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Dynamics of the vacuolar H⁺-ATPase in the contractile vacuole complex and the endosomal pathway of Dictyostelium cells

¹ Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, USA
² Max-Planck-Institut für Biochemie, D82152 Martinsried, Germany
³ Washington University School of Medicine, St Louis, Missouri, USA

View larger version (82K): [in a new window]   Fig. 3. Dynamics of the contractile vacuole system revealed by VatM-GFP and interference reflection microscopy. An AX3 cell expressing VatM-GFP was viewed simultaneously by fluorescence microscopy (A-C) and IRM (D-F). These images correspond to frames 14 (A,D), 36 (B,E) and 51 (C,F) from Movie 1. Images were captured at 1 second intervals, and so these images span a total of 36 seconds. Three vacuoles in the upper right portion of the cell emptied during this time interval and were replaced by a tracery of tubules. The arrowhead marks the same position in each frame. Two other vacuoles (upper left) fused during this same interval and later emptied (see Movie 1).

View larger version (52K): [in a new window]   Fig. 1. Effect of VatM-GFP expression on the distribution of VatA in bacterially grown VatMpr cells. VatMpr and VatMpr/VatM-GFP cells were grown for two days on K. aerogenes, then fixed and stained with the N4 monoclonal antibody, which recognizes the A subunit of the V-ATPase, followed by Cy3-labeled anti-mouse antibodies. A and B show VatMpr cells (A, Cy3; B, phase contrast), and C to E show VatMpr/VatM-GFP cells (C, Cy3; D, phase contrast; E, GFP). VatA is diffusely distributed in VatMpr cells (A), but, as seen in C, colocalizes with VatM (E) on contractile vacuole membranes in VatMpr/VatM-GFP cells. In cells with poor expression of VatM-GFP, VatA remains diffuse (two cells at the lower left in C and E). Bar, 10 µm.

View larger version (102K): [in a new window]   Fig. 2. Orientation of GFP on the C-terminus of VatM. Cells expressing VatM-GFP were fed TRITC-dextran, and endosomes were isolated on a Percoll gradient. Endosomes, identified by TRITC-dextran fluorescence (A), were rimmed with green fluorescence (B) from VatM-GFP in the endosomal membranes; C shows the two images overlaid. Following brief treatment with Proteinase K, TRITC-dextran was still present in the endosomes (D), although the green fluorescence (E) had disappeared from endosomal membranes; F shows the two images overlaid.

View larger version (52K): [in a new window]   Fig. 4. Labeling of contractile vacuole and endosomal membranes with VatM-GFP. Dictyostelium cells expressing VatM-GFP were observed following a 70 minute incubation in one-third strength nutrient medium containing TRITC-dextran (2 mg/ml). Three signals were recorded: green (VatM-GFP), red (TRITC-dextran), and blue (phase contrast). All three channels are shown in A and B, whereas C shows the same image as B minus the red channel. A is a focal plane near the substratum showing the contractile vacuole complex, and B and C are a focal plane higher in the cell, showing several endosomes and also some elements of the contractile vacuole complex including several vacuoles, which are phase lucent. The arrows mark endosomes whose periphery is not labeled with VatM-GFP (presumptive late endosomes). Bar, 10 µm.

View larger version (58K): [in a new window]   Fig. 5. Clustering of acidic endosomal vesicles labeled with VatM-GFP at the membrane of new phagosomes, followed by the appearance of VatM-GFP in the phagosome membrane. Cells expressing VatM-GFP were fed yeast particles in the presence of Neutral Red (A) or TRITC-dextran (B-D). The TRITC-dextran was present for at least 30 minutes before addition of the yeast. (A) Neutral Red-positive vesicles rimmed with VatM-GFP clustered next to a phagosome (2 minutes after uptake of the yeast particle). The yeast particle is marked with a white diamond. (B-D) A large cell containing several yeast particles taking up a new yeast particle (marked with a white diamond); (B) phagosome membrane closing around the new yeast particle; (C) clustering of TRITC-dextran-positive, VatM-GFP-rimmed vesicles at the membrane of the new phagosome (70 seconds later); (D) similar intensity of VatM-GFP label in membranes of new and old phagosomes (4 minutes after uptake). Bars, 10 µm.

View larger version (64K): [in a new window]   Fig. 6. Delivery of endosomal content to the phagosome. (A) A cell fed yeast in the presence of Neutral Red. Within the cell are three phagosomes containing yeast particles, all with Neutral Red accumulation at the bud neck. The upper phagosome also has mounds of Neutral-Red-positive material at sites along its membrane. (There are also two free yeast particles just above the cell; these are not stained.) (B,C) A cell whose endosomes were labeled with TRITC-dextran prior to addition of yeast particles. Mounds of TRITC-dextran are present along the phagosome membrane (B). Subtraction of the red channel shows that VatM-GFP is continuous around the outside of these mounds (C), suggesting that they are sites of fusion between endosomal vesicles and the phagosome membrane. Bar, 10 µm.

View larger version (43K): [in a new window]   Fig. 7. Time course of VatM-GFP delivery to a new phagosome. This cell already contained several yeast particles in a multi-particle phagosome whose membrane was strongly labeled with VatM-GFP. The cell had been pre-labeled with TRITC-dextran, most of which was also localized in this large phagosomal compartment. The paucity of free endosomes correlated with slow delivery of VatM-GFP to the new phagosome. (A) Uptake of the new yeast particle; (B) 3 minutes and 15 seconds after uptake; (C) 6 minutes and 20 seconds after uptake; (D) 9 minutes after uptake. In C and D, TRITC-dextran is visible at the bud neck of the new phagosome and also at a point of contact between the new phagosome and the pre-existing phagosomal compartment, suggesting that fusion has occurred. Bar, 10 µm. (These frames are taken from Movie 2.)

View larger version (106K): [in a new window]   Fig. 8. Digestion of a yeast particle indicated by Neutral Red staining of the yeast cytoplasm. Neutral Red is excluded from living yeast particles but floods and stains the cytoplasm of phagocytosed yeast particles when digestion has progressed sufficiently. This transition takes place suddenly. VatM-GFP is still present in the phagosome membrane at this time. The image in B was recorded 30 seconds after the image in A. Bar, 10 µm.

View larger version (76K): [in a new window]   Fig. 9. Late phagosomes without membrane labeling by VatM-GFP. The membrane of the multi-particle phagosome in the upper part of this cell is labeled with VatM-GFP. TRITC-dextran has accumulated between yeast particles in this phagosome as well as at the bud necks. Below this large compartment are three phagosomes without VatM-GFP in their membranes (arrowheads); the presence of TRITC-dextran indicates that these are late phagosomes. The image in B is the same as that in A, but without the blue (phase contrast) channel. Bar, 10 µm.

View larger version (47K): [in a new window]   Fig. 10. Exocytosis of a digested yeast particle. (A-D) A cell containing two phagosomes with VatM-GFP-labeled membranes and a third with an unlabeled membrane (arrowheads). (A) A focal plane showing all three phagosomes; (B) a focal plane showing that the bud neck of the yeast particle in the unlabeled phagosome has a TRITC-dextran collar and is therefore a late phagosome; (C,D) two frames 5 seconds apart showing expulsion of the yeast particle from the unlabeled phagosome. Bar, 10 µm. (These frames are taken from Movie 3.)