis (0 degrees C, 30 min) and the epithelium re-incubated to allow membrane movement for 15 and 30 min (37 degrees C, 2nd incubation). After each step of the protocol, tissue pieces were fixed and processed for electron microscope autoradiography and the results studied by morphometric analysis. Following labeling, 99% of the silver grains were associated with the apical domain of the cell membrane (AD). After the 1st incubation at 37 degrees C, 30% of the grains were inside the cells in association with the cytoplasmic vesicles (Cyt ves), secretory vacuoles (SV), Golgi vesicles (GV), Golgi cisternae (GC), multivesicular bodies (MVB), lysosomes (LYS), and the cell membrane basolateral domain (BLD). About 58% of non-internalized radioactivity was removed by hydrolysis. During the 2nd incubation at 37 degrees C the concentration of label increased in BLD and LYS, decreased in SV and MVB, and fluctuated in GC, GV and AD. The distribution of grains observed at 15 min, as compared using the chi-square test, was highly significantly different from that expected without recycling. The results show that cell membrane glycoproteins internalized at the cell apex recycle back to the membrane apical domain and are consistent with the involvement of GC and SV in the recycling pathway. Membrane shuttle between the apical and basolateral domains of the cell membrane is also suggested by these observations.
本研究旨在阐明分泌细胞从顶端区域内化的膜成分的去向，特别是其可能的再循环以及参与其中的区室。用³H-半乳糖在体外（0℃，20分钟）对豚鼠精囊分泌细胞顶端膜的糖蛋白进行共价标记，并将上皮细胞孵育15分钟（37℃，第一次孵育）以允许内吞作用。然后将未内化的标记物进行酶水解（0℃，30分钟），并将上皮细胞再次孵育以允许膜移动15分钟和30分钟（37℃，第二次孵育）。在该实验方案的每个步骤之后，将组织块固定并进行电子显微镜放射自显影处理，并通过形态计量分析研究结果。标记后，99%的银颗粒与细胞膜的顶端区域（AD）相关。在37℃第一次孵育后，30%的颗粒位于细胞内，与细胞质小泡（Cyt ves）、分泌泡（SV）、高尔基体小泡（GV）、高尔基体潴泡（GC）、多泡体（MVB）、溶酶体（LYS）和细胞膜基底外侧区域（BLD）相关。约58%未内化的放射性通过水解去除。在37℃第二次孵育期间，标记物的浓度在BLD和LYS中增加，在SV和MVB中降低，在GC、GV和AD中波动。使用卡方检验比较，在15分钟时观察到的颗粒分布与无再循环时预期的分布高度显著不同。结果表明，在细胞顶端内化的细胞膜糖蛋白再循环回到膜顶端区域，并且与GC和SV参与再循环途径一致。这些观察结果还提示了细胞膜顶端和基底外侧区域之间的膜穿梭。
