Pseudopod growth and evolution during cell movement is controlled through SCAR/WAVE dephosphorylation

Abstract

Background: SCAR/WAVE is a principal regulator of pseudopod growth in crawling cells. It exists in a stable pentameric complex, which is regulated at multiple levels that are only beginning to be understood. SCAR/WAVE is phosphorylated at multiple sites, but how this affects its biological activity is unclear. Here we show that dephosphorylation of Dictyostelium SCAR controls normal pseudopod dynamics.

Results: We demonstrate that the C-terminal acidic domain of most Dictyostelium SCAR is basally phosphorylated at four serine residues. A small amount of singly phosphorylated SCAR is also found. SCAR phosphorylation site mutants cannot replace SCAR's role in the pseudopod cycle, though they rescue cell size and growth. Unphosphorylatable SCAR is hyperactive-excessive recruitment to the front results in large pseudopods that fail to bifurcate because they continually grow forward. Conversely, phosphomimetic SCAR is weakly active, causing frequent small, disorganized pseudopods. Even in its regulatory complex, SCAR is normally held inactive by an interaction between the phosphorylated acidic and basic domains. Loss of basic residues complementary to the acidic phosphosites yields a hyperactive protein similar to unphosphorylatable SCAR.

Conclusions: Regulated dephosphorylation of a fraction of the cellular SCAR pool is a key step in SCAR activation during pseudopod growth. Phosphorylation increases autoinhibition of the intact complex. Dephosphorylation weakens this interaction and facilitates SCAR activation but also destabilizes the protein. We show that SCAR is specifically dephosphorylated in pseudopods, increasing activation by Rac and lipids and supporting positive feedback of pseudopod growth.

Figure 1. Phosphorylation of the SCAR acidic region

A. Phosphorylation-dependent bandshift. Lysates of vegetative and developed wild type (AX3) cells were treated with calf intestine phosphatase (CIP) then blotted and probed with anti-SCAR. CIP treated (+) SCAR shows higher mobility than untreated (-) SCAR. B. Isoelectric point focusing. Vegetative and developed wild type (AX3) cells were separated by IEF, blotted and probed with anti-SCAR. Left panel - normal exposure; right panel – overexposed to show endogenous unphosphorylated SCAR. C. IEF analysis of partially dephosphorylated SCAR. AX3 lysates were partially (+) and completely (+++) phosphatase treated, then separated by IEF. Focused band positions are indicated (*); numbers represent deduced numbers of phosphates. Right panel contrast-enhanced to show endogenous unphosphorylated SCAR. D. Loss of bandshift following acidic region deletion. Above – schematic of SCAR primary structure, showing SCAR homology domain (SHD), basic region (B), proline rich region (P), WH2 domain (W), central region (C), acidic region (A). Below: Full length and SCAR lacking the acidic region (ΔA) were expressed in SCAR null cells (IR46), and lysates were examined with or without CIP treatment. E. Acidic region mutations. Above: acidic regions of human SCAR/WAVE2 and Dictyostelium SCAR. Phosphorylatable serine residues are indicated (*). All were substituted with alanine in the unphosphorylatable mutant (SA), and with aspartic acid in the phosphomimetic mutant (SD). Below: Wild type and SA & SD mutant SCARs were expressed in SCAR knockout (IR46) cells, and examined with and without CIP treatment. Neither mutant shows a band-shift due to phosphorylation.

Figure 2. Phenotypes caused by SCAR mutants.

