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. 2010 Jan 5;5(1):e8585.
doi: 10.1371/journal.pone.0008585.

Retrieval of the vacuolar H-ATPase from phagosomes revealed by live cell imaging

Margaret Clarke  1 , Lucinda Maddera, Ulrike Engel, Günther Gerisch
Affiliations

Retrieval of the vacuolar H-ATPase from phagosomes revealed by live cell imaging

Margaret Clarke et al. PLoS One. .

Abstract

Background: The vacuolar H+-ATPase, or V-ATPase, is a highly-conserved multi-subunit enzyme that transports protons across membranes at the expense of ATP. The resulting proton gradient serves many essential functions, among them energizing transport of small molecules such as neurotransmitters, and acidifying organelles such as endosomes. The enzyme is not present in the plasma membrane from which a phagosome is formed, but is rapidly delivered by fusion with endosomes that already bear the V-ATPase in their membranes. Similarly, the enzyme is thought to be retrieved from phagosome membranes prior to exocytosis of indigestible material, although that process has not been directly visualized.

Methodology: To monitor trafficking of the V-ATPase in the phagocytic pathway of Dictyostelium discoideum, we fed the cells yeast, large particles that maintain their shape during trafficking. To track pH changes, we conjugated the yeast with fluorescein isothiocyanate. Cells were labeled with VatM-GFP, a fluorescently-tagged transmembrane subunit of the V-ATPase, in parallel with stage-specific endosomal markers or in combination with mRFP-tagged cytoskeletal proteins.

Principal findings: We find that the V-ATPase is commonly retrieved from the phagosome membrane by vesiculation shortly before exocytosis. However, if the cells are kept in confined spaces, a bulky phagosome may be exocytosed prematurely. In this event, a large V-ATPase-rich vacuole coated with actin typically separates from the acidic phagosome shortly before exocytosis. This vacuole is propelled by an actin tail and soon acquires the properties of an early endosome, revealing an unexpected mechanism for rapid recycling of the V-ATPase. Any V-ATPase that reaches the plasma membrane is also promptly retrieved.

