The contractile vacuole network of Dictyostelium as a distinct organelle: its dynamics visualized by a GFP marker protein

The contractile vacuole system is an osmoregulatory organelle composed of c isternae and interconnecting ducts. Large cisternae act as bladders that pe riodically fuse with the plasma membrane, forming pores to expel water, To visualize the entire network in vivo and to identify constituents of the va cuolar complex in cell fractions, we introduced a specific marker into Dict yostelium cells, GFP-tagged dajumin, The C-terminal, GFP-tagged region of t his transmembrane protein is responsible for sorting to the contractile vac uole complex. Dajumin-GFP negligibly associates with the plasma membrane, i ndicating its retention during discharge of the bladder. Fluorescent labele d cell-surface constituents are efficiently internalized by endocytosis, wh ile no significant cycling through the contractile vacuole is observed, End osomes loaded with yeast particles or a fluid-phase marker indicate sharp s eparation of the endocytic pathway from the contractile vacuole compartment . Even after dispersion of the contractile vacuole system during mitosis, d ajumin-GFP distinguishes the vesicles from endosomes, and visualizes post-m itotic re-organization of the network around the nucleus. Highly discrimina tive sorting and membrane fusion mechanisms are proposed to account for the sharp separation of the contractile vacuole and endosomal compartments, Ev idence for a similar compartment in other eukaryotic cells is discussed.