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The contractile actin cortex is a thin layer of filamentous actin, myosin motors, and regulatory proteins beneath the plasma membrane crucial to cytokinesis, morphogenesis, and cell migration. However, the factors regulating actin assembly in this compartment are not well understood. Using the Dictyostelium model system, we show that the three Diaphanous-related formins (DRFs) ForA, ForE, and ForH are regulated by the RhoA-like GTPase RacE and synergize in the assembly of filaments in the actin cortex. Single or double formin-null mutants displayed only moderate defects in cortex function whereas the concurrent elimination of all three formins or of RacE caused massive defects in cortical rigidity and architecture as assessed by aspiration assays and electron microscopy. Consistently, the triple formin and RacE mutants encompassed large peripheral patches devoid of cortical F-actin and exhibited severe defects in cytokinesis and multicellular development. Unexpectedly, many forA(−)/E(−)/H(−) and racE(−) mutants protruded efficiently, formed multiple exaggerated fronts, and migrated with morphologies reminiscent of rapidly moving fish keratocytes. In 2D-confinement, however, these mutants failed to properly polarize and recruit myosin II to the cell rear essential for migration. Cells arrested in these conditions displayed dramatically amplified flow of cortical actin filaments, as revealed by total internal reflection fluorescence (TIRF) imaging and iterative particle image velocimetry (PIV). Consistently, individual and combined, CRISPR/Cas9-mediated disruption of genes encoding mDia1 and -3 formins in B16-F1 mouse melanoma cells revealed enhanced frequency of cells displaying multiple fronts, again accompanied by defects in cell polarization and migration. These results suggest evolutionarily conserved functions for formin-mediated actin assembly in actin cortex mechanics.

Formins and Arp2/3 complex are major actin assembly factors in eukaryotic cells. While Arp2/3 complex induces branching of the existing filaments, thus creating a dense actin meshwork at the cell's leading edge, formins promote nucleation and elongation of linear actin filaments, mostly in filopodia, stress fibers and cytokinetic rings1. Formins are defined by the presence of a highly conserved C-terminal formin homology 2 (FH2) domain preceded by an FH1 domain, which are necessary and sufficient to nucleate actin polymerisation and elongate filaments2. Diaphanous-related formins (DRFs) constitute a conserved subfamily in which core FH1 and FH2 domains are flanked by regulatory domains: a GTPase binding domain (GBD), a Diaphanous-inhibitory domain (DID) and a dimerization domain (DD) at the N terminus, and a C-terminal Diaphanous-autoregulatory domain (DAD). DRFs are autoinhibited through interaction between DID and DAD and binding of a Rho GTPase to GBD releases this inhibition. We have characterized formin G (ForG) from the professional phagocyte Dictyostelium discoideum as a Diaphanous-related formin that fully conforms to the canonical domain organization of DRFs, and described its role in large-scale endocytosis3. Biochemical assays identified ForG as a rather week actin nucleator that efficiently elongates actin filaments. Live cell imaging demonstrated that ForG was enriched at the plasma membrane during the formation and maturation of macropinocytic cups. Analysis of phagocytosis also revealed localization to nascent phagocytic cups and ForG remained associated with the membrane over the entire course of engulfment and internalization of yeast particle (Fig. 1). Real-time assays of the nutrient uptake showed markedly decreased fluid-phase uptake of TRITC-dextran and phagocytosis of TRITC-labeled yeast particles in forG null cells compared with control cells. Interestingly, yeast-two-hybrid (Y2H) assay failed to identify any interaction of ForG with Rho family GTPases, although DRFs have been previously shown to act exclusively as effectors of Rho GTPases1. Strikingly, another Y2H screen and bimolecular fluorescence complementation (BiFC) in vivo identified specifically activated forms of RasB and RasG, but not their dominant negative variants, as ForG interactors (Fig. 2). The role of RasG in macropinocytosis has already been well established. To assess the role of RasB in this process, we have created rasB null cells and tested their nutrient uptake. The uptake of large particles was substantially impaired in rasB deficient cells, while pinocytosis rates were only slightly diminished. Taken together, these data demonstrate the significance of ForG for actin assembly in endocytic structures, with RasB being mainly responsible for its regulation during phagocytosis of large particles, and RasG being the main regulator of ForG in macropinocytosis.

