C2GAP2 is a common regulator of Ras signaling for chemotaxis, phagocytosis, and macropinocytosis.

Phagocytosis, macropinocytosis, and G protein coupled receptor-mediated chemotaxis are Ras-regulated and actin-driven processes. The common regulator for Ras activity in these three processes remains unknown. Here, we show that C2GAP2, a Ras GTPase activating protein, highly expressed in the vegetative growth state in model organism Dictyostelium. C2GAP2 localizes at the leading edge of chemotaxing cells, phagosomes during phagocytosis, and macropinosomes during micropinocytosis. c2gapB- cells lacking C2GAP2 displayed increased Ras activation upon folic acid stimulation and subsequent impaired chemotaxis in the folic acid gradient. In addition, c2gaB- cells have elevated phagocytosis and macropinocytosis, which subsequently results in faster cell growth. C2GAP2 binds multiple phospholipids on the plasma membrane and the membrane recruitment of C2GAP2 requires calcium. Taken together, we show a shared negative regulator of Ras signaling that mediates Ras signaling for chemotaxis, phagocytosis, and macropinocytosis.

Forty-five years of cGMP research in Dictyostelium: understanding the regulation and function of the cGMP pathway for cell movement and chemotaxis.

In Dictyostelium, chemoattractants induce a fast cGMP response that mediates myosin filament formation in the rear of the cell. The major cGMP signaling pathway consists of a soluble guanylyl cyclase sGC, a cGMP-stimulated cGMP-specific phosphodiesterase, and the cGMP-target protein GbpC. Here we combine published experiments with many unpublished experiments performed in the past 45 years on the regulation and function of the cGMP signaling pathway. The chemoattractants stimulate heterotrimeric Gαßγ and monomeric Ras proteins. A fraction of the soluble guanylyl cyclase sGC binds with high affinity to a limited number of membrane binding sites, which is essential for sGC to become activated by Ras and Gα proteins. sGC can also bind to F-actin; binding to branched F-actin in pseudopods enhances basal sGC activity, whereas binding to parallel F-actin in the cortex reduces sGC activity. The cGMP pathway mediates cell polarity by inhibiting the rear: in unstimulated cells by sGC activity in the branched F-actin of pseudopods, in a shallow gradient by stimulated cGMP formation in pseudopods at the leading edge, and during cAMP oscillation to erase the previous polarity and establish a new polarity axis that aligns with the direction of the passing cAMP wave.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria.

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

Rapid and efficient genetic engineering of both wild type and axenic strains of Dictyostelium discoideum.

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.

GPCR-controlled membrane recruitment of negative regulator C2GAP1 locally inhibits Ras signaling for adaptation and long-range chemotaxis.

Eukaryotic cells chemotax in a wide range of chemoattractant concentration gradients, and thus need inhibitory processes that terminate cell responses to reach adaptation while maintaining sensitivity to higher-concentration stimuli. However, the molecular mechanisms underlying inhibitory processes are still poorly understood. Here, we reveal a locally controlled inhibitory process in a GPCR-mediated signaling network for chemotaxis in Dictyostelium discoideum We identified a negative regulator of Ras signaling, C2GAP1, which localizes at the leading edge of chemotaxing cells and is activated by and essential for GPCR-mediated Ras signaling. We show that both C2 and GAP domains are required for the membrane targeting of C2GAP1, and that GPCR-triggered Ras activation is necessary to recruit C2GAP1 from the cytosol and retains it on the membrane to locally inhibit Ras signaling. C2GAP1-deficient c2gapA- cells have altered Ras activation that results in impaired gradient sensing, excessive polymerization of F actin, and subsequent defective chemotaxis. Remarkably, these cellular defects of c2gapA- cells are chemoattractant concentration dependent. Thus, we have uncovered an inhibitory mechanism required for adaptation and long-range chemotaxis.

A plasma membrane template for macropinocytic cups.

Macropinocytosis is a fundamental mechanism that allows cells to take up extracellular liquid into large vesicles. It critically depends on the formation of a ring of protrusive actin beneath the plasma membrane, which develops into the macropinocytic cup. We show that macropinocytic cups in Dictyostelium are organised around coincident intense patches of PIP3, active Ras and active Rac. These signalling patches are invariably associated with a ring of active SCAR/WAVE at their periphery, as are all examined structures based on PIP3 patches, including phagocytic cups and basal waves. Patch formation does not depend on the enclosing F-actin ring, and patches become enlarged when the RasGAP NF1 is mutated, showing that Ras plays an instructive role. New macropinocytic cups predominantly form by splitting from existing ones. We propose that cup-shaped plasma membrane structures form from self-organizing patches of active Ras/PIP3, which recruit a ring of actin nucleators to their periphery.