Conclusions/significance: Thus, live cell microscopy has revealed both a usual route and alternative means of recycling the V-ATPase in the endocytic pathway.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Delivery of the V-ATPase to new phagosomes.
A, uptake of living S. cerevisiae. Living yeast were added to Dictyostelium cells expressing VatM-GFP and mRFP-LimEΔ. At 0 seconds, a phagosome containing a newly ingested yeast (marked with a circle) is surrounded by actin filaments as it moves into the cell. At 37 seconds, the actin filaments have disappeared and VatM-GFP-positive vesicles have clustered around the phagosome; by 97 seconds their numbers have increased. By 144 seconds, transfer of VatM-GFP to the phagosome membrane is evident. Movie S1 contains the complete time series. B, uptake of heat-killed S. cerevisiae. Dictyostelium cells expressing VatM-GFP were incubated with TRITC-dextran for 4 hours to label all endocytic compartments (endocytic transit time for fluid phase markers being about 1 hour [53]), then heat-killed yeast were added. Some TRITC-dextran penetrated the dead yeast, labeling them as well. At 0 seconds and 46 seconds, a phagocytic cup is extending around a yeast (marked with a circle). In the 110 and 173-second panels, TRITC-dextran content (arrowheads) is evident in several of the VatM-GFP-positive vesicles clustered about the phagosome. Movie S2 contains the complete time series. Perkin-Elmer Ultra View microscope. Bars, 5 µm.
Figure 2
Figure 2. Labeling of early endosomes in Dictyostelium cells with GFP-2FYVE, a biosensor for PI(3)P.
The cells are expressing GFP-2FYVE and mRFP-LimEΔ. A, a macropinosome (marked by arrowheads in each panel). At 0 seconds, a nascent macropinosome is surrounded by actin filaments. After the actin filaments have disappeared, GFP-2FYVE begins to label the macropinosome, the strongest labeling being seen as the originally amorphous macropinosome assumes a spherical shape about 1 minute after uptake. During the next 2 to 3 minutes, the GFP-2FYVE label gradually fades as the macropinosome becomes increasingly elongated and fragmented. (See Movie S3.) B, phagosomes containing bacteria. The Dictyostelium cells were mixed with E. coli expressing a low level of a red cytoplasmic marker, and the uptake of two bacteria was tracked. In panel 0, the phagosome containing the first bacterium has already been internalized and has begun binding GFP-2FYVE. A second phagosome is beginning to form (arrowhead), surrounded by actin filaments (red); the completion and internalization of the phagosome is seen in the 43-second and 90-second panels. In subsequent panels, labeling by GFP-2FYVE reveals expansion and morphological changes in the phagosome, including tubular extensions in the 264-second panel. During this period, the GFP-2FYVE labeling fluctuates in intensity, being weaker at 275 seconds than at 319 seconds, and it has largely faded by 409 seconds. (See Movie S4.) A, Perkin-Elmer Ultra View; B, Zeiss LSM510 microscope. Bars, 5 µm.
Figure 3
Figure 3. Exocytosis of phagosomes from which the V-ATPase has been removed by vesiculation.
A and B, Dictyostelium cells expressing VatM-GFP and mRFP-LimEΔ were mixed with living S. cerevisiae 2 to 4 hours earlier. In both time series, the cells contain several phagosomes whose membranes are rich in VatM-GFP. A, at 0 seconds, the membrane of a phagosome containing a budded yeast (marked with a circle) is brightly labeled with VatM-GFP. By 71 seconds, the VatM-GFP content of the phagosome membrane has diminished and there is a collection of labeled vesicles nearby. At 196 seconds, VatM-GFP can no longer be detected in the phagosome membrane. At 278 seconds, actin assembly is evident at several points about the phagosome membrane, and by 364 seconds, exocytosis of yeast carcass has been mostly completed. The complete time series is shown in Movie S5. B, at 0 seconds, the upper Dictyostelium cell has four yeast-containing phagosomes labeled with VatM-GFP, one of which (marked with a circle) is more weakly labeled than the others. The cell is also beginning to form a phagocytic cup to take up another yeast, marked with an asterisk; at 94 seconds that phagosome, surrounded by actin filaments, has sealed and is moving into the cell. Meanwhile the VatM-GFP content of the phagosome marked with a circle has diminished further, and by 202 seconds VatM-GFP is no longer detectable. At 310 seconds, actin assembly is seen at several points about that phagosome, and its exocytosis is mostly complete at 455 seconds. Meanwhile the membrane of the new phagosome (asterisk) is becoming enriched in VatM-GFP, although that process occurs rather slowly in this cell because so much of the V-ATPase is already in use in the membrane of other phagosomes . The complete time series is shown in Movie S6. Perkin-Elmer Ultra View microscope. Bars, 5 µm.
Figure 4
Figure 4. Exocytosis of a FITC-yeast from a phagosome whose membrane has been depleted of VatM-GFP by vesiculation.
