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Actin-based protrusions are reinforced through positive feedback, but it is unclear what restricts their size, or limits positive signals when they retract or split. We identify an evolutionarily conserved regulator of actin-based protrusion: CYRI (CYFIP-related Rac interactor) also known as Fam49 (family of unknown function 49). CYRI binds activated Rac1 via a domain of unknown function (DUF1394) shared with CYFIP, defining DUF1394 as a Rac1-binding module. CYRI-depleted cells have broad lamellipodia enriched in Scar/WAVE, but reduced protrusion-retraction dynamics. Pseudopods induced by optogenetic Rac1 activation in CYRI-depleted cells are larger and longer lived. Conversely, CYRI overexpression suppresses recruitment of active Scar/WAVE to the cell edge, resulting in short-lived, unproductive protrusions. CYRI thus focuses protrusion signals and regulates pseudopod complexity by inhibiting Scar/WAVE-induced actin polymerization. It thus behaves like a 'local inhibitor' as predicted in widely accepted mathematical models, but not previously identified in cells. CYRI therefore regulates chemotaxis, cell migration and epithelial polarization by controlling the polarity and plasticity of protrusions.

Dictyostelium has a mature technology for molecular-genetic manipulation based around transfection using several different selectable markers, marker re-cycling, homologous recombination and insertional mutagenesis, all supported by a well-annotated genome. However this technology is optimized for mutant, axenic cells that, unlike non-axenic wild type, can grow in liquid medium. There is a pressing need for methods to manipulate wild type cells and ones with defects in macropinocytosis, neither of which can grow in liquid media. Here we present a panel of molecular genetic techniques based on the selection of Dictyostelium transfectants by growth on bacteria rather than liquid media. As well as extending the range of strains that can be manipulated, these techniques are faster than conventional methods, often giving usable numbers of transfected cells within a few days. The methods and plasmids described here allow efficient transfection with extrachromosomal vectors, as well as chromosomal integration at a 'safe haven' for relatively uniform cell-to-cell expression, efficient gene knock-in and knock-out and an inducible expression system. We have thus created a complete new system for the genetic manipulation of Dictyostelium cells that no longer requires cell feeding on liquid media., Competing Interests: The authors have declared that no competing interests exist.

Phagocytes locate microorganisms via chemotaxis and then consume them using phagocytosis. Dictyostelium amoebas are stereotypical phagocytes that prey on diverse bacteria using both processes. However, as typical phagocytic receptors, such as complement receptors or Fcγ receptors, have not been found in Dictyostelium, it remains mysterious how these cells recognize bacteria. Here, we show that a single G-protein-coupled receptor (GPCR), folic acid receptor 1 (fAR1), simultaneously recognizes the chemoattractant folate and the phagocytic cue lipopolysaccharide (LPS), a major component of bacterial surfaces. Cells lacking fAR1 or its cognate G-proteins are defective in chemotaxis toward folate and phagocytosis of Klebsiella aerogenes. Computational simulations combined with experiments show that responses associated with chemotaxis can also promote engulfment of particles coated with chemoattractants. Finally, the extracellular Venus-Flytrap (VFT) domain of fAR1 acts as the binding site for both folate and LPS. Thus, fAR1 represents a new member of the pattern recognition receptors (PRRs) and mediates signaling from both bacterial surfaces and diffusible chemoattractants to reorganize actin for chemotaxis and phagocytosis., Competing Interests: The authors have declared that no competing interests exist.

The steps leading to constitutive exocytosis are poorly understood. In Dictyostelium WASH complex mutants, exocytosis is blocked, so cells that take up fluorescent dextran from the medium retain it and remain fluorescent. Here, we establish a FACS-based method to select cells that retain fluorescent dextran, allowing identification of mutants with disrupted exocytosis. Screening a pool of random mutants identified members of the WASH complex, as expected, and multiple mutants in the conserved HEAT-repeat-containing protein Mroh1. In mroh1 mutants, endosomes develop normally until the stage where lysosomes neutralize to postlysosomes, but thereafter the WASH complex is recycled inefficiently, and subsequent exocytosis is substantially delayed. Mroh1 protein localizes to lysosomes in mammalian and Dictyostelium cells. In Dictyostelium , it accumulates on lysosomes as they mature and is removed, together with the WASH complex, shortly before the postlysosomes are exocytosed. WASH-generated F-actin is required for correct subcellular localization; in WASH complex mutants, and immediately after latrunculin treatment, Mroh1 relocalizes from the cytoplasm to small vesicles. Thus, Mroh1 is involved in a late and hitherto undefined actin-dependent step in exocytosis., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

