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The WASp-like protein Scar regulates macropinocytosis, phagocytosis and endosomal membrane flow in Dictyostelium

David J. Seastone^1,, Ed Harris¹, Lesly A. Temesvari², James E. Bear^3,*, Charles L. Saxe³ and James Cardelli^1,2,¶

¹ Department of Microbiology and Immunology, Louisiana State University Health Sciences Center Shreveport, LA 71130, USA
² The Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center Shreveport, LA 71130, USA
³ Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322-3030, USA
^* Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA
Present address: Department of Biochemistry, Pikeville College School of Osteopathic Medicine, Pikeville, KY 41501, USA
^¶ Author for correspondence (e-mail: jcarde{at}Isumc.edu )

View larger version (72K): [in a new window]   Fig. 1. scar- cells are defective in phagocytosis, fluid phase pinocytosis and macropinocytosis. To determine the rates of phagocytosis, cells were incubated with 1 µm fluorescent latex beads or FITC-dextran for the indicated times and the intracellular fluorescence was calculated using a spectrofluorimeter. The fluorescence value at each time point was normalized to protein load to account for any difference in cell size among the strains. (A) scar- cells internalized beads at a rate two to three times less than that of control cells, indicating that the mutant was defective in phagocytosis (n=6). (B) scar- cells internalized FITC-dextran at half the rate of control cells, indicating that the mutant also defective in fluid phase pinocytosis (n=5). (C-F) Cells were incubated with 2 mg ml-1 FITC-dextran for 10 minutes, washed twice in fresh HL5 growth medium, spotted onto plastic coverslips and examined using phase contrast (C,E) or fluorescence (D,F) microscopy. Control cells (C,D) contained many large macropinosomal vesicles (arrows), whereas the scar- cells (E,F) contained no large macropinosomes. Bar, 2.5 µm.

View larger version (13K): [in a new window]   Fig. 2. scar- cells are defective in fluid phase exocytosis. To examine exocytosis, wild-type and scar- cells were loaded with FITC-dextran for 3 hours, washed and allowed to efflux FITC-dextran for the indicated times prior to harvesting and fluorescence measurement. The percentage FITC-dextran remaining inside the cell was calculating by comparing the fluorescence values at each of the time points to the value at time T=0. (A) The average of four independent experiments, showing that scar- cells are defective in exocytosis. Whereas wild-type cells have released 50% of the FITC-dextran from the cell by 50 minutes, scar- cells required nearly twice as long (90 minutes) to exocytose 50% of the FITC-dextran. (B) The vesicular pH was calculated over time as described in Materials and Methods. Fluid phase entered acidic vesicles rapidly (within 10 minutes into the chase period in both control Ax3 and scar- cells). However, the fluid phase only slowly left lysosomes in scar- cells and did not reach more neutral pH post-lysosomes until after 60 minutes into the chase period, whereas, in wild-type cells, the fluid phase entered the post-lysosomes within 45 minutes.

View larger version (68K): [in a new window]   Fig. 3. scar- cells contain mostly small acidic vesicles. Phase contrast (A,C) and fluorescent (B,D) microscopic images of control (A,B) and scar- cells (C,D). (A-D) Cells were incubated with FITC-dextran for 1 hour, washed and spotted onto coverslips prior to examination. In control cells (A,B), vesicles of many different sizes were present, including large post-lysosomal and macropinosomal vesicles (arrows). By contrast, scar- cells contained primarily smaller vesicles. (E,F) scar- cells were incubated with RITC-dextran for 1 hour, washed and further incubated with Lysosensor Green for 10 minutes. After spotting cells on coverslips, they were examined using the red channel (E) or the green channel (F) of the fluorescence microscope. Most of the RITC-dextran-positive vesicles (E) also stained with Lysosensor Green (F), indicating that most of the vesicles in the scar- cells were acidic. Bar, 5 µm.