Dictyostelium cells expressing VatM-GFP and DdmCherry-LimEΔ were mixed with FITC-yeast 4 hours earlier. In the 0 and 113-second panels, a phagosome containing a budded yeast is surrounded by a cloud of VatM-GFP-positive vesicles as it undergoes vigorous movement about the cell. This activity extended over a period of about 5 minutes. At 238 seconds, there are no longer vesicles associated with the phagosome membrane, and the FITC-yeast is visible. By 281 seconds, the yeast has been exocytosed with no change in the intensity of the fluorescent signal, indicating that it was not in an acidic compartment at the time of exocytosis. (See Movie S7.) Perkin-Elmer Ultra View microscope. Bar, 5 µm.
Figure 5
Figure 5. Retrieval of the V-ATPase following premature exocytosis.
A and B, Dictyostelium cells expressing GFP-α-tubulin and mRFP-LimEΔ were mixed with living S. cerevisiae 6 hours earlier. In both time series, a phagosome marked with a circle is exocytosed prematurely, prior to removal of the V-ATPase. A, at 0 seconds, the VatM-GFP-positive phagosome is close to the plasma membrane. A small vacuole brightly labeled with VatM-GFP (arrow) is in the process of budding off from the phagosome membrane. At 119 and 174 seconds, exocytosis is in progress. At 217 seconds, a patch of VatM-GFP is present in the plasma membrane at the site of exocytosis, and a microtubule is lying along the inner surface of the plasma membrane in that area. By 238 seconds, the VatM-GFP signal in plasma membrane is diminishing. The complete time series is shown in Movie S8. B, at 0 seconds, a VatM-GFP-positive phagosome is present near the plasma membrane. At 82 seconds, a large vacuole (V) enriched in VatM-GFP is separating from the phagosome. At 114 seconds, exocytosis of the phagosome is in progress, and at 188 seconds, it has been completed, leaving a bright patch of VatM-GFP in the plasma membrane (arrowhead). This label is much reduced by 271 seconds. (See Supplemental Figure S1 for quantitation.) Meanwhile, the VatM-GFP-positive vacuole (v) has moved about within the cell and undergone a series of morphological changes, including inward budding, which is evident in the 188 and 271-second panels. The complete time series is shown in Movie S9. Perkin-Elmer Ultra View microscope. Bars, 5 µm.
Figure 6
Figure 6. Acidic nature of prematurely exocytosed phagosomes and actin-powered vacuole movement.
A and B show Dictyostelium cells expressing VatM-GFP and DdmCherry-LimEΔ; the cells were mixed with living FITC-labeled S. cerevisiae 5 hours earlier. A, at 0 seconds, VatM-GFP is visible in the phagosome membrane, but the FITC-yeast inside can be seen only faintly. At 2 seconds, a VatM-GFP-positive vacuole (V) is beginning to form and the FITC-yeast has become bright; the yeast bud is now visible. At 11 seconds, the vacuole is separating from the phagosome membrane, and at 18 seconds, it is moving away. New actin assembly detected with DdmCherry-LimEΔ at the rear of the moving vacuole (arrowhead) appears to propel it. The complete time series is shown in Movie S10. B, at 0 seconds, a phagosome containing a budded yeast is faintly green but is more heavily labeled with the red probe for actin filaments. By 8 seconds, the green signal is stronger (appearing yellow where it overlaps the red) as the FITC-yeast has brightened, and a VatM-GFP-positive vacuole (V) has begun to separate from the phagosome. In the 15 and 35-second panels, actin filaments labeled with DdmCherry-LimEΔ can be seen at the rear of the moving vacuole (arrowheads), and by 103 seconds, the vacuole has become an irregular, elongated compartment. The complete time series is shown in Movie S11. Perkin-Elmer Ultra View microscope. Bars, 5 µm.
Figure 7
Figure 7. Schematic showing retrieval of the V-ATPase and fluorescence of FITC-yeast during normal and premature exocytosis.
Normal exocytosis: Near the end of endocytic transit, the V-ATPase (detected as VatM-GFP) is removed from the phagosome membrane in the form of small vesicles over a period of a few minutes. About two minutes after the V-ATPase has been removed, actin assembles at several points on the phagosome membrane, and exocytosis follows. After removal of the V-ATPase, the FITC-yeast is visible through its own fluorescence, which does not increase further upon exocytosis. Premature exocytosis: The V-ATPase (detected as VatM-GFP) is present in the phagosome membrane. A combination of cell movement and trapping of a bulky phagosome bring the phagosome close to the plasma membrane. An abrupt increase in both the volume of the phagosome and the fluorescence of the FITC-yeast occurs, and a large vacuole whose membrane is rich in the V-ATPase separates from the phagosome and moves away. Actin assembly (shown in red) is instrumental in both the separation of the vacuole and its propulsion through the cytoplasm. Upon exocytosis of the FITC-yeast, the V-ATPase remaining in the phagosome membrane is transferred to the plasma membrane, from which it is soon retrieved. (In the diagram, FITC fluorescence is depicted as a lighter shade of green than GFP fluorescence. However, under our experimental conditions the two signals were not distinguishable.)
Figure 8
Figure 8. Rapid reacidification of the remaining portion of a multi-particle phagosome following premature exocytosis.