The individual molecular pathways downstream of Cdc42, Rac, and Rho GTPases are well documented, but we know surprisingly little about how these pathways are coordinated when cells move in a complex environment in vivo. In the developing embryo, melanoblasts originating from the neural crest must traverse the dermis to reach the epidermis of the skin and hair follicles. We previously established that Rac1 signals via Scar/WAVE and Arp2/3 to effect pseudopod extension and migration of melanoblasts in skin. Here we show that RhoA is redundant in the melanocyte lineage but that Cdc42 coordinates multiple motility systems independent of Rac1. Similar to Rac1 knockouts, Cdc42 null mice displayed a severe loss of pigmentation, and melanoblasts showed cell-cycle progression, migration, and cytokinesis defects. However, unlike Rac1 knockouts, Cdc42 null melanoblasts were elongated and displayed large, bulky pseudopods with dynamic actin bursts. Despite assuming an elongated shape usually associated with fast mesenchymal motility, Cdc42 knockout melanoblasts migrated slowly and inefficiently in the epidermis, with nearly static pseudopods. Although much of the basic actin machinery was intact, Cdc42 null cells lacked the ability to polarize their Golgi and coordinate motility systems for efficient movement. Loss of Cdc42 de-coupled three main systems: actin assembly via the formin FMNL2 and Arp2/3, active myosin-II localization, and integrin-based adhesion dynamics., (Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Macropinocytosis is an ancient mechanism that allows cells to harvest nutrients from extracellular media, which also allows immune cells to sample antigens from their surroundings. During macropinosome formation, bulk plasma membrane is internalized with all its integral proteins. It is vital for cells to salvage these proteins before degradation, but the mechanisms for sorting them are not known. Here we describe the evolutionarily conserved recruitment of the WASH (WASP and SCAR homolog) complex to both macropinosomes and phagosomes within a minute of internalization. Using Dictyostelium, we demonstrate that WASH drives protein sorting and recycling from macropinosomes and is thus essential to maintain surface receptor levels and sustain phagocytosis. WASH functionally interacts with the retromer complex at both early and late phases of macropinosome maturation, but mediates recycling via retromer-dependent and -independent pathways. WASH mutants consequently have decreased membrane levels of integrins and other surface proteins. This study reveals an important pathway enabling cells to sustain macropinocytosis without bulk degradation of plasma membrane components., Competing Interests: The authors declare no conflict of interest.

Collective cell movement is a key component of many important biological processes, including wound healing, the immune response and the spread of cancers. To understand and influence these movements, we need to be able to identify and quantify the contribution of their different underlying mechanisms. Here, we define a set of six candidate models-formulated as advection-diffusion-reaction partial differential equations-that incorporate a range of cell movement drivers. We fitted these models to movement assay data from two different cell types: Dictyostelium discoideum and human melanoma. Model comparison using widely applicable information criterion suggested that movement in both of our study systems was driven primarily by a self-generated gradient in the concentration of a depletable chemical in the cells' environment. For melanoma, there was also evidence that overcrowding influenced movement. These applications of model inference to determine the most likely drivers of cell movement indicate that such statistical techniques have potential to support targeted experimental work in increasing our understanding of collective cell movement in a range of systems., (© 2016 The Authors.)

The five-subunit WASH complex generates actin networks that participate in endocytic trafficking, migration and invasion in various cell types. Loss of one of the two subunits WASH or strumpellin in mice is lethal, but little is known about their role in mammals in vivo. We explored the role of strumpellin, which has previously been linked to hereditary spastic paraplegia, in the mouse melanocytic lineage. Strumpellin knockout in melanocytes revealed abnormal endocytic vesicle morphology but no impairment of migration in vitro or in vivo and no change in coat colour. Unexpectedly, WASH and filamentous actin could still localize to vesicles in the absence of strumpellin, although the shape and size of vesicles was altered. Blue native PAGE revealed the presence of two distinct WASH complexes, even in strumpellin knockout cells, revealing that the WASH complex can assemble and localize to endocytic compartments in cells in the absence of strumpellin., (© 2016 The Authors. Pigment Cell & Melanoma Research Published by John Wiley & Sons Ltd.)

We describe three chemotaxis assays-Insall chambers, circular invasion assays, and 3D organotypic assays-that are particularly appropriate for measuring migration of cancer cells in response to gradients of soluble attractants. Each assay has defined advantages, and together they provide the best possible quantitative assessment of cancer chemotaxis.