A. Growth rate. SCAR SA and SD mutants were expressed in SCAR knockouts (IR46), and their growth rates in suspension were compared. Cells expressing SCAR-SA and SCAR-SD grew as fast as wild type. Data show mean ±SD from 3 independent cultures. B. Cell size. SCAR, SA and SD mutants were expressed in SCAR knockouts (IR46), grown in suspension, and their cell size was measured using a CASY cell counter. scar^- cells are 64% of wild type (WT) volume. Data are shown as mean ±SD from 3 24h time points of 3 independent cultures (n=9). C. Migration speed. Average speeds were quantified from movies of development on agar. Data are shown as mean ±SD from 9 independent cell tracks. SCAR-SA and SCAR-SD mutants rescued cell speed significantly, but less well than wild type SCAR. D. Cell morphology. SCAR knockouts expressing normal and mutated SCARs were developed on agar, and observed by phase contrast microscopy. Expression of SCAR, but not mutant forms, restores normal pseudopod splitting. SCAR-SA cells are excessively long and polarised, with no pseudopod splitting. SCAR-SD cells generate new pseudopods, but they are small and uncoordinated, yielding a rounded morphology.

Figure 3. Pseudopod extension caused by unphosphorylatable SCAR

A. Pseudopod dynamics during chemotaxis. Chemotaxis to cAMP under agar was examined by DIC microscopy in developed IR46 (scar^-) cells expressing wild type SCAR (WT) or unphosphorylatable SCAR-SA. The outline of the leading edge in the image at 0s is shown. scar^- cells mostly move using blebs (arrows) under these conditions. In contrast, pseudopods from cells expressing SCAR-WT and SCAR-SA grow by smooth extension (arrowheads). B. Kymographs of pseudopod dynamics. The leading edges of scar^-, WT, and SCAR-SA were tracked in DIC movies, and the results plotted as kymographs. The X axis of each kymograph represents pseudopod extension (rightward), and the Y axis indicates time. Scale bars, 1 minute. Inset images are 2 × magnified images of indicated area. The leading edge of scar^- cells advances in small, sudden steps, reflecting movement through blebs. WT edges intersperse smooth forward movement with pauses; SA mutant edges move forward at the same rate as WT cells, but each period of smooth movement lasts longer. C. Bleb frequency. Blebs were counted from 3 independent DIC movies, and shown as mean ±SD.

Figure 4. Aberrant pseudopod extension caused by phosphomimetic SCAR

A. Small pseudopods. Developed scar^- (IR46) cells expressing SCAR-SD were observed on agar by DIC microscopy at 1 frame/2s. Thready pseudopods (arrowheads) are seen splitting off the leading edge. B. Uncoordinated pseudopod generation. Developed scar^- (IR46) cells expressing SCAR-SD were observed under agar by DIC microscopy at 1 frame/2s. The leading edge at 0s is indicated. Smoothgrowing pseudopods (arrowheads), and frequent blebbing (arrows) combine to give an irregular edge.

Figure 5. SCAR accumulation in mutants.

A. Increased SCAR recruitment in unphosphorylatable mutants. Developed scar^- (IR46) cells expressing HSPC300-GFPand the indicated SCAR mutant were allowed to chemotax under agar and observed by wide-field fluorescence microscopy at 1 frame/5s. Arrowheads indicate SCAR complex accumulation. B. Size of SCAR patches. The width of each patch of membrane-localised SCAR was measured from the same movies as Figure 5A. In each case, the peak length along the cell perimeter of each separate accumulation event was recorded. Data shown as mean ± SD (n^{SCAR WT} = 110, n^{SCAR SA} = 138, n^{SCAR SD} = 147). C. Lifetime of SCAR patches. The survival of SCAR accumulation for each mutant were measured directly from the movies. The mean lifetimes of SCAR-SA patches was significantly longer, and of SCAR-SD significantly shorter than SCAR-WT. Data shown as mean ± SD (n^{SCAR WT} = 101, n^{SCAR SA} = 123, n^{SCAR SD} = 140). D. Frequency of SCAR patch generation. SD mutants showed a significant increase in the frequency of new SCAR patches when compared with SCAR-WT; SA was not significantly different. Data shown as mean ± SD (n^{SCAR WT} = 12, n^{SCAR SA} = 12, n^{SCAR SD} = 11). E. Unphosphorylatable SCAR still causes actin polymerization. Developed scar^- (IR46) cells coexpressing HSPC300-GFP, mRFP-actin and unphosphorylatable SCAR-SA were allowed to chemotax under agar and observed by wide-field fluorescence microscopy at 1 frame/5s. Arrowheads indicate accumulation of SCAR complex (green) and actin (red). Arrows indicate loss of SCAR complex or actin patches.

