Contractile Vacuole in Amoeba Is Meant for

The contractile vacuole, as the name suggests, expels water from the cell by contraction. The growth (water collection) and contraction (water outlet) of contractile vacuoles are periodic. A cycle lasts several seconds, depending on the type and osmolarity of the environment. The stage at which water flows into the CV is called diastole. The contraction of the contractile vacuoles and the expulsion of water from the cell are called systoles. 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. 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). Disgorgin shares some similarities with drainin, another CV discharge regulator. Both contain areas of tuberculosis and the removal of one of the two proteins causes an increase in CV formation. However, cell morphologies and CV flow differ in the two zero strains. CVs are less enlarged and vacuol sizes are more uniform in disgorginous cells than in drainin cells (Figure 4A and Additional Figure S2A). Drainin− cells are partially hypoosmotically sensitive and have two types of abnormal discharge, one of which is similar to that of disgorgine− cells (vacuole forms a bleb) (Becker et al., 1999).

Although disgorgine− cells do not lack active CV plasma fusion as described above, cells can discharge and are insensitive to hypotonic stress (Figure 2C; Data not displayed). Full expression of disgorgin or disgorgin without the F-Box (DisgorginΔF-Box; Figure 1A) in cells Disgorgin− completes the large vacuol phenotype and does not cause an overexpression phenotype when expressed in wild-type cells (Figure 1D, data not shown). Disgorgin with amino acid substitutions in one of the conserved residues necessary for GAP activity (DisgorginR515A; DisgorginQ551A) does not complete zero phenotypes and produces even larger vacuoles when placed in disgorgin or wild type cells (up to 11 μm; Figure 1D and Additional Figure S2A), suggesting that mutated proteins exert an effect as dominant-negative mutants, possibly by further blocking the intrinsic activity of GTPase Rab and/or competing with common substrates or essential components of the signaling pathway. These results suggest that disgorgin RabGAP activity is necessary in the signaling pathway that regulates vacuoles and that the F-box domain is not essential to this process. In wild-type cells, we suggest, as Heuser (2006), that CV vacuoles are released by fusion of CV and plasma membranes. In disgorginous cells, CV flow is defective and we suggest that the fusion of the bladder and plasma membrane is poorly regulated. In the absence of disgorging, the fully charged bubbles press against the plasma membrane and form leaves under hypotonic conditions. Once some of the contents are released from the bladder, the bladder withdraws from the plasma membrane, although the discharge is not complete. The formation and kinetics of these vesicles suggest that membrane tension may be involved in CV discharge in disgorginous cells. We suggest that in disgorginous cells, a possible tension of the bubbles against the plasma membrane can lead to small fractures localized in the plasma membrane that allow the release of CV content. Once some of the contents are released, the tension of the membrane can be reduced, the bubbles then retract and the cell closes the spaces in the CV and plasma membranes. Such a resealing process has already been proposed (Becker et al., 1999).

To date, there is no direct evidence as to whether membrane tension is necessary for the normal discharge process in wild-type dictyostelium cells, as proposed for paramecium cells (Tani et al., 2001). It has been suggested that membrane tension is the main force that drives CV discharge (Heuser, 2006). However, our data suggest that membrane tension alone is not sufficient for CV flow, as large CVs accumulate in disgorginous cells under isotonic conditions when CV activity is reduced, and discharge in the absence of disgorgine is ineffective under hypotonic conditions. Our data support that disgorgement-mediated Rab8A-GTP hydrolysis mediates the melting event. To identify other potential components in the disgorgine signaling pathway, we performed INSERTIONSMutagenesis (REMI) screening for suppressors and amplifiers at the second site of the vacuolar phenotype of the disgorgin cell by identifying strains that exhibited a change in vacuolar size. Visual screening of ∼7000 clones revealed five candidates with altered vacuolar morphology. After cloning the insertion site of the suppressor and amplifier clones, we found that lvsA (DDB0191124) was disturbed in the strain that did not have vacuoles and lvsD (DDB0185108) in both strains with larger vacuoles. We were unable to clone the insertion site of the other two strains.

To confirm the phenotypes of the REMI clone, we disrupted lvsA and lvsD in disgorginous cells. As shown in Figure 6A, the disruption of lvsA in disgorginous cells suppressed the large vacuol phenotype of disgorgine cells, while the phenotype of disgorgine- cells was improved when lvsD was disturbed (Figure 6A and Supplementary Figure S2A). 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. 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.

However, not all species that have a CV are freshwater organisms; some marine and soil microorganisms also have a CV. Cv is prevalent in species that do not have a cell wall, but there are exceptions. Through the evolutionary process, CV has been largely eliminated in multicellular organisms; However, there are still in the single-celled stage of several multicellular fungi and in different types of cells in sponges, including amoebocytes, pinacocytes and choanocytes. To understand the role of disgorgine, we compared the CV cycle in wild-type cells and disgorgine cells by marking the cells with RFP dajumine and placing the cells in water to activate the CV system. As previously described (Gabriel et al., 1999; Heuser, 2006), when cells are placed under hypotonic conditions, CV network activity increases significantly and the dynamic load and discharge of vesicles can be easily monitored (Figure 2B). After emptying the vacuole, dajumine RFP is visible as a patch on the plasma membrane, suggesting that the CV membrane is flattened against the plasma membranes. In addition, new CV bladders are preferably formed at these sites, suggesting that the collapsed CV bladder is different from the majority of the plasma membrane, after which it regenerates into a new CV (Figure 2B) (Heuser, 2006). To study this in more detail, we used the styryl dye FM4-64, which characterizes both the plasma membrane and CVs (Heuser et al., 1993). In wild-type cells, we observed the complete collapse of the CV against the plasma membrane. .