This cell is expressing mRFP-LimEΔ, and it was mixed with FITC-yeast 3 hours earlier. At time 0, the yeast are dim, indicating an acidic environment. At 4 seconds, both yeast brighten at the onset of premature exocytosis, indicating contact with the higher pH external medium and demonstrating that they share a single compartment. The phagosome membrane seals behind the first yeast as exocytosis is completed, and the phagosome becomes reacidified, indicating that the V-ATPase remains in active form. Time is shown in seconds. Zeiss LSM510 microscope. Bar, 10 µm.
Figure 9
Figure 9. Recruitment of myosin-IB at the onset of premature exocytosis.
This cell is expressing GFP-MyoB and mRFP-LimEΔ; it was mixed with unlabeled yeast two hours earlier. The cell is initially migrating (0 time), but the bulky phagosome soon becomes immobilized. At 84 seconds, the immobilized phagosome has brightened (arrowhead), indicating binding of GFP-MyoB, and a vacuole (tailed arrow) is just separating from the phagosome. The vacuole travels across the cell, labeled with GFP-MyoB and trailed by an actin tail (103 and 106 seconds). Perkin-Elmer Ultra View microscope. Bar, 10 µm.
Figure 10
Figure 10. Increase in phagosome volume and dilution of fluid phase marker prior to premature exocytosis.
This cell was incubated in the presence of both TRITC-dextran and yeast for 3 hours, then rinsed with buffer, covered with a layer of agarose, and observed immediately. This time series (Movie S12) was captured 4 minutes after observation began, so all endosomal compartments except new macropinosomes should be filled with TRITC-dextran. The cell has two V-ATPase-positive phagosomes, each containing two yeast particles and some TRITC-dextran. The phagosome at the upper right expands, a vacuole separates from it, and the two yeast are sequentially exocytosed (although only the first exocytosis is shown). The graphics below the images display the intensity per pixel in the red, green, and brightfield channels along the blue line in each image, as determined by the Zeiss AIM software. The maximum possible intensity value is 255 for these 8-bit images. Although the intensity of the TRITC-dextran (red) signal has saturated the detector in the first image (A), it is clear that the red pixel intensity diminishes in the second image (B) as the phagosome expands, indicating that at least part of the new fluid has come from an unlabeled source. The two small endosomes in the process of fusing at the bottom of the phagosome in A also had saturated peak pixel intensities (not shown); these were incorporated when the phagosome expanded, contributing some additional TRITC-dextran. As a control for photobleaching, a second phagosome with TRITC-dextran content at the bud neck was also scanned (dashed arrows); its TRITC-dextran signal is shown by the dashed lines on the plot. There was no change in pixel intensity between the two time points for the TRITC-dextran in this phagosome. Movie S12 shows the expansion of the phagosome and the dynamic behavior of the vacuole that separates from the phagosome, including the formation of intraluminal vesicles. In the bottom right quadrant of this cell, one can also see the action of the contractile vacuole complex, an osmoregulatory organelle found on the cytoplasmic surface of the substratum-attached plasma membrane; contractile vacuole membranes are also rich in the V-ATPase. Additional time points and a second example are shown in Supplemental Figures S2 and S3. Zeiss LSM 510 microsope.
Figure 11
Figure 11. Changes in the phosphoinositide composition of the phagosome and vacuole in premature exocytosis.
A, these Dictyostelium cells, expressing PHcrac-GFP and mRFP-LimEΔ, were mixed with unlabeled yeast one and a half hours earlier. The upper, more weakly labeled cell is migrating. Arrowheads indicate the direction of cell movement or attempted movement in the first four panels. A phagosome containing a budded phagosome (marked with a circle) is initially pushed along by actin assembly (red), but the phagosome becomes increasingly constrained and presently ceases to move. At the same time, PHcrac-GFP begins to bind to the phagosome membrane (tailed arrow in 250-second panel), indicating the presence of PI(3,4,5)P3 and/or PI(3,4)P2. This biosensor also labels the expanded phagosome (704 seconds) and the vacuole that separates from it (720 and 735 seconds) (tailed arrows). Meanwhile, the phagosome is exocytosed. B, this Dictyostelium cell, expressing GFP-2FYVE and mRFP-LimEΔ, was mixed with FITC-yeast 4 hours earlier. At 0 seconds it contains a phagosome in which the FITC-yeast is barely visible, indicating that the phagosome is acidic. At 14 seconds the FITC-yeast has brightened, indicating a rise in pH within the phagosome, and a vacuole (V) has separated from the phagosome. The vacuole moves rapidly away from the phagosome at the head of a trail of actin filaments, creating a protrusion in plasma membrane at 35 seconds, then rebounding to the cytoplasm at 68 seconds. By 100 seconds the vacuole is binding GFP-2FYVE and has assumed the elongated morphology of an early endosome. Meanwhile the FITC-yeast has been exocytosed. Movie S13 shows the complete time series. A, Zeiss LSM510 microscope; B, Perkin-Elmer Ultra View microscope. Bars, 5 µm.
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