The vacuole contractile (CV) system is the osmoregulatory organelle necessary for the survival of many free cells under hypotonic conditions. We identified a novel CV regulator, Disgorgin, a protein containing a TBC domain that translocates into the CV membrane at the advanced stage of CV loading and regulates the fusion and discharge of the CV plasma membrane. Disgorginous cells produce large CVs due to impaired fusion of the CV plasma membrane. Disgorgin is a specific GAP for Rab8A-GTP, which is also located on the CV and whose hydrolysis is necessary for discharge. We show that drainin, a protein containing a previously identified TBC domain, is located in this signaling pathway before Disgorgin. Unlike Disgorgin, drainin lacks GAP activity but acts as a Rab11A effector. The proteins of the BEACH LvsA and LvsD family have been identified in a suppressor/amplifier screening of the DISgorgine-sized CV phenotype and have been shown to have different functions in regulating CV formation. Our studies help define the pathways that control the CV function. Most freshwater Chlamydomonas species have two contractile vacuoles, some species have only one, and others have four or more (Ettl 1976a; Luykx, 2000).
They are not seen in marine species or in freshwater species maintained in hypertonic environments. The number of contractile vacuoles was used by Gerloff (1940) as the main taxonomic criterion for subdividing the subgenus, but this practice was not followed by Ettl (1976a). In species with two vacuoles, for example C. moewusii (Guillard, 1960) or C. reinhardtii (Luykx et al., 1997a), the pulse of vacuoles alternately, depending on conditions, usually at intervals of about 10 to 15 seconds. Figure 14.3. Living cell imaging of contractile vacuoles. T. cruzi epimastigotes were exposed to hyposmotic stress (150 mOsm) to detect swelling of the vacuol contractile bladder (arrows).
Note that the cells are rounded after stress and there is also swelling of the smaller vacuoles at the back of the cells. The images were taken at intervals of 5 s after hyposmotic stress for a total time of 6 min. The first 20 seconds of the response are shown in the figure. Bar = 10 μm. The contractile vacuole complex (CV) is an osmoregulatory organelle of free amoebae and protozoa that controls intracellular fluid balance by accumulating and expelling excess water from the cell so that the cells can survive under hypotonic stress as in pond water. In the absence of a functional CV complex, cells cannot expel water, swell sharply and lyse. Important work has been done to characterize the characteristics and function of the CV system. In Dictyostelium, the CV system consists of tubules and vacuoles (or bubbles) that are interconvertible (Gerisch et al., 2002).
Tubular structures act as collecting channels to accumulate excess water while bubbles fuse with the plasma membrane, allowing bladder contents to be released into the extracellular medium, thus expelling water from the cell body (Heuser et al., 1993). When the cells are in an isotonic medium, the CV system shows limited activity, but when the cells are placed in a hypotonic medium, the CV system is quickly activated: the bubbles fill with water and then fuse with the plasma membrane, discharging their contents (Heuser et al., 1993; Gabriel et al., 1999). Heuser (2006) proposed that the CV should not disappear during the discharge phase, but should collapse and flatten against the plasma membrane, thus preserving its various membrane components. To identify the properties of large vacuoles, we labeled disgorginous cells with markers for different types of organelles: TRITC-dextran (endosomes), lysotrackers (lysosomes) or RFP-dajumin (CV system) (Gabriel et al., 1999; Insall et al., 2001). Dajumine RFP, but not the other markers, clearly marked the large vacuol structures corresponding to those observed in phase contrast microscopy, suggesting that the large vacuoles in the disgorginous cells are enlarged CVs (Figure 2A and Additional Film S1). Interestingly, the large vacuoles were no longer present when we placed the disgorgin cells in a low-salt buffer; Instead, we observed many smaller bladder structures (Figure 2A and additional film S1), suggesting that CV activity in disgorginous cells changed dramatically under hypotonic stress. On the other hand, the tone of the medium did not affect the CV structures in the wild-type cells (Figure 2A and Additional Film S1). In summary, the contractile vacuole system now appears as an unexpected dynamical system – far beyond its impressive systole/diastole cycle. PtSyb2 and PtSyx2 are SNAREs found exclusively in this complex organelle, in all its parts except the decorated spongioma (where ATPase H+ is exclusively found). There is no evidence of actin in this organelle. D is very correct, yes, it helps the setting to get rid of excess water, since everyone knew that paramecium lives in water. In a genetic screening of cell morphology mutants, we identified a new CV regulator that we called Disgorgin (DDB0218275, Dictybase).