A Gα-Stimulated RapGEF Is a Receptor-Proximal Regulator of Dictyostelium Chemotaxis.

Chemotaxis, or directional movement toward extracellular chemical gradients, is an important property of cells that is mediated through G-protein-coupled receptors (GPCRs). Although many chemotaxis pathways downstream of Gßγ have been identified, few Gα effectors are known. Gα effectors are of particular importance because they allow the cell to distinguish signals downstream of distinct chemoattractant GPCRs. Here we identify GflB, a Gα2 binding partner that directly couples the Dictyostelium cyclic AMP GPCR to Rap1. GflB localizes to the leading edge and functions as a Gα-stimulated, Rap1-specific guanine nucleotide exchange factor required to balance Ras and Rap signaling. The kinetics of GflB translocation are fine-tuned by GSK-3 phosphorylation. Cells lacking GflB display impaired Rap1/Ras signaling and actin and myosin dynamics, resulting in defective chemotaxis. Our observations demonstrate that GflB is an essential upstream regulator of chemoattractant-mediated cell polarity and cytoskeletal reorganization functioning to directly link Gα activation to monomeric G-protein signaling.

Chemotaxis of a model organism: progress with Dictyostelium.

Model organisms have been key to understanding many core biological processes. Dictyostelium amoebae have the attributes required to perform this role for chemotaxis, and by providing an evolutionary distant reference point to mammalian cells, they allow the central features of chemotaxis to be discerned. Here we highlight progress with Dictyostelium in understanding: pseudopod and bleb driven movement; the role of the actin cytoskeleton; chemotactic signal processing, including how cells adapt to background stimulation, and the controversial role of PIP3. Macropinocytosis and the axenic mutations are raised as potential confounding factors, while the identification of new players through proteomics holds great promise.

Neurofibromin controls macropinocytosis and phagocytosis in Dictyostelium.

Cells use phagocytosis and macropinocytosis to internalise bulk material, which in phagotrophic organisms supplies the nutrients necessary for growth. Wildtype Dictyostelium amoebae feed on bacteria, but for decades laboratory work has relied on axenic mutants that can also grow on liquid media. We used forward genetics to identify the causative gene underlying this phenotype. This gene encodes the RasGAP Neurofibromin (NF1). Loss of NF1 enables axenic growth by increasing fluid uptake. Mutants form outsized macropinosomes which are promoted by greater Ras and PI3K activity at sites of endocytosis. Relatedly, NF1 mutants can ingest larger-than-normal particles using phagocytosis. An NF1 reporter is recruited to nascent macropinosomes, suggesting that NF1 limits their size by locally inhibiting Ras signalling. Our results link NF1 with macropinocytosis and phagocytosis for the first time, and we propose that NF1 evolved in early phagotrophs to spatially modulate Ras activity, thereby constraining and shaping their feeding structures.

Drink or drive: competition between macropinocytosis and cell migration.

The cytoskeleton is utilized for a variety of cellular processes, including migration, endocytosis and adhesion. The required molecular components are often shared between different processes, but it is not well understood how the cells balance their use. We find that macropinocytosis and cell migration are negatively correlated. Heavy drinkers move only slowly and vice versa, fast cells do not take big gulps. Both processes are balanced by the lipid phosphatidylinositol 3,4,5-trisphosphate (PIP3). Elevated PIP3 signalling causes a shift towards macropinocytosis and inhibits motility by redirecting the SCAR/WAVE complex, a major nucleator of actin filaments. High resolution microscopy shows that patches with high levels of PIP3 recruit SCAR/WAVE on their periphery, resulting in circular ruffle formation and engulfment. Results shed new light on the role of PIP3, which is commonly thought to promote cell motility.

Melanoma cells break down LPA to establish local gradients that drive chemotactic dispersal.

