Figure 1.
Characteristics of Dictyostelium AdcA.
(A) Detailed domain structure of AdcA. Are indicated the H domain that contains the histidine-rich tri-repeat (H1-H2-H3), the hydrophobic region (Ф domain), the arrestin N- and C domains, the zinc-finger FYVE domain and the C-terminal extension rich in tyrosine residues (Y domain). (B) Protein sequence of AdcA. The limits of the H, Ф and arrestin domains are indicated by a thick gray or black underlining. Underlined AdcA residues correspond to putative clathrin- (dotted line) and adaptin- (continuous line) binding motifs. Stars indicate the two peptides used to produce the rAb-AdcA antibody. (C) Homology modeling of the Dictyostelium AdcA core (amino acids 140–460). Residues are shown in ribbon format, with the color code running from blue (N-terminal) to red (C-terminal) along the polypeptide chain. The 2WTR 3D-structure corresponding to bovine β-arrestin 1 is shown for visual comparison with AdcA's modeled structure. (D) Alignment of the histidine-rich repeats of the H domain. Identical amino acids in the three repeats H1, H2, and H3 are shaded in black. Conservative changes or residues conserved in only two repeats are shaded in gray. Dashes indicate gaps. (E) Alignment of FYVE domains present in Dictyostelium AdcA, human EEA1 and human SARA proteins. Identical amino acids in the three sequences are shaded in black. Conservative changes or residues conserved in only two sequences are shaded in gray. Dashes indicate gaps and (+) stands for lysine or arginine.
Figure 2.
AdcA is docked to endocytic compartments.
(A) Subcellular fractionation. KAx-3 and KAx-3 AdcAGFP cells were broken in different buffers (Mes-Na pH 6.5 or Na-carbonate pH 11) as mentioned in Materials and Methods. The postnuclear supernatant was spun by high-speed centrifugation (30 min, 135,000×g, TL100 Beckman) to separate membranes and soluble fractions. Samples were analyzed by Western blot using either gAb-AdcA or anti-GFP antibodies. The adcA null strain was used as a control. T, total before centrifugation; S, soluble fraction; M, membranes. (B), (C) AdcA is present on endocytic vesicles. KAx-3 and KAx-3 expressing AdcAmyc were fixed in methanol and processed for immunofluorescence with an anti-myc antibody (B) or the gAb-AdcA antibody (C). The scale bar represents 3 µm. (D) Cells expressing AdcAGFP were let to internalize the fluid-phase marker TRITC-dextran for 2 hr. Live imaging was then performed on a Leica TCS-SP2 confocal microscope. Panels illustrate the location of AdcAGFP, TRITC-dextran and the overlay in false colors of both fluorescences. The full arrowhead points to a vesicle positive for both TRITC-dextran and AdcAGFP. Open arrowheads point to vesicles positive for TRITC-dextran but negative for AdcAGFP. (E) Cells expressing AdcAGFP were let to internalize Texas Red-labeled zymosan BioParticles® for 2 hr. Live imaging was then performed on a Leica TCS-SP2 confocal microscope. Panels illustrate the location of AdcAGFP, Texas Red-labeled zymosan and the overlay in false colors of both fluorescences. The full arrowhead points to a vesicle positive for both Texas Red-labeled zymosan BioParticles® and AdcAGFP. Open arrowheads point to vesicles positive for BioParticles® but negative for AdcAGFP. A dashed line delineates cell outlines. The scale bar in (D) and (E) represents 4 µm.
Figure 3.
AdcAGFP is present on early endocytic compartments.
(A) Cells expressing AdcAGFP were pulsed with TRITC-dextran for 5 min. After several washes to remove the external marker, cells were resuspended in fresh culture medium and visualized after 1 min chase (top) or 15 min chase (bottom) on a Zeiss Axiovert 200 M microscope. During the chase period, cells accumulated compartments positive for TRITC-dextran but devoid of AdcAGFP (full arrowheads). New endosomes formed during the chase period were positive for AdcA and negative for TRITC-dextran (empty arrowhead). (B–E) Cells expressing AdcAGFP were fixed in methanol and processed for immunofluorescence with anti-cathepsin D (B), anti-vacuolin (C, top), anti-p80 (C, bottom) and anti-actin (D, E) antibodies as well as DAPI to stain the nucleus. In panel (C), a strongly p80-positive, AdcA-negative post-lysosome is indicated by a full arrowhead. p80-positive, AdcA-positive endosomes are indicated by empty arrowheads. Panel (E) illustrates the concentration difference of AdcA on a macropinocytic cup and an early macropinosome identified by actin staining. For panels (B), (C), (D) and (E), in order to enhance resolution, optical sections were taken every 0.250 µm throughout the cell and digitally deconvolved using Zeiss Axiovision software. A median z section is shown. The scale bar represents 2 µm.