Disgorgin contains an F-box domain near its N terminus and a TBC domain (RabGAP) with the conserved Arg and Gln catalytic residues necessary for the GAP activity (Figure 1A and B; Pan et al., 2006). Disgorginous knockout strains generated by homologous recombination and confirmed by southern and northern transfer analyses (additional figures S1A and B) have a significant vacuole morphology (up to 7 μm) that is easily observed by phase contrast or DIC microscopy, whereas in most wild-type cells (Ax2 strain; Figure 1C and additional figure S2A). Given the high-discharge phenotype we observed in Disgorgin cells, we suggest that Disgorgin mediates CV discharge by regulating the effective fusion between CV and plasma membranes. We hypothesize that the mechanism underlying the formation of large vacuoles in disgorginous cells in isotonic media is that vacuoles continue to grow (and eventually fuse) because they cannot fuse with the plasma membrane. The best understood contractile vacuoles include the protists Paramecium, Amoeba, Dictyostelium and Trypanosoma and, to a lesser extent, the green algae Chlamydomonas. Not all species that possess a contractile vacuole are freshwater organisms; some marine, soil and parasite microorganisms also have a contractile vacuole. Contractile vacuole is widespread in species that do not have a cell wall, but there are exceptions (especially chlamydomonas) that have a cell wall. During evolution, the contractile vacuole has generally been lost in multicellular organisms, but it still exists in the single-celled stage of several multicellular fungi, as well as in different types of cells in sponges (amoebocytes, pinacocytes and choanocytes). [1] Gruber and Rosario (1979) suggested that vesicles emerge from Golgi, increase in size and eventually fuse with the contractile vacuole or plasma membrane. The same vesicles have also been postulated to be involved in the formation of new membranes during cytokinesis. No contractile elements were observed in conjunction with the vacuole, which led these authors to believe that the vacuole discharge should be considered a collapse on the cell surface rather than a repeated contraction, followed by the formation of a new vacuole by fusion of cytoplasmic vesicles.
Although Disgorgin is a Rab8A-GAP, overexpression of Rab8A in Disgorgin cells suppresses steady-state accumulation of large vacuoles (i.e., large vacuoles do not accumulate; Data not presented), possibly by providing a sufficient share of Rab8A-GDP to the CV system, which could compete with Rab8A-GTP for work on CV membranes. However, Rab8A does not suppress the abnormal discharge of CV into disgorginous cells (data not shown), suggesting that the transition from the GTP-related form to the GDP-related form of Rab8A is important for fulfilling its function in the fusion of the CV plasma membrane. It was noted that since no CV of a previously characterized organism is significantly acidic (p. e.g., one study (Stock et al., 2002) calculated the pH of CV in Paramecium multimicronucleatum at 6.4), H+ ATPases most likely provide the primary electrochemical gradient for the movement of other ions (Allen and Naitoh, 2002). Interestingly, T. cruzi`s HVAC also has a vacuolar pyrophosphatase H+ (Rohloff et al., 2004), which would provide a redundant mechanism for generating electrochemical potential. However, the role of VCs in protists extends beyond regulating cell volume to maintaining Ca2+ homeostasis (Malchow et al., 2006; Moniakis et al., 1999; Xie et al., 1996) and protein transport to the plasma membrane (Sesaki et al., 1997), although these functions have not been studied in T. cruzi. Recently, Hasne et al. (2010) showed that the CV of T. cruzi hosts a polyamine transporter that can be transferred to the plasma membrane if the incubation medium is deficient in polyamines. T.
cruzi`s CVC also has an aquaporin involved in its periodic filling (Montalvetti et al., 2004; Rohloff et al., 2004). A contractile vacuole (CV) is an organelle or subcellular structure involved in osmoregulation and waste disposal. Previously, a CV was known as a pulsed or pulsed vacuole. CVs should not be confused with vacuoles that store food or water. A CV is found mainly in protists and single-celled algae. In freshwater environments, the concentration of solutes inside the cell is higher than outside the cell. Under these conditions, water flows from the environment into the cell by osmosis. Thus, the CV acts as a protective mechanism against cell expansion (and possibly explosion) due to too much water; it expels excess water from the cell by contracting.. .