The data presented in this article show that Dictyostelium Scar is present in a large-molecular-weight complex and interacts with Abi and HSPC300 and that the N-terminal 96 amino acids are necessary and sufficient for this interaction. The physiological consequences of removing the Abi and HSPC300 binding site(s) of Scar leads to disruption of the Scar containing complex, loss of Scar localization, increased stability of the truncated protein, abnormal cellular actin dynamics, aberrant cell adhesion and motility, and failure of normal cytokinesis.
We determined that the first 44 amino acids of Scar are necessary for binding to both HSPC300 and Abi, and the first 96 amino acids were sufficient for this binding in vitro. It is quite possible that the first 44 amino acids of Scar are also sufficient for binding to Abi and HSPC300, but for technical reasons we were unable to establish that unequivocally. Consistent with that idea, the phenotype of cells expressing ScarΔ44-GFP is virtually identical to that of ScarΔ96-GFP–expressing cells (data not shown). We observed that the in vitro binding of Scar to Abi may be subtly enhanced in the presence of HSPC300. Although our data are only suggestive, others have also reported an enhanced recruitment of Scar to the complex when HSPC300 and Abi are present (Gautreau et al., 2004
). In contrast, the presence or absence of Abi had no effect on the binding between Scar and HSPC300.
The first 96 amino acids of Scar are contained within the SHD (Bear et al., 1998
). The SHD region has been implicated in binding to Abi (Echarri et al., 2004
; Innocenti et al., 2004
; Leng et al., 2005
), and overexpression studies in Cos-7 cells indicate that amino acids 32–66 of Scar2/WAVE2 are necessary for Abi binding (Leng et al., 2005
). In addition, expressing Scar/WAVE in cultured cells depleted for Abi result in loss of Scar/WAVE localization (Kunda et al., 2003
; Rogers et al., 2003
). Our data show that the first 96 amino acids of Scar are not only necessary to bind Abi, consistent with Leng et al. (2005)
, but are sufficient for that binding. The SHD region also appears to play a role in the localization of Scar2/WAVE2 to lamellipodia and filopodia (Nakagawa et al., 2001
; Nozumi et al., 2003
; Leng et al., 2005
; Mitsushima et al., 2006
). When overexpressed in NG108 neuroblastoma cells Scar2/WAVE2 fragments containing only amino acids 1–83 localize to filopodial tips, but fragments containing amino acids 1–54 do not (Nozumi et al., 2003
). We report here that Scar missing the first 96 amino is unable to localize properly. This is consistent if Abi binding is required for proper Scar localization (Leng et al., 2005
). An iso/leucine-rich region is present in the conserved SHD region of Scar family members (Bear et al., 1998
) and may play a role in protein-protein interactions (Miki et al., 1998
; Tu et al., 2004
). In Dictyostelium
the iso/leucine-rich region spans approximately amino acids 28–90, overlapping the region that we find critical for binding to HSPC300 and Abi. There is no direct evidence linking any of the hydrophobic residues to Scar interactions and/or localization, but it is quite possible. More specific mutagenesis studies are needed to establish involvement of these residues in interactions between Scar, Abi, and HSPC300.
