Cell type-specific filamin complex regulation by a novel class of HECT ubiquitin ligase is required for normal cell motility and patterning.

Differential cell motility, which plays a key role in many developmental processes, is perhaps most evident in examples of pattern formation in which the different cell types arise intermingled before sorting out into discrete tissues. This is thought to require heterogeneities in responsiveness to differentiation-inducing signals that result in the activation of cell type-specific genes and 'salt and pepper' patterning. How differential gene expression results in cell sorting is poorly defined. Here we describe a novel gene (hfnA) that provides the first mechanistic link between cell signalling, differential gene expression and cell type-specific sorting in Dictyostelium. HfnA defines a novel group of evolutionarily conserved HECT ubiquitin ligases with an N-terminal filamin domain (HFNs). HfnA expression is induced by the stalk differentiation-inducing factor DIF-1 and is restricted to a subset of prestalk cells (pstO). hfnA(-) pstO cells differentiate but their sorting out is delayed. Genetic interactions suggest that this is due to misregulation of filamin complex activity. Overexpression of filamin complex members phenocopies the hfnA(-) pstO cell sorting defect, whereas disruption of filamin complex function in a wild-type background results in pstO cells sorting more strongly. Filamin disruption in an hfnA(-) background rescues pstO cell localisation. hfnA(-) cells exhibit altered slug phototaxis phenotypes consistent with filamin complex hyperactivity. We propose that HfnA regulates filamin complex activity and cell type-specific motility through the breakdown of filamin complexes. These findings provide a novel mechanism for filamin regulation and demonstrate that filamin is a crucial mechanistic link between responses to differentiation signals and cell movement in patterning based on 'salt and pepper' differentiation and sorting out.

Nap1 regulates Dictyostelium cell motility and adhesion through SCAR-dependent and -independent pathways.

SCAR--also known as WAVE--is a key regulator of actin dynamics. Activation of SCAR enhances the nucleation of new actin filaments through the Arp2/3 complex, causing a localized increase in the rate of actin polymerization . In vivo, SCAR is held in a large regulatory complex, which includes PIR121 and Nap1 proteins, whose precise role is unclear. It was initially thought to hold SCAR inactive until needed , but recent data suggest that it is essential for SCAR function . Here, we show that disruption of the gene that encodes Nap1 (napA) causes loss of SCAR function. Cells lacking Nap1 are small and rounded, with diminished actin polymerization and small pseudopods. Furthermore, several aspects of the napA phenotype are more severe than those evoked by the absence of SCAR alone. In particular, napA mutants have defects in cell-substrate adhesion and multicellular development. Despite these defects, napA(-) cells move and chemotax surprisingly effectively. Our results show that the members of the complex have unexpectedly diverse biological roles.

PIR121 regulates pseudopod dynamics and SCAR activity in Dictyostelium.

BACKGROUND: The WASP/SCAR family of adaptor proteins coordinates actin reorganization by coupling different signaling molecules, including Rho-family GTPases, to the activation of the Arp2/3 complex. WASP binds directly to Cdc42 through its GTPase binding domain (GBD), but SCAR does not contain a GBD, and no direct binding has been found. However, SCAR has recently been found to copurify with four other proteins in a complex. One of these, PIR121, binds directly to Rac. RESULTS: We have identified four of the members of this complex in Dictyostelium and disrupted the pirA gene, which encodes PIR121. The resulting mutant cells are unusually large, maintain an excessive proportion of their actin in a polymerized state and display severe defects in movement and chemotaxis. They also continually extend new pseudopods by widening and splitting existing leading edges rather than by initiating new pseudopods. Comparing these cells to scar null mutants shows behavior that is broadly consistent with overactivation of SCAR. Deletion of the pirA gene in a scar(-) mutant resulted in cells resembling their scar(-) parents with no obvious changes, confirming that PIR121 mainly acts through SCAR in vivo. Surprisingly given their hyperactive phenotype, we find that pirA(-) mutants contain very little intact SCAR protein despite normal levels of mRNA, suggesting a posttranscriptional downregulation of activated SCAR. CONCLUSIONS: Our results demonstrate a genetic connection between the pirA and scar genes. PIR121 appears to inhibit the activity of SCAR in the absence of activating signals. The location of the newly formed protrusions indicates that unregulated SCAR is acting at the edges of existing pseudopods, not elsewhere in the cell. We suggest that active SCAR protein released from the inhibitory complex is rapidly removed and that this is an important and novel mechanism for controlling actin dynamics.

Cell motility and SCAR localisation in axenically growing Dictyostelium cells.

Dictyostelium is a popular experimental organism, in particular for studies of actin dynamics, cell motility and chemotaxis. We find that the motility of axenic cells is unexpectedly different from other strains during growth. In particular, vegetative AX3 cells do not show detectable localisation of SCAR and its regulatory complex to actin-rich protrusions such as filopodia and pseudopodia. Similarly, a range of different mutations, in particular knockouts of members of the SCAR complex and Ras proteins, cause different phenotypes during vegetative growth in different parental strains. Development reverses this unusual behaviour; aggregation-competent AX3 cells localise SCAR in the same way as cells of other strains and species. Studies on cell motility using vegetative cells should therefore be interpreted with caution.

bZIP transcription factor interactions regulate DIF responses in Dictyostelium.

The signalling molecule DIF-1 is required for normal cell fate choice and patterning in Dictyostelium. To understand how these developmental processes are regulated will require knowledge of how cells receive and respond to the DIF-1 signal. Previously, we have described a bZIP transcription factor, DimA, which is required for cells to respond to DIF-1. However, it was unknown whether DimA activity is required to activate the DIF response pathway in certain cells or is a component of the response pathway itself. In this study, we describe the identification of a DimA-related bZIP transcription factor, DimB. Rapid changes in the subcellular localisation of both DimA and DimB in response to DIF-1 suggest that they are directly downstream of the DIF-1 signal. Genetic and biochemical interactions between DimA and DimB provides evidence that their ability to regulate diverse targets in response to DIF-1 is partly due to their ability to form homo- and heterodimeric complexes. DimA and DimB are therefore direct regulators of cellular responses to DIF-1.