View larger version (84K): [in a new window]   Fig. 4. F-Actin rings endo-lysosomes in control but not in scar- cells. Cells expressing GFP-ABD (A) were incubated with RITC-dextran in growth medium for 1 hour (B), recovered by centrifugation and fixed with formaldehyde prior to visualization using a fluorescence microscope. These two panels show that all the vesicles ringed with F-actin were endo-lysosomal in nature. (C-F) Control cells (C,D) and scar- cells (E,F) were fixed and decorated with fluorescent phalloidin to visualize F-actin. (C,E) Fluorescent images; (D,F) Differential interference contrast (DIC) images. Bar, 5 µm.

View larger version (63K): [in a new window]   Fig. 5. Cells with the pI-/II-/scar- triple mutation are tight aggregate mutants and show abnormal F-actin staining. As described in Materials and Methods, strains were produced that were null for profilin I and profilin II and Scar. (A,B) Development of profilin double mutants (pI-/II-) and triple mutants (pI-/II-/scar-), respectively. The absence of Scar does not significantly alter the profilin-null developmental phenotype. However, cortical F-actin staining as visualized with TRITC-phalloidin was significantly reduced in the triple mutant (D) relative to the double profilin mutant (C). TRITC-phalloidin staining of wild-type (E) and Scar null cells (F) are included for comparison. The arrow identifies a forming F-actin-rich macropinosome in control cells (E), structures that are absent from all the mutant cell lines. Bar, 10 µm.

View larger version (14K): [in a new window]   Fig. 6. Cells with the pI-/II-/scar- triple mutation are severely defective in fluid phase pinocytosis, exocytosis and lysosomal enzyme secretion. Cells were incubated with FITC-dextran and, at various times, the intracellular fluorescence was calculated as described in Materials and Methods; the averages of four independent experiments are shown (A). Alternatively, cells were loaded with FITC-dextran for 3 hours, washed and placed back in fresh growth medium. At various times, cells were collected, washed and the remaining intracellular FITC-dextran was measured (B). Finally, cells growing exponentially were collected by centrifugation and the intracellular and extracellular levels of -mannosidase were measured (C). (A) pI-/II-/scar- cells displayed a defect in pinocytosis compared with control cells, and pI-/II-/scar- cells showed an additive defect compared with Scar and profilin null mutants alone, supporting the hypothesis that these two proteins interact to regulate fluid internalization. (B) pI-/II- cells displayed an exocytic defect: 50% of the fluid phase remained inside the cell after 90 minutes post-chase (compared with 45 minutes for release of one half of the fluid phase from control cells). Cells with the pI-/II-/scar- triple mutation showed an additive exocytic defect: 50% of the fluid phase remained inside the cell after 150 minutes into the chase period. After 3 hours into the chase period, none of the fluid phase remained in control cells, whereas 20% remained in the pI-/II- cells and 40% remained inside the pI-/II-/scar- cells. (C) The steady state secretion rate of -mannosidase was calculated by comparing the extracellular enzymatic activity with the total enzymatic activity of the cells and supernatant from pelleted cells. At steady state, only 20% of the -mannosidase activity remained inside the cell, whereas 80% of the activity resided in the supernatant, owing to secretion of the lysosomal hydrolase. In scar- cells, 40% of the -mannosidase activity remained inside the cell, and for the pI-/II- cells, 30% of the enzymatic activity remained intracellular, indicating that there was a secretion defect in both of these strains. The pI-/II-/scar- cells displayed an additive secretion defect: only 15% of the -mannosidase activity was found in the supernatant.

View larger version (93K): [in a new window]   Fig. 7. Cells with the pI-/II-/scar- triple mutation contain mainly small acidic endosomes. To examine the morphology of the endo-lysosomal system of pI-/II- and pI-/II-/scar- mutants, cells were incubated with FITC-dextran for 1 hour, spotted on coverslips and prepared for phase contrast (A,C,E) or fluorescence (B,D,F) microscopy. Control cells (A,B) contained vesicles of many different sizes, representing pinosomes, macropinosomes, lysosomes and post-lysosomes. By contrast, pI-/II- and pI-/II-/scar- cells (C-F) contained only smaller vesicles that are presumed to be lysosomes, because they stained with an acidic fluorophore (data not shown). Bar, 5 µm.