We have studied fluid secretion by the contractile vacuole apparatuss of the trypanosomatid flagellate Leptomonas collosoma with thin sections and freeze-fracture replicas of cells stabilized by ultrarapid freezing without prior fixation or cryoprotection. The ultrarapid freezing has revealed membrane specializations related to fluid segregation and transport as well as membrane rearrangements which may accompany water expulsion at systole. This osmoregulatory apparatu consists of the spongiome, the contractile vacuole, and the fluid discharge site. The coated tubules of the spongiome converge on the contractile vacuole from all directions. These 60- to 70-nm tubules contain characteristic double rows of 11-nm intramembrane particles in a helical configuration which fracture predominantly with the E face. Short double rows of similar particles are also frequently found on both faces of the contractile vacuole itself, in addition to many smaller particles on the P face. The spongiome tubules fuse with the vacuole during the filling stage of each cycle and then detach before secretion. The contractile vacuole membrane is permanently attached to the plasma membrane of the flagellar pocket by a dense adhesion plaque. In some ultrarapidly frozen cells, 20- to 40-nm perforations can be visualized within the plaque and the adjacent membranes during the presumptive time of discharge. The formation of the plaque perforations and the membrane channels occurs without fusion of the vacuole and the plasma membrane and does not require extracellular calcium. On the basis of our results, we have developed a model for water secretion which suggests that the adhesion plaque may induce pore formation in the adjoining lipid bilayers, thereby allowing bulk expulsion of the fluid.

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