Immunosenescence is the functional deterioration of the immune system during natural aging. Despite increased susceptibility to bacterial infections in older adults, age-associated changes to neutrophil responses are only partially understood, and neutrophil migration has not been characterized in detail. Here we describe reduced chemotaxis but preserved chemokinesis toward a range of inflammatory stimuli in migrating neutrophils isolated from healthy older subjects. Cross-sectional data indicate that migratory behavior changes in the sixth decade of life. Crucially, aberrant migration may increase "bystander" tissue damage and heighten inflammation as a result of excess proteinase release during inaccurate chemotaxis, as well as reducing pathogen clearance. We show evidence of increased neutrophil proteinase activity in older adults, namely, raised levels of neutrophil proteinase substrate-derived peptides and evidence of primary granule release, associated with increased systemic inflammation. Inaccurate migration was causally associated with increased constitutive phosphoinositide 3-kinase (PI3K) signaling; untreated neutrophils from old donors demonstrated significant PI3K activation compared with cells from young donors. PI3K-blocking strategies, specifically inhibition of PI3Kγ or PI3Kδ, restored neutrophil migratory accuracy, whereas SHIP1 inhibition worsened migratory flaws. Targeting PI3K signaling may therefore offer a new strategy in improving neutrophil functions during infections and reduce inappropriate inflammation in older patients.

The SCAR/WAVE complex drives the actin polymerisation that underlies protrusion of the front of the cell and thus drives migration. However, it is not understood how the activity of SCAR/WAVE is regulated to generate the infinite range of cellular shape changes observed during cell motility. What are the relative roles of the subunits of the SCAR/WAVE complex? What signaling molecules do they interact with? And how does the complex integrate all this information in order to control the temporal and spatial polymerisation of actin during protrusion formation? Unfortunately, the interdependence of SCAR complex members has made genetic dissection hard. In our recent paper,(1) we describe stabilization of the Dictyostelium SCAR complex by a small fragment of Abi. Here we summarize the main findings and discuss how this approach can help reveal the inner workings of this impenetrable complex.

Phosphorylation of the actin-related protein 2 (Arp2) subunit of the Arp2/3 complex on evolutionarily conserved threonine and tyrosine residues was recently identified and shown to be necessary for nucleating activity of the Arp2/3 complex and membrane protrusion of Drosophila cells. Here we use the Dictyostelium diploid system to replace the essential Arp2 protein with mutants that cannot be phosphorylated at Thr-235/6 and Tyr-200. We found that aggregation of the resulting mutant cells after starvation was substantially slowed with delayed early developmental gene expression and that chemotaxis toward a cAMP gradient was defective with loss of polarity and attenuated F-actin assembly. Chemotaxis toward cAMP was also diminished with reduced cell speed and directionality and shorter pseudopod lifetime when Arp2 phosphorylation mutant cells were allowed to develop longer to a responsive state similar to that of wild-type cells. However, clathrin-mediated endocytosis and chemotaxis under agar to folate in vegetative cells were only subtly affected in Arp2 phosphorylation mutants. Thus, phosphorylation of threonine and tyrosine is important for a subset of the functions of the Arp2/3 complex, in particular an unexpected major role in regulating development.

Direct visualization chambers are considered the gold standard for measuring and analyzing chemotactic responses, because they allow detailed analysis of cellular behavior during the process of chemotaxis. We have previously described the Insall chamber, an improved chamber for measuring cancer cell chemotaxis. Here, we describe in detail how this system can be used to perform two key assays for both fast- and slow-moving mammalian and nonmammalian cell types. This allows for the detailed analysis of chemotactic responses in linear gradients at the levels of both overall cell behavior and subcellular dynamics.

Rationale: There are increased neutrophils in the lungs of patients with chronic obstructive pulmonary disease (COPD), but it is unclear if this is due to increased inflammatory signal or related to the inherent behavior of the neutrophils. This is critical, because inaccurate or excessive neutrophil chemotaxis could drive pathological accumulation and tissue damage., Objectives: To assess migratory dynamics of neutrophils isolated from patients with COPD compared with healthy smoking and nonsmoking control subjects and patients with α(1)-antitryspin deficiency., Methods: Migratory dynamics and structure were assessed in circulating neutrophils, using phase and differential interference contrast microscopy and time-lapse photography. The effect of COPD severity was studied. Surface expression of receptors was measured using flow cytometry. The in vitro effects of a phosphoinositide 3-kinase inhibitor (LY294002) were studied., Measurements and Main Results: COPD neutrophils moved with greater speed than cells from either control group but with reduced migratory accuracy, in the presence of IL-8, growth-related oncogene α, formyl-methionyl-leucyl-phenylalanine, and sputum. This was present across all stages of COPD. Structurally, COPD neutrophils formed fewer pseudopods during migration. There were no differences in surface expression of the receptors CXCR1, CXCR2, or FPR1. LY294002 reduced COPD neutrophil migratory speed while increasing chemotactic accuracy, returning values to normal. The inhibitor did not have these effects in healthy control subjects or patients with a similar degree of lung disease., Conclusions: COPD neutrophils are intrinsically different than cells from other studied populations in their chemotactic behavior and migratory structure. Differences are not due to surface expression of chemoattractant receptors but instead appear to be due to differences in cell signaling.