The high mortality of melanoma is caused by rapid spread of cancer cells, which occurs unusually early in tumour evolution. Unlike most solid tumours, thickness rather than cytological markers or differentiation is the best guide to metastatic potential. Multiple stimuli that drive melanoma cell migration have been described, but it is not clear which are responsible for invasion, nor if chemotactic gradients exist in real tumours. In a chamber-based assay for melanoma dispersal, we find that cells migrate efficiently away from one another, even in initially homogeneous medium. This dispersal is driven by positive chemotaxis rather than chemorepulsion or contact inhibition. The principal chemoattractant, unexpectedly active across all tumour stages, is the lipid agonist lysophosphatidic acid (LPA) acting through the LPA receptor LPAR1. LPA induces chemotaxis of remarkable accuracy, and is both necessary and sufficient for chemotaxis and invasion in 2-D and 3-D assays. Growth factors, often described as tumour attractants, cause negligible chemotaxis themselves, but potentiate chemotaxis to LPA. Cells rapidly break down LPA present at substantial levels in culture medium and normal skin to generate outward-facing gradients. We measure LPA gradients across the margins of melanomas in vivo, confirming the physiological importance of our results. We conclude that LPA chemotaxis provides a strong drive for melanoma cells to invade outwards. Cells create their own gradients by acting as a sink, breaking down locally present LPA, and thus forming a gradient that is low in the tumour and high in the surrounding areas. The key step is not acquisition of sensitivity to the chemoattractant, but rather the tumour growing to break down enough LPA to form a gradient. Thus the stimulus that drives cell dispersal is not the presence of LPA itself, but the self-generated, outward-directed gradient.

PIP3-dependent macropinocytosis is incompatible with chemotaxis.

In eukaryotic chemotaxis, the mechanisms connecting external signals to the motile apparatus remain unclear. The role of the lipid phosphatidylinositol 3,4,5-trisphosphate (PIP3) has been particularly controversial. PIP3 has many cellular roles, notably in growth control and macropinocytosis as well as cell motility. Here we show that PIP3 is not only unnecessary for Dictyostelium discoideum to migrate toward folate, but actively inhibits chemotaxis. We find that macropinosomes, but not pseudopods, in growing cells are dependent on PIP3. PIP3 patches in these cells show no directional bias, and overall only PIP3-free pseudopods orient up-gradient. The pseudopod driver suppressor of cAR mutations (SCAR)/WASP and verprolin homologue (WAVE) is not recruited to the center of PIP3 patches, just the edges, where it causes macropinosome formation. Wild-type cells, unlike the widely used axenic mutants, show little macropinocytosis and few large PIP3 patches, but migrate more efficiently toward folate. Tellingly, folate chemotaxis in axenic cells is rescued by knocking out phosphatidylinositide 3-kinases (PI 3-kinases). Thus PIP3 promotes macropinocytosis and interferes with pseudopod orientation during chemotaxis of growing cells.

Actin dynamics: cell migration takes a new turn with arpin.

Accurate cell migration requires intricate control over the actin cytoskeleton. Recent work has identified an Arp2/3-interacting protein called Arpin, which restricts the rate of actin polymerization and is the latest component in the steadily expanding protein repertoire that controls cell migration.

Measuring chemotaxis using direct visualization microscope chambers.

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.

Extrachromosomal inducible expression.

Inducible expression systems are very convenient for proteins that induce strong side effects such as retardation of growth or development and are essential for the expression of toxic proteins. In this chapter we describe the doxycycline-inducible expression system, optimized for the controlled expression in. Two types of inducible plasmids are presented, in which transcription is induced by either adding or removing doxycycline, respectively. Detailed protocols are provided for the construction of the plasmids and the inducible expression of the target protein.

GxcC connects Rap and Rac signaling during Dictyostelium development.

BACKGROUND: Rap proteins belong to the Ras family of small G-proteins. Dictyostelium RapA is essential and implicated in processes throughout the life cycle. In early development and chemotaxis competent cells RapA induces pseudopod formation by activating PI3K and it regulates substrate attachment and myosin disassembly via the serine/threonine kinase Phg2. RapA is also important in late development, however so far little is known about the downstream effectors of RapA that play a role in this process. RESULTS: Here we show that cells expressing constitutively active RapA exhibit a high level of Rac activation. With a pull-down screen coupled to mass spectrometry, we identified the Rac specific guanine nucleotide exchange factor, GxcC, as Rap binding partner. GxcC binds directly and specifically to active RapA and binds to a subset of Dictyostelium Rac proteins. Deletion studies revealed that this pathway is involved in regulating Dictyostelium development. CONCLUSIONS: GxcC provides a novel link between Rap and Rac signalling and is one of the Rap effectors regulating the progression of multicellular development.

