SCAR knockouts in Dictyostelium: WASP assumes SCAR's position and upstream regulators in pseudopods

Abstract

Under normal conditions, the Arp2/3 complex activator SCAR/WAVE controls actin polymerization in pseudopods, whereas Wiskott-Aldrich syndrome protein (WASP) assembles actin at clathrin-coated pits. We show that, unexpectedly, Dictyostelium discoideum SCAR knockouts could still spread, migrate, and chemotax using pseudopods driven by the Arp2/3 complex. In the absence of SCAR, some WASP relocated from the coated pits to the leading edge, where it behaved with similar dynamics to normal SCAR, forming split pseudopods and traveling waves. Pseudopods colocalized with active Rac, whether driven by WASP or SCAR, though Rac was activated to a higher level in SCAR mutants. Members of the SCAR regulatory complex, in particular PIR121, were not required for WASP regulation. We thus show that WASP is able to respond to all core upstream signals and that regulators coupled through the other members of SCAR's regulatory complex are not essential for pseudopod formation. We conclude that WASP and SCAR can regulate pseudopod actin using similar mechanisms.

Figure 1.

SCAR complex and WASP localization in wild-type D. discoideum cells. (A) TIRF microscopy of SCAR complex (labeled with HSPC300-GFP) in pseudopods, filopods, cell–cell contact, and cell spreading of wild-type cells. Numbers indicate the time in seconds. The position of the asterisks is fixed across different time points. Arrows show SCAR-rich protrusions. (B and C) Colocalization of GFP-tagged SCAR complex (B) and WASP (C) with RFP-tagged Arp2/3 complex (subunit ARPC4) during cell migration. Quantitations of the fluorescence intensity along the indicated lines through the pseudopod are displayed on the right. Images are representative of ≥50 cells observed. (D) Colocalization of GFP-WASP and RFP-clathrin light chain. (E) Kymograph from a video similar to D showing arrival and disappearance of clathrin and WASP during a single clathrin-mediated endocytosis event. a.u., arbitrary unit. Bars, 5 µm.

Figure 2.

WASP compensates for the loss of SCAR. (A) Colocalization of WASP and Arp2/3 complex in SCAR-null cells. (B) Kymograph showing distribution of GFP-tagged WASP and SCAR complex with RFP-tagged Arp2/3 complex in a wild-type cell protrusion. The protrusion extends upwards, and time is along the horizontal axis. (C) Kymograph of WASP and the Arp2/3 complex in a protrusion of a SCAR-null cell. (D) Quantification of the number of pseudopods with SCAR complex and with WASP and the number of blebs in wild-type and SCAR-null cells. Over 200 pseudopods/blebs were counted in a total of 40 cells that were recorded with time-lapse microscopy for a length of 10 min each. (E) Western blot of whole-cell lysates of the indicated strains with an anti-WASP antibody. After immunodetection, the blot was stained with Coomassie brilliant blue. Molecular mass (Mm) size markers are indicated on the right in kilodaltons. (F) TIRF microscopy image of GFP-WASP in a SCAR-null cell that is dropping out of solution and spreading on the glass substratum. Time is indicated in seconds. Bars, 5 µm.

Figure 3.

WASP dynamics in SCAR complex mutants. (A) TIRF image series of splitting pseudopods in a wild-type cell labeled with HSPC300-GFP and a SCAR knockout cell labeled with GFP-WASP. Cells are moving to the right, and time is indicated in seconds. (B) TIRF image series of WASP waves in a SCAR-null cell. In the top images, this image is overlaid with the localization of WASP 2 s before the current frame, and in the bottom images, this image is overlaid with the Arp2/3 complex. A single-pass Gaussian blur was applied to the full image to smooth background fluorescence. The arrow tracks a single WASP wave. (C) TIRF images of GFP-WASP and RFP-tagged Arp2/3 complex in strains deleted for PIR121, Nap1, Abi, and HSPC300. Bars, 5 µm.

Figure 4.

Correlation between WASP, SCAR, and active Rac. (A) Visualization of the active Rac marker CRIB-GFP in a migrating cell in a confocal slice at a position 1 µm above the basal membrane (confocal) and directly on the basal membrane (TIRF). Arrowheads mark active protrusions. (B) Schematic drawing of the distribution of active Rac in a migrating cell. (C) TIRF image of a migrating cell coexpressing the active Rac marker CRIB-RFP and a marker for the SCAR complex (HSPC300-GFP) or GFP-WASP. (D) Quantification of the number of visible SCAR and WASP protrusions that are located on active Rac patches and outside of active Rac patches, respectively (n = 130 pseudopods from two experiments). (E) CRIB-GFP and free RFP were coexpressed in the indicated cells and visualized using TIRF microscopy. Active Rac distribution was normalized by dividing the CRIB-GFP signal by the free RFP signal. Background signal outside of the cell was masked. Arrow indicates cell’s direction. (F) Short videos of cells coexpressing CRIB-GFP and free RFP were recorded. Active Rac levels were normalized as described in E. The fold enrichment of active Rac in pseudopods in the normalized image was calculated by dividing the mean pixel value of the pseudopod by the mean pixel value of the cytosol. For each cell, the fold enrichment in pseudopods of each cell is plotted. Each data point is the mean of at least three pseudopods. The difference between the means is significant (Student’s t test, P < 0.001). Lines and error bars indicate means ± SEM. Bars, 5 µm.

Figure 5.

Model of protrusion formation by SCAR and WASP in wild-type cells. Rac1 activation of the SCAR complex leads to new actin filament formation. We predict the following: (a) Feedback through actin filaments results in excitable behavior and a normal pseudopod cycle. (b) The Rac signal is turned off through negative feedback as the pseudopod matures. (c) In the absence of SCAR, protrusion formation is diminished, and active Rac levels increase until they engage WASP, which then replaces SCAR in essentially all respects. (d) In SCAR-null cells, as in wild-type cells, evolving pseudopods are formed through feedback, and normal protrusions are formed, though at a reduced frequency.