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Altered expression of the 100 kDa subunit of the Dictyostelium vacuolar proton pump impairs enzyme assembly, endocytic function and cytosolic pH regulation

¹ Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, 825 N.E. 13th Street, Oklahoma City, OK 73104, USA
² Zoologisches Institut, Ludwig-Maximilians-Universität, Munich, Germany

View larger version (16K): [in a new window]   Fig. 1. Replacement of the vatM promoter with the act6 promoter. Panel A shows the plasmid pVATM-act6, which contains segments of the vatM promoter and coding region separated by the selectable marker pyr5-6 and the act6 promoter. Panel B illustrates the predicted outcome of homologous recombination between the chromosomal vatM gene and a linearized segment of pVATM-act6, following its transformation into DH1 cells. Panel C shows a Southern blot of genomic DNA from DH1 (D) and one of the transformants (V). The genomic DNA was digested with BclI and probed to detect vatM. This transformant, which was given the name VatMpr, displayed an increased restriction fragment size consistent with the double crossover event illustrated in B.

View larger version (36K): [in a new window]   Fig. 2. (A) Effect of growth conditions on the level of VatM in VatMpr and DH1. Amoebae of DH1 (lanes a,b) and VatMpr (lanes c,d) were grown axenically (lanes a,c) or in association with K. aerogenes (lanes b,d); cells were harvested from exponentially growing cultures. Each lane of the SDS polyacrylamide gel received the lysate from 1x106 cells. Following electrophoresis, the proteins were transferred to a membrane, and the upper portion of the membrane was stained with N2 antibodies, which recognize the 100 kDa V-ATPase subunit, VatM. The lower portion of the membrane was stained with anti-actin antibodies, as a control for equal loading of the lanes. (B) Immunoblot showing levels of VatM in VMop cells as a function of growth conditions. VMop-11 cells were harvested from axenic culture (lane a) or after two days of growth on a suspension of bacteria (lane b) and were immunoblotted to detect VatM and actin as described above. Similar levels of VatM were present under the two growth conditions.

View larger version (132K): [in a new window]   Fig. 3. Growth of VatMpr, DH1 and VMop amoebae on a bacterial lawn. Cells from clonal axenic populations of VatMpr (A,B), DH1 (C,D), and VMop (E,F) were plated at low density with K. aerogenes on nutrient agar plates. The plates were photographed after 5 (left column) and 6 (right column) days of growth at 22°C. By day 5, fruiting bodies began to appear in the plaques formed by DH1 and VMop cells; arrowheads mark some of the fruiting bodies on day 6. The very slow growth rate of VatMpr amoebae was evident from their tiny plaque size. A few aggregates could be seen on day 6; these eventually formed slugs, but never fruiting bodies. All micrographs are at the same magnification. Bar, 2 mm.

View larger version (180K): [in a new window]   Fig. 4. Visualization of V-ATPase subunits in VatMpr, DH1 and VMop cells. Bacterially grown DH1 (A,D,G,J), VatMpr (B,E,H,K), and VMop cells (C,F,I,L) were fixed and stained with either N2 (A,B,C) or N4 (G,H,I) monoclonal antibodies, recognizing VatM and VatA, respectively. Phase contrast images are shown below their corresponding immunofluorescence images. Fixation and staining conditions (described in Materials and Methods) were identical for all cell types. Bar, 10 µm.

View larger version (71K): [in a new window]   Fig. 5. Effect of growth conditions on accumulation of the A subunit of the V-ATPase. VatMpr (a,b) and DH1 (c,d) cells were grown either axenically (b,d) or for 2 days on a suspension of K. aerogenes (a,c). Cells were harvested from exponentially growing cultures, and the lysate from 1x106 cells was loaded in each lane of an SDS polyacrylamide gel. After electrophoresis, the proteins were transferred to a membrane and stained with antibodies that recognize VatA and VatM (upper portion of membrane) and actin (lower portion). In bacterially grown VatMpr cells (lane a), the amount of VatA protein (normalized for actin levels) was 133% of the amount in the other samples.

View larger version (108K): [in a new window]   Fig. 6. Visualization of proton pumps in contractile vacuole membranes of VatMpr and DH1 cells. Cells grown on a suspension of K. aerogenes for 2 days were washed free of bacteria and plated on glass. The cells were ruptured and freeze-dried, and platinum replicas were prepared as described (Heuser et al., 1993). The proton pumps appear as 15 nm diameter `studs' on the cisternal and tubular elements of the contractile vacuole system; the density of the studs is reduced in VatMpr cells.

View larger version (16K): [in a new window]   Fig. 7. Uptake of fluorescent yeast particles by VatMpr () and AX3 ([UNK]) cells. Cells were cultured on a suspension of K. aerogenes for 2 days prior to the assay. Bacteria were washed away and the cells were swirled on a rotary shaker for 30-40 minutes before TRITC-yeast particles were added (see Materials and Methods for details). At T0 and 20 minute intervals thereafter, duplicate samples were taken, the fluorescence of uningested yeast particles was quenched and the fluorescence of the internalized particles was determined. Uptake was linear throughout the 2 hour assay for mutant cells and during the first 80 minutes for wild-type cells. At longer times wild-type values reached a steady state that presumably reflected complete transit of particles through the endo/lysosomal pathway and exocytosis of the undigested remnants. Comparison of linear regression plots for the first 80 minutes showed that the rate of particle uptake by wild-type cells was twice that of VatMpr cells.

View larger version (60K): [in a new window]   Fig. 8. Microscopy of living VatMpr and AX3 cells in the presence of TRITC-yeast. The images show AX3 (A) and VatMpr cells (B,C) from bacterially grown (A,B) or axenically grown (C) cultures. The images shown are single frames from time-lapse movies. The time after addition of yeast particles is (A) 40 minutes, (B) 70 minutes, and (C) 34 minutes. The relative motion of cells and yeast particles in the movies revealed that one particle in panel A, all particles in panel B, and two particles in panel C had not been ingested. There are six cells in the cluster at the right of panel A.

View larger version (21K): [in a new window]   Fig. 9. Dynamic response of the cytosolic pH to an increase in acid loading in wild-type cells and VatMpr cells. Cells expressing ratiometric pHluorin were washed and placed in a fluorimeter cuvette, and the fluorescence of the `acid' pHluorin peak (ex=475 nm, em=510 nm) was monitored continuously while acetate was added to the medium without a change in extracellular pH. An increase in fluorescence indicates a decrease in cytosolic pH. (A) Bacterially grown VatMpr and AX2 cells at pH 6. Acetate was added at T0 to a final concentration of 15 mM. The pH decreased over at least 200 seconds in AX2, but reached an abrupt plateau at 30-45 seconds in VatMpr. (B) AX2 cells grown axenically at pH 7. Half of the cells were shifted to pH 5 at T minus 4 hours; acetate was added at T0 (2 mM final concentration). The pH decreased for both cultures, but in cells that had been pre-incubated at pH 5, the curve reached an abrupt plateau, similar to that observed for bacterially grown VatMpr. The behavior of axenically grown VatMpr is identical to that of AX2 (data not shown).

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