Here, we analyzed the single inverse Bin/Amphiphysin/Rvs (I-BAR) family member IBARa from Dictyostelium discoideum. The X-ray structure of the N-terminal I-BAR domain solved at 2.2 A resolution revealed an all-α-helical structure that self-associates into a 165-A zeppelin-shaped antiparallel dimer. The structural data are consistent with its shape in solution obtained by small-angle X-ray scattering. Cosedimentation, fluorescence anisotropy, and fluorescence and electron microscopy revealed that the I-BAR domain bound preferentially to phosphoinositide-containing vesicles and drove the formation of negatively curved tubules. Immunofluorescence labeling further showed accumulation of endogenous IBARa at the tips of filopodia, the rim of constricting phagocytic cups, in foci connecting dividing cells during the final stage of cytokinesis and most prominently at the osmoregulatory contractile vacuole (CV). Consistently, IBARa-null mutants displayed defects in CV formation and discharge, growth, phagocytosis and mitotic cell division, whereas filopodia formation was not compromised. Of note, IBARa-null mutants were also strongly impaired in cell spreading. Taken together, these data suggest that IBARa constitutes an important regulator of numerous cellular processes intimately linked with the dynamic rearrangement of cellular membranes.

Diaphanous-related formins (DRFs) act as downstream effectors of Rho family GTPases and drive the formation and elongation of linear actin filaments in various cellular processes. Here we analyzed the DRF dDia1 from Dictyostelium cells. The biochemical characterization of recombinant dDia1-FH1FH2 by bulk polymerization assays and single filament TIRF microscopy revealed that dDia1 is a rather weak nucleator. Addition of any of the three Dictyostelium profilin isoforms, however, markedly accelerated formin-mediated actin filament barbed end elongation in TIRF assays. Interestingly, filament elongation was significantly faster in presence of DdPFN I (profilin I) when compared to the other two isoforms, suggesting selectivity of dDia1 for DdPFN I. Additionally, we frequently observed dissociation of the formin from growing barbed ends. These findings are consistent with dilution-induced depolymerization assays in presence of dDia1-FH1FH2 showing that dDia1 is a weak capper in comparison with heterodimeric capping protein. To study the physiological role of this formin, we created cell lines lacking dDia1 or overexpressing GFP-tagged dDia1. Of note, constitutively active dDia1 accumulated homogenously in the entire pseudopod suggesting that it controls microfilament architecture to regulate cell migration. Comparison of wild type and dDia1-null cells in random cell migration and chemotaxis toward a cAMP gradient revealed no major differences. By contrast, phototaxis of dDia1-deficient cells during the multicellular stage was markedly impaired. While wild type slugs moved with high directionality toward the light source, the trails of dDia1-null slugs displayed a characteristic V-shaped profile and deviated in angles between 50° and 60° from the path of the incident light. Possibly in conjunction with this defect, dDia1-null cells also formed substantially smaller fruiting bodies. These findings demonstrate dDia1 to be critically involved in collective cell migration during terminal differentiation.

Dictyostelium discoideum is an exceptionally powerful eukaryotic model to study many aspects of growth, development, and fundamental cellular processes. Its small-sized, haploid genome allows highly efficient targeted homologous recombination for gene disruption and knock-in epitope tagging. We previously described a robust system for the generation of multiple gene mutations in Dictyostelium by recycling the Blasticidin S selectable marker after transient expression of the Cre recombinase. We have now further optimized the system for higher efficiency and, additionally, coupled it to both, knock-out and knock-in gene targeting, allowing the characterization of multiple and cooperative gene functions in a single cell line.