Scar and PIR121 are found in complexes of ~400–600 kDa, consistent with the existing model of Scar/WAVE regulation and with the Insall laboratory's demonstration of coimmunoprecipitation of endogenous Scar and PIR121 (Ibarra et al., 2006
; Stradal and Scita, 2006
). ScarΔ96-GFP is found in a smaller sized complex(es). Neither full-length Scar nor PIR121 comigrate with ScarΔ96-GFP in the smaller complex(es), arguing that there is no interaction in vivo. The other components of the ScarΔ96-GFP–containing complex(es) are unknown and are presently being pursued. Suetsugu et al. (2006)
reported that overexpression of full-length tagged WAVE2, in the presence of endogenous WAVE2, remains largely as monomers, though they did not directly establish that the WAVE in lower fractions was monomeric (Suetsugu et al., 2006
). The same, tagged, WAVE2 is found in the high-molecular-weight complex when expressed in cells devoid of endogenous WAVE2. Consistent with their observations, and with our Scar null rescue data, we find full-length Scar-GFP, expressed in Scar null cells, present in fractions representing the high-molecular-weight complex (not shown). We have been unable to stably express full-length Scar-GFP in cells expressing endogenous Scar. Transient expression has been detected, but only at very low levels and for short periods of time. This is consistent with the idea that this protein is more unstable that ScarΔ96. The presence in cells of ScarΔ96-GFP does not interfere with the ability of endogenous Scar or PIR121 to participate in a high-molecular-weight complex. This is consistent with the in vitro data that amino acids 1–96 of Scar are needed to bind Abi and HSPC300 and that Abi is necessary for the assembly of the Scar-containing macromolecular protein complex (Gautreau et al., 2004
; Innocenti et al., 2004
Expression of ScarΔ96-GFP has a dramatic effect on cell morphology, cytoskeletal organization, substrate adhesion, motility, and cytokinesis. This is true whether it is expressed in the presence (HPS400) or absence (scar−
) of the normal Scar-containing complex and is likely to reflect inability of ScarΔ96-GFP to respond to normal Scar regulation. Although ScarΔ96-GFP does not associate with the pentapeptide complex, it is expected to still bind actin and Arp2/3 and stimulate F-actin production, potentially with little or no regulation. It was, therefore, somewhat surprising to see the overall level of F-actin in ScarΔ96-GFP–expressing cells to be the same as in control cells. This is in contrast to expression of a construct, Scar PWA-GFP, which does result in cells having an increased basal level of F-actin. Those same Scar PWA-GFP cells do not produce a phenotype at all like ScarΔ96-GFP ( and Supplementary Figure S1). The data suggest that ScarΔ96-GFP does not act like a constitutively active Arp2/3 activator. It may be that there is transient, localized activation of Arp2/3 and F-actin production but not enough to be detected on a cell-wide basis. Consistent with that is the abnormal distribution of F-actin into patches around the periphery of the cell, a different pattern from that seen in either parental or scar null cells. Whether the control of ScarΔ96-GFP is through the protein complex(es) seen in the sucrose gradients or by some other means awaits further characterization of ScarΔ96-GFP. There is also an increase in the number of vesicles present in ScarΔ96-GFP cells. Dictyostelium
scar null cells are known to have defects in vesicle maturation and export (Seastone et al., 2001
) and the phenotype of ScarΔ96-GFP–expressing cells may reflect a misregulation of a normal role for Scar in vesicle morphogenesis or trafficking. Excessive formation of long, thin protrusions is seen in Drosophila
S2 cells when Scar, Abi, Nap1, or PIR121 are RNA interference depleted (Kunda et al., 2003
; Rogers et al., 2003
). A similar increase is seen when ScarΔ96-GFP is expressed. In both systems this increase occurs when one or more of the complex members is absent. In the absence of PIR121, Abi, or Nap1 there is a decrease in the level of wild-type Scar within the cells (Blagg et al., 2003a
; Kunda et al., 2003
; Rogers et al., 2003
; Ibarra et al., 2006
). This is likely to be, at least in part, proteasome-mediated degradation (Rogers et al., 2003
; Mitsushima et al., 2006
). In contrast to wild-type Scar, ScarΔ96-GFP is not rapidly degraded. This is true in parental as well as in null backgrounds for Scar, PIR121, Nap1, and HSPC300. This strongly suggests that in addition to ScarΔ96-GFP being resistant to regulation by the pentapeptide complex, it is resistant to degradation. It is possible that the N-terminal 96 amino acids of Scar contain a targeting site for degradation or is important for masking such a site. It was recently reported that an Arabidopsis
ortholog of HSPC300, BRICK1, plays a role in stabilizing SCAR2 in that organism (Le et al., 2006
). They suggest that BRICK1 binds Scar and contributes to the masking of a degradation sequence. It is also possible that regulated degradation of Scar is dependent on proper localization. It may be that the presence of Scar in the peptapeptide complex not only targets it to sites of new F-actin assembly, but is responsible for presenting Scar to the degradation machinery. It is also possible that the deletion of the first 44 or 96 amino acids of Scar changes the conformation of the protein such that abi and/or HSPC300 no longer have an intact recognition site. Although the experiments showing amino acids 1–96 of Scar are sufficient to bind HSPC300, we cannot rule out that possibility for abi.
