Proton pumps populate the contractile vacuoles of Dictyostelium amoebae.

Abstract

Abstract. Amoebae of the eukaryotic microorganism Dictyostelium discoideum were found to contain an interconnected array of tubules and cisternae whose membranes were studded with 15-nm-diameter "pegs" Comparison of the ultrastructure and freeze-fracture behavior of these pegs with similar structures found in other cells and tissues indicated that they were the head domains of vacuolar-type proton pumps. Supporting this identification, the pegs were observed to decorate and clump when broken amoebae were exposed to an antiserum against the B subunit of mammalian vacuolar H+-ATPase. The appearance of the peg-rich cisternae in quick-frozen amoebae depended on their osmotic environment: under hyperosmotic conditions, the cisternae were fiat with many narrow tubular extensions, while under hypo-osmotic conditions the cisternae ranged from bulbous to spherical. In all cases, however, their contents deep etched like pure water. These properties indicated that the interconnected tubules and cisternae comprise the contractile vacuole system of Dictyostelium. Earlier studies had demonstrated that contractile vacuole membranes in Dictyostelium are extremely rich in calmodulin (Zhu, Q., and M. Clarke. 1992. J. Cell Biol. 118: 347-358). Light microscopic immunofluorescence confirmed that antibodies against the vacuolar proton pump colocalized with anti-calmodulin antibodies on these organelles. Time-lapse video recording of living amoebae imaged by interference-reflection microscopy, or by fluorescence microscopy after staining contractile vacuole membranes with potential-sensitive styryl dyes, revealed the extent and dynamic interrelationship of the cisternal and tubular elements in Dictyostelium's contractile vacuole system. The high density of proton pumps throughout its membranes suggests that the generation of a proton gradient is likely to be an important factor in the mechanism of fluid accumulation by contractile vacuoles.

Identification and Characterization of a Novel α-Kinase with a von Willebrand Factor A-like Motif Localized to the Contractile Vacuole and Golgi Complex inDictyostelium discoideum

“…This suggested that these circular bodies surrounded by GFP-VwkA might be contractile vacuoles, dynamic organelles involved in osmoregulation (Gerisch et al, 2002). To explore this localization further, live GFP-VwkAexpressing cells were stained with FM2-10, a lipophylic styryl fluorescent dye that preferentially labels contractile vacuoles in living Dictyostelium cells (Heuser et al, 1993;Gerisch et al, 2002). As shown in the Figure 10B, one of the circular shaped bodies is stained with FM2-10 dye, indicating that VwkA is localized to contractile vacuole in the cells ( Figure 10B, top arrowhead).…”

Localization of cyclic‐AMP receptors with acidosomes in Dictyostelium discoideum

“…Well characterized acidosomes also have a tubulo-vesicular morphology in vitro ( Fig. 4; also see [8,9]) and in vivo [9,12,21], further supporting their putative function as an early endosomal compartment. Also note the smaller vesicles accumulated inside acidosomes (Fig.…”

“…It has been recently suggested that the tubulo-vesicular structure of the acidosome [22] in Dictyostelium is the contractile vacuole [12]. For the following reasons, we believe that contractile vacuoles and acidosomes are in fact two distinct organelles.…”

“…We suggested that the acidosome is an endocytic organelle and functions in the acidification of the endocytic circuit [10,11]. Others have suggested its role in contractile vacuole function [12,13].…”

GIANT VACUOLES OBSERVED IN DICTYOSTELIUM DISCOIDEUM

“…The giant vacuoles may be part of the CV since they apparently occurred in response to the changing osmolarity of the nutrient medium. Since excretion of the vacuolar contents presumably would occur through the CV complex, the giant vacuoles may be composite structures related to both endosomes and the CV, which are evidently different organelles (Gabriel et al, 1999) but share some common molecules (e.g., the vacuolar-H + -ATPase [Heuser et al, 1993;Nolta et al, 1993] and a Rab4-like GTPase [Bush et al, 1994]). These two organelle systems may be connected under certain osmotic conditions (Padh, 1995;Padh et al, 1991a,b;Steck et al, 1997).…”