Diaphanous-related formins (DRFs) drive the nucleation and elongation of linear actin filaments downstream of Rho GTPase signalling pathways. Dictyostelium formin C (ForC) resembles a DRF, except that it lacks a genuine formin homology domain 1 (FH1), raising the questions whether or not ForC can nucleate and elongate actin filaments. We found that a recombinant ForC-FH2 fragment does not nucleate actin polymerization, but moderately decreases the rate of spontaneous actin assembly and disassembly, although the barbed-end elongation rate in the presence of the formin was not markedly changed. However, the protein bound to and crosslinked actin filaments into loose bundles of mixed polarity. Furthermore, ForC is an important regulator of morphogenesis since ForC-null cells are severely impaired in development resulting in the formation of aberrant fruiting bodies. Immunoblotting revealed that ForC is absent during growth, but becomes detectable at the onset of early aggregation when cells chemotactically stream together to form a multicellular organism, and peaks around the culmination stage. Fluorescence microscopy of cells ectopically expressing a GFP-tagged, N-terminal ForC fragment showed its prominent accumulation in the leading edge, suggesting that ForC may play a role in cell migration. In agreement with its expression profile, no defects were observed in random migration of vegetative mutant cells. Notably, chemotaxis of starved cells towards a source of cAMP was severely impaired as opposed to control. This was, however, largely due to a marked developmental delay of the mutant, as evidenced by the expression profile of the early developmental marker csA. In line with this, chemotaxis was almost restored to wild type levels after prolonged starvation. Finally, we observed a complete failure of phototaxis due to abolished slug formation and a massive reduction of spores consistent with forC promoter-driven expression of beta-galactosidase in prespore cells. Together, these findings demonstrate ForC to be critically involved in signalling of the cytoskeleton during various stages of development.

Dictyostelium discoideum is an exceptionally powerful eukaryotic model to study many aspects of growth, development, and fundamental cellular processes. Its small-sized, haploid genome allows highly efficient targeted homologous recombination for gene disruption and knock-in epitope tagging. We previously described a robust system for the generation of multiple gene mutations in Dictyostelium by recycling the Blasticidin S selectable marker after transient expression of the Cre recombinase. We have now further optimized the system for higher efficiency and, additionally, coupled it to both, knock-out and knock-in gene targeting, allowing the characterization of multiple and cooperative gene functions in a single cell line.

The inactivation of proteins in cells is inevitable to study their physiological role in various cellular processes. In contrast to strategies to alter the amount of active proteins in cells, only a gene knockout guarantees complete removal of the protein of interest. For Dictyostelium discoideum cells, the gene replacement construct typically consists of a Blasticidin S resistance (Bsr) cassette flanked by fragments of the target gene to allow insertion by homologous recombination. More advanced knockout constructs additionally carry loxP sites on both sides of the Bsr cassettes for subsequent removal of the selection marker by transient expression of Cre recombinase, thus allowing generation of multiple knockouts using just a single selection marker. However, due to its design, the available neomycin selection-based Cre expression plasmid occasionally tends to integrate into the genome and also yield only a moderate number of transfectants in liquid media. In some cases, for instance in SCAR-null cells, it was not possible to remove the Bsr cassette without stable integration of the Cre expression vector into the genome. To circumvent these difficulties we designed the extrachromosomal Cre-recombinase expression vector pTX-NLS-Cre. We verified the greatly improved efficacy of this novel Cre-loxP approach by removal of the Bsr cassette in five different cell lines including the SCAR-null mutant. As a consequence, this vector will be a highly valuable means for the rapid generation of single or multiple mutants remaining sensitive to the most reliable selection markers Blasticidin S and neomycin.

Phagocytosis and macropinocytosis are Ras-regulated and actin-driven processes that depend on the dynamic rearrangements of the plasma membrane that protrudes and internalizes extracellular material by cup-shaped structures. However, the regulatory mechanisms underlying actin assembly in large-scale endocytosis remain elusive. Here, we show that the Diaphanous-related formin G (ForG) from the professional phagocyte Dictyostelium discoideum localizes to endocytic cups. Biochemical analyses revealed that ForG is a rather weak nucleator but efficiently elongates actin filaments in the presence of profilin. Notably, genetic inactivation of ForG is associated with a strongly impaired endocytosis and a markedly diminished F-actin content at the base of the cups. By contrast, ablation of the Arp2/3 (actin-related protein-2/3) complex activator SCAR (suppressor of cAMP receptor) diminishes F-actin mainly at the cup rim, being consistent with its known localization. These data therefore suggest that ForG acts as an actin polymerase of Arp2/3-nucleated filaments to allow for efficient membrane expansion and engulfment of extracellular material. Finally, we show that ForG is directly regulated in large-scale endocytosis by RasB and RasG, which are highly related to the human proto-oncogene KRas.