Background: SCAR/WAVE proteins couple signalling to actin polymerization, and are thus fundamental to the formation of pseudopods and lamellipods. They are controlled as part of a five-membered complex that includes the tiny HSPC300 protein. It is not known why SCAR/WAVE is found in such a large assembly, but in Dictyostelium the four larger subunits have different, clearly delineated functions., Results: We have generated Dictyostelium mutants in which the HSPC300 gene is disrupted. As has been seen in other regulatory complex mutants, SCAR is lost in these cells, apparently by a post-translational mechanism, though PIR121 levels do not change. HSPC300 knockouts resemble scar mutants in slow migration, roundness, and lack of large pseudopods. However hspc300-colonies on bacteria are larger and more similar to wild type, suggesting that some SCAR function can survive without HSPC300. We find no evidence for functions of HSPC300 outside the SCAR complex., Conclusion: HSPC300 is essential for most SCAR complex functions. The phenotype of HSPC300 knockouts is most similar to mutants in scar, not the other members of the SCAR complex, suggesting that HSPC300 acts most directly on SCAR itself.

Entamoeba histolytica cells, the cause of amoebic dysentery, are highly motile, and this motility is an essential feature of the pathogenesis and morbidity of amoebiasis. However, the control of E. histolytica motility within the gut and during invasion is poorly understood. We have used an improved chemotaxis assay to identify the key extracellular signals mediating Entamoeba chemotaxis. The dominant responses we observe are caused by factors generated by E. histolytica cells themselves. Medium that has been conditioned by E. histolytica growth causes both chemokinesis and negative chemotaxis. The speed of random movement is more than doubled in conditioned compared with fresh medium, and cells move efficiently away from conditioned medium by negative chemotaxis. Ethanol, the product of Entamoeba glucose metabolism, is the principal component of the chemokinetic response. The closely related but nonpathogenic Entamoeba dispar shows no change in motility in response to conditioned medium implying that these responses are central to E. histolytica pathogenesis.

Background: Dictyostelium discoideum is a eukaryote with a simple lifestyle and a relatively small genome whose sequence has been fully determined. It is widely used for studies on cell signaling, movement and multicellular development. Ras guanine-nucleotide exchange factors (RasGEFs) are the proteins that activate Ras and thus lie near the top of many signaling pathways. They are particularly important for signaling in development and chemotaxis in many organisms, including Dictyostelium., Results: We have searched the genome for sequences encoding RasGEFs. Despite its relative simplicity, we find that the Dictyostelium genome encodes at least 25 RasGEFs, with a few other genes encoding only parts of the RasGEF consensus domains. All appear to be expressed at some point in development. The 25 genes include a wide variety of domain structures, most of which have not been seen in other organisms. The LisH domain, which is associated with microtubule binding, is seen particularly frequently; other domains that confer interactions with the cytoskeleton are also common. Disruption of a sample of the novel genes reveals that many have clear phenotypes, including altered morphology and defects in chemotaxis, slug phototaxis and thermotaxis., Conclusion: These results suggest that the unexpectedly large number of RasGEF genes reflects an evolutionary expansion of the range of Ras signaling rather than functional redundancy or the presence of multiple pseudogenes.

Cdc42 is a key regulator of cell polarity and actin dynamics. One of its effectors, WASP, initiates the assembly of new actin filaments. In this issue of Cell, show that a previously unknown regulator named Toca-1 is required for Cdc42 to activate WASP. This discovery changes our picture of how small GTPases and multiple other signals converge to stimulate actin polymerization and cell motility.

Various chemicals with harmful effects are not themselves toxic, but are metabolized in vivo to produce toxic products. One example is methanol in Dictyostelium, which is lethal to cells containing the acrA gene, but relatively harmless to acrA mutants. This makes methanol resistance one of the tightest genetic selections in DICTYOSTELIUM: Loss of acrA also confers cross-resistance to unrelated compounds such as acriflavine and thiabendazole. We have used insertional mutagenesis to demonstrate that the acrA locus encodes the peroxisomal catalase A enzyme. Disruption of the catA gene results in parallel resistance to acriflavine. Molecular and biochemical studies of several previously characterized methanol-resistant strains reveal that each lacks catalase activity. One allele, acrA2, contains a 13 bp deletion which introduces a frameshift in the middle of the gene. The involvement of catalase in methanol resistance in Dictyostelium compares with its role in methanol metabolism in yeast and rodents. However, this is the first study to show that catalase is required for the toxicity of acriflavine. Our results imply that acriflavine and thiabendazole are precursors which must be oxidized to generate biologically active species. The catA/acrA gene is also a potentially invaluable negative selectable marker for Dictyostelium molecular genetics.