Cyclical action of the WASH complex: FAM21 and capping protein drive WASH recycling, not initial recruitment.

WASH causes actin to polymerize on vesicles involved in retrograde traffic and exocytosis. It is found within a regulatory complex, but the physiological roles of the other four members are unknown. Here we present genetic analysis of the subunits' individual functions in Dictyostelium. Mutants in each subunit are completely blocked in exocytosis. All subunits except FAM21 are required to drive actin assembly on lysosomes. Without actin, lysosomes never recycle vacuolar-type H(+)-adenosine triphosphatase (V-ATPase) or neutralize to form postlysosomes. However, in FAM21 knockout lysosomes, WASH generates excessive, dynamic streams of actin. These successfully remove V-ATPase, neutralize, and form huge postlysosomes. The distinction between WASH and FAM21 phenotypes is conserved in human cells. Thus, FAM21 and WASH act at different steps of a cyclical pathway in which FAM21 mediates recycling of the complex back to acidic lysosomes. Recycling is driven by FAM21's interaction with capping protein, which couples the WASH complex to dynamic actin on vesicles.

Loss of Scar/WAVE complex promotes N-WASP- and FAK-dependent invasion.

BACKGROUND: The Scar/WAVE regulatory complex (WRC) drives lamellipodia assembly via the Arp2/3 complex, whereas the Arp2/3 activator N-WASP is not essential for 2D migration but is increasingly implicated in 3D invasion. It is becoming ever more apparent that 2D and 3D migration utilize the actin cytoskeletal machinery differently. RESULTS: We discovered that WRC and N-WASP play opposing roles in 3D epithelial cell migration. WRC depletion promoted N-WASP/Arp2/3 complex activation and recruitment to leading invasive edges and increased invasion. WRC disruption also altered focal adhesion dynamics and drove FAK activation at leading invasive edges. We observed coalescence of focal adhesion components together with N-WASP and Arp2/3 complex at leading invasive edges in 3D. Unexpectedly, WRC disruption also promoted FAK-dependent cell transformation and tumor growth in vivo. CONCLUSIONS: N-WASP has a crucial proinvasive role in driving Arp2/3 complex-mediated actin assembly in cooperation with FAK at invasive cell edges, but WRC depletion can promote 3D cell motility.

Zizimin and Dock guanine nucleotide exchange factors in cell function and disease.

Zizimin proteins belong to the Dock (Dedicator of Cytokinesis) superfamily of Guanine nucleotide Exchange Factor (GEF) proteins. This family of proteins plays a role in the regulation of Rho family small GTPases. Together the Rho family of small GTPases and the Dock/Zizimin proteins play a vital role in a number of cell processes including cell migration, apoptosis, cell division and cell adhesion. Our recent studies of Zizimin proteins, using a simple biomedical model, the eukaryotic social amoeba Dictyostelium discoideum, have helped to elucidate the cellular role of these proteins. In this article, we discuss the domain structure of Zizimin proteins from an evolutionary viewpoint. We also compare what is currently known about the mammalian Zizimin proteins to that of related Dock proteins. Understanding the cellular functions of these proteins will provide a better insight into their role in cell signaling, and may help in treating disease pathology associated with mutations in Dock/Zizimin proteins.

SCAR knockouts in Dictyostelium: WASP assumes SCAR's position and upstream regulators in pseudopods.

Under normal conditions, the Arp2/3 complex activator SCAR/WAVE controls actin polymerization in pseudopods, whereas Wiskott-Aldrich syndrome protein (WASP) assembles actin at clathrin-coated pits. We show that, unexpectedly, Dictyostelium discoideum SCAR knockouts could still spread, migrate, and chemotax using pseudopods driven by the Arp2/3 complex. In the absence of SCAR, some WASP relocated from the coated pits to the leading edge, where it behaved with similar dynamics to normal SCAR, forming split pseudopods and traveling waves. Pseudopods colocalized with active Rac, whether driven by WASP or SCAR, though Rac was activated to a higher level in SCAR mutants. Members of the SCAR regulatory complex, in particular PIR121, were not required for WASP regulation. We thus show that WASP is able to respond to all core upstream signals and that regulators coupled through the other members of SCAR's regulatory complex are not essential for pseudopod formation. We conclude that WASP and SCAR can regulate pseudopod actin using similar mechanisms.