Figure 4.
AdcA harbors a functional FYVE domain.
(A) Lipid overlay assay. AdcA FYVE domain was expressed as a MBP-fusion protein and tested for its binding to lipids immobilized on a membrane. MBP alone was used as a control. LPA, lysophosphatidic acid; LPC, lysophosphocholine; PI, phosphatidylinositol; PI3P, phosphatidylinositol 3-phosphate; PI4P, phosphatidylinositol 4-phosphate; PI5P, phosphatidylinositol 5-phosphate; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphingosine 1-phosphate; PI3,4P2, phosphatidylinositol 3,4-bisphosphate; PI3,5P2, phosphatidylinositol 3,5-bisphosphate; PI4,5P2, phosphatidylinositol 4,5-bisphosphate; PI3,4,5P3, phosphatidylinositol 3,4,5-trisphosphate; PA, phosphatidic acid; PS, phosphatidylserine; no, blank. (B) Binding of Ins(1,3)P2. Variations of tryptophan intrinsic fluorescence (λex 290 nm) of MBP-FYVE (o) and MBP (•) were measured between 300 and 400 nm in response to addition of Ins(1,3)P2, a soluble analog of PI(3)P. Variations were expressed as ΔF/F0 in percent. (C) AdcA membrane association is pH dependent. Cells expressing AdcAGFP were broken in different buffers at different pHs and treated as in panel (A).
Figure 5.
Role of AdcA subdomains in its endosomal location.
(A) Deletion constructs. To test the contribution of AdcA subdomains to the protein subcellular location, various truncated constructs were generated, tagged with the GFP at the N- or C-terminus and expressed in KAx-3 cells under act15 promoter. Numbers correspond to the constructs borders. (B) The distribution of AdcAΔFYGFP after fractionation was analyzed by Western Blot as described in Figure 2. (C) The location of the GFP-tagged truncated proteins was observed in live cells by confocal microscopy to evaluate the effect of the truncation on the endosomal docking during macropinocytosis. (D) Cells expressing the mentioned constructs were let to internalize Texas Red-labeled zymosan BioParticles® for 2 hr prior observation. The upper panel illustrates the subcellular location of the AdcAGFP constructs and the lower panel corresponds to the overlay of green (AdcA constructs) and red (phagocytic probe) fluorescences. The scale bar represents 2 µm.
Figure 6.
The C domain of AdcA arrestin core interacts with Dd-ArfA in vitro.
AdcAC was expressed and purified as a GST-fusion protein. ArfA was tagged with a C-terminal His6 tag and purified on a Ni-NTA column. GST-AdcC and GST were kept bound to the glutathione Sepharose beads and incubated overnight at 4°C with ArfA in the presence of 100 µM GDPβS, GTPγS or no extra nucleotide. After several washes in incubation buffer, beads were resuspended in Laemmli denaturating gel buffer and proteins were analyzed by Western blot for the presence of ArfA.
Figure 7.
Subcellular localization of ArfA.
(A) KAx-3 cells expressing ArfAGFP were let to adhere in Labtek chambers. DMSO was added (t = 0) at a final concentration of 5% and cells were observed on a Zeiss Axiovert 200 M microscope immediately or after 10 min or 45 min of incubation. (B) KAx-3 cells expressing ArfAGFP were fixed in 4% PFA, permeabilized in methanol and stained with the monoclonal antibody 1/39 described to label the Golgi apparatus [55]. The overlay panel correspond to the overlay of the green and red fluorescences in addition to the nuclear DAPI staining. (C) Cells expressing both ArfAGFP and RFPAdcA were fixed and observed. The arrow indicates an AdcA positive vesicle in close proximity with the Golgi apparatus. (D) Live cells expressing ArfAGFP were observed by time-lapse microscopy. Individual pictures corresponding to the indicated frames (obtained every 0.25 sec) were extracted from the movie (Movie S1). ArfA-positive tubules are indicated by empty arrowheads. The full arrowhead in frames 11–24 shows an ArfA-positive vesicle associated with an ArfA-positive tubule, first moving away from the Golgi apparatus (centrifugal direction) and finally reversing its movement (centripetal direction).