Expression of ScarΔ96-GFP has a dominant, gain-of-function effect on cell motility. Chemotaxis is not rescued in Scar null cells by expression of ScarΔ96-GFP, and chemotaxis of parental cells is severely compromised. The effects do not seem to be on the ability of the cells to detect the chemoattractant, because movies show that cells are able to identify the direction of the source. Rather the defect in motility may be an indirect consequence of enhanced substrate adhesion and an inability of cells to polarize properly. Movies show many cells expressing ScarΔ96-GFP adhering to the substrate, flattening out, and moving very little. These cells can convert back to less adherent cells but still extend multiple, poorly directed protrusion resulting in inefficient directed motion. The lack of chemotactic response appears to reflect more on the cell's general inability to organize its actin cytoskeleton, as seen by phalloidin F-actin staining, rather than on its ability to orient correctly. It is possible that ScarΔ96-GFP is affecting the Phg2-Adrm pathway and/or SadA-mediated adhesion as both play prominent roles in regulating F-actin organization, adhesion, and motility (Fey et al., 2002
; Gebbie et al., 2004
; Cherix et al., 2006
). Further studies are underway to investigate these possibilities.
Perhaps the most surprising effect of expressing ScarΔ96-GFP was on cytokinesis. Unlike myosin II null mutants that are multinucleate in suspension but can recover a form of cell division when returned to a solid surface (De Lozanne and Spudich, 1987
; Knecht and Loomis, 1987
; Fukui et al., 1990
), ScarΔ96-GFP–expressing cells become highly multinucleate on solid support. In that sense the ScarΔ96-GFP–expressing cells are more reminiscent of null mutants in coronin (de Hostos et al., 1993
), SAPKα (Sun et al., 2003
), or the multiple mutants in PI3-kinase and PTEN involved in the phosphatidyl 4,5-bisphosphate synthetic pathway (Janetopoulos et al., 2005
). The latter connection is particularly interesting given the proposed relationship between phophoinositide signaling and members of the Scar/WASp family of proteins (e.g., Oikawa et al., 2004
; Weiner et al., 2006
). Janetopoulos et al. (2005)
and others have suggested a connection between G-protein–coupled receptor (GPCR) signaling, PI3K/PTEN and cytokinesis. Because Scar was originally identified as a suppressor of GPCR signaling, it is possible that expression of the truncated Scar is interfering with that normal regulation. It is also possible that the cytokinesis defect is due to the abnormal substrate adhesion seen in ScarΔ96-GFP– expressing cells. This could result in new cells being unable to effectively make a division furrow and move apart. SadA mutants also show a cytokinesis defect, again suggesting a possible connection between the abnormal actin regulation of ScarΔ96-GFP–expressing cells and SadA-mediated adhesion.
Expression of N-terminally truncated Scar results in a wide array of deleterious consequences including effects on cell morphology, substrate adhesion, motility, and cytokinesis. This protein is defective in at least two different ways: it is not regulated by the normal involvement in a large multiprotein complex and it is unusually stable. These two things may be related as targeting to the plasma membrane by the complex may be required for both activation and degradation of Scar. The findings reported here underline the importance of maintaining strict regulation over Arp2/3 activators like Scar and WASp. Localized activation and rapid degradation of Scar/WASp proteins provide mechanisms for both spatial and temporal regulation of Arp2/3-mediated actin polymerization. Our data suggest that necessary and perhaps sufficient components of that regulation are contained within the first 100 amino acids of Scar. The ability to perform site-directed mutagenesis on the N-terminus of Scar and return it to an in vivo context provides us with a means to more precisely define the role of the N-terminus of Scar provide insight into its regulation.