Figure 6. Instability of SCAR mutants

A. Instability of SCAR mutants during migration. Cells expressing normal and mutant SCAR were harvested at the indicated stages (vegetative, starvation 2 hrs and shaking-developed 2hrs, streaming 4 hrs) and SCAR levels were measured from 3 independent blots (see Fig. S4; shown as mean ± SD). Levels of SCAR-SA drop by about 60%, while normal SCAR levels remain approximately constant. B. Cycloheximide treatment. Starved cells were treated with or without 100 μM cycloheximide and allowed to stream. Normal SCAR levels dropped gradually, but most protein remained after 3 hrs. In contrast, unphosphorylatable SA levels dropped rapidly, indicating rapid degradation of unphosphorylated SCAR.

Figure 7. Autoinhibition caused by phosphorylation-dependent interaction between basic and acidic motifs of complexed SCAR.

A. Proximity between the basic and acidic motifs of complexed SCAR/WAVE. The structure of the mini-WRC (PDB ID: 3P8C; a derivative of the human SCAR/WAVE1 complex; from [26]) was replotted to show proximity between the basic domain (B) and the end of the C region (C). The C-terminal acidic domain was not resolved; its approximate position is shown (A). B. Orientation of basic residues in the basic domain. The interaction between basic and acidic motifs shown above was replotted to show location of basic side chains. 5/8 of the basic residues (red) face the region of the acidic domain; 3/8 face outwards. C. The Dictyostelium SCAR basic region. Most basic residues are found in a pattern implying localisation at one side of an predicted α-helix. Substitutions in the neutralized (BN) mutant are indicated in red, and unaltered basic residues in blue. D. Phosphorylation-dependent interaction between isolated B and WCA domains. Recombinant SCAR-WCA fused to GST was bound to glutathione-agarose beads, with or without phosphorylation by CK2. Beads were then mixed with FITC-labelled B region peptide, and bound peptide was quantified by fluorimetry. Data shown mean ±SD from 3 independent experiments. Phosphorylated WCA interacted more strongly with B peptide than unphosphorylated WCA. E. Similar migration defect caused by basic domain and acidic domain SCAR mutants. Developed cells expressing basic domain neutralized (BN) mutant SCAR were viewed on agar by phase contrast. The BN-expressing cells resemble cells expressing SA mutant SCAR in their hyperpolarization and movement. F. Dominant pseudopods caused by basic domain mutations. Developed cells expressing SCAR-BN were examined by DIC chemotaxing under agar (1 frame/2s). The initial leading edge is indicated. The edge of the pseudopod (arrows) extended smoothly like SCAR-SA pseudopods. See Movie S9. G. Kymography of SCAR-BN mutant. Cells were examined as in F. The X-axis of the kymograph shows the pseudopod extension distance (rightward), and the Y-axis shows time. Scale bars, 1 minute. Inset shows 2 × magnified image of the indicated area. The SCAR-BN kymograph shows smooth extension as was seen for SCAR-SA. H. Excessive leading edge localization of SCAR-BN. Developed cells coexpressing SCAR-BN and GFP-Nap1 observed during chemotaxis by wide-field fluorescence. Images were recorded every 5 seconds. Arrows indicate SCAR complex accumulation in pseudopod tips. I. Sizes of SCAR-BN patches. The width of each patch of SCAR accumulation was measured from the same movies as shown in H. In each case, the peak length along the cell perimeter of each separate accumulation event was recorded. Data shown as mean ± s.d. (n^{SCAR WT} = 30, n^{SCAR BN} = 27).