Gabriel D, Hacker U, Köhler J, Müller-Taubenberger A, Schwartz J M, Westphal M, Gerisch G
Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany.
J Cell Sci. 1999 Nov;112 ( Pt 22):3995-4005. doi: 10.1242/jcs.112.22.3995.
The contractile vacuole system is an osmoregulatory organelle composed of cisternae and interconnecting ducts. Large cisternae act as bladders that periodically fuse with the plasma membrane, forming pores to expel water. To visualize the entire network in vivo and to identify constituents of the vacuolar complex in cell fractions, we introduced a specific marker into Dictyostelium cells, GFP-tagged dajumin. The C-terminal, GFP-tagged region of this transmembrane protein is responsible for sorting to the contractile vacuole complex. Dajumin-GFP negligibly associates with the plasma membrane, indicating its retention during discharge of the bladder. Fluorescent labeled cell-surface constituents are efficiently internalized by endocytosis, while no significant cycling through the contractile vacuole is observed. Endosomes loaded with yeast particles or a fluid-phase marker indicate sharp separation of the endocytic pathway from the contractile vacuole compartment. Even after dispersion of the contractile vacuole system during mitosis, dajumin-GFP distinguishes the vesicles from endosomes, and visualizes post-mitotic re-organization of the network around the nucleus. Highly discriminative sorting and membrane fusion mechanisms are proposed to account for the sharp separation of the contractile vacuole and endosomal compartments. Evidence for a similar compartment in other eukaryotic cells is discussed.
收缩泡系统是一种渗透调节细胞器，由扁平囊和相互连接的管道组成。大型扁平囊起到囊泡的作用，它们会定期与质膜融合，形成孔隙以排出水分。为了在体内观察整个网络，并识别细胞组分中液泡复合体的成分，我们将一种特定的标记物——绿色荧光蛋白（GFP）标记的dajumin引入了盘基网柄菌细胞中。这种跨膜蛋白的C末端GFP标记区域负责将其分选到收缩泡复合体。dajumin-GFP与质膜的结合可以忽略不计，这表明它在囊泡排出过程中被保留了下来。荧光标记的细胞表面成分通过内吞作用有效地内化，而未观察到其通过收缩泡进行显著的循环。装载有酵母颗粒或液相标记物的内体表明内吞途径与收缩泡区室明显分离。即使在有丝分裂期间收缩泡系统分散后，dajumin-GFP也能将这些囊泡与内体区分开来，并显示出有丝分裂后围绕细胞核的网络重新组织情况。本文提出了高度特异性的分选和膜融合机制来解释收缩泡和内体区室的明显分离。文中还讨论了其他真核细胞中类似区室的证据。
