The Amoebal Model for Macropinocytosis.

Macropinocytosis is a relatively unexplored form of large-scale endocytosis driven by the actin cytoskeleton. Dictyostelium amoebae form macropinosomes from cups extended from the plasma membrane, then digest their contents and absorb the nutrients in the endo-lysosomal system. They use macropinocytosis for feeding, maintaining a high rate of fluid uptake that makes assay and experimentation easy. Mutants collected over the years identify cytoskeletal and signalling proteins required for macropinocytosis. Cups are organized around plasma membrane domains of intense PIP3, Ras and Rac signalling, proper formation of which also depends on the RasGAPs NF1 and RGBARG, PTEN, the PIP3-regulated protein kinases Akt and SGK and their activators PDK1 and TORC2, Rho proteins, plus other components yet to be identified. This PIP3 domain directs dendritic actin polymerization to the extending lip of macropinocytic cups by recruiting a ring of the SCAR/WAVE complex around itself and thus activating the Arp2/3 complex. The dynamics of PIP3 domains are proposed to shape macropinocytic cups from start to finish. The role of the Ras-PI3-kinase module in organizing feeding structures in unicellular organisms most likely predates its adoption into growth factor signalling, suggesting an evolutionary origin for growth factor signalling.

Triparental inheritance in Dictyostelium.

Sex promotes the recombination and reassortment of genetic material and is prevalent across eukaryotes, although our knowledge of the molecular details of sexual inheritance is scant in several major lineages. In social amoebae, sex involves a promiscuous mixing of cytoplasm before zygotes consume the majority of cells, but for technical reasons, sexual progeny have been difficult to obtain and study. We report here genome-wide characterization of meiotic progeny in Dictyostelium discoideum We find that recombination occurs at high frequency in pairwise crosses between all three mating types, despite the absence of the Spo11 enzyme that is normally required to initiate crossover formation. Fusions of more than two gametes to form transient syncytia lead to frequent triparental inheritance, with haploid meiotic progeny bearing recombined nuclear haplotypes from two parents and the mitochondrial genome from a third. Cells that do not contribute genetically to the Dictyostelium zygote nucleus thereby have a stake in the next haploid generation. D. discoideum mitochondrial genomes are polymorphic, and our findings raise the possibility that some of this variation might be a result of sexual selection on genes that can promote the spread of individual organelle genomes during sex. This kind of self-interested mitochondrial behavior may have had important consequences during eukaryogenesis and the initial evolution of sex.

Akt and SGK protein kinases are required for efficient feeding by macropinocytosis.

Macropinocytosis is an actin-driven process of large-scale and non-specific fluid uptake used for feeding by some cancer cells and the macropinocytosis model organism Dictyostelium discoideum In Dictyostelium, macropinocytic cups are organized by 'macropinocytic patches' in the plasma membrane. These contain activated Ras, Rac and phospholipid PIP3, and direct actin polymerization to their periphery. We show that a Dictyostelium Akt (PkbA) and an SGK (PkbR1) protein kinase act downstream of PIP3 and, together, are nearly essential for fluid uptake. This pathway enables the formation of larger macropinocytic patches and macropinosomes, thereby dramatically increasing fluid uptake. Through phosphoproteomics, we identify a RhoGAP, GacG, as a PkbA and PkbR1 target, and show that it is required for efficient macropinocytosis and expansion of macropinocytic patches. The function of Akt and SGK in cell feeding through control of macropinosome size has implications for cancer cell biology.

Amplification of PIP3 signalling by macropinocytic cups.

In a role distinct from and perhaps more ancient than that in signal transduction, PIP3 and Ras help to spatially organize the actin cytoskeleton into macropinocytic cups. These large endocytic structures are extended by actin polymerization from the cell surface and have at their core an intense patch of active Ras and PIP3, around which actin polymerizes, creating cup-shaped projections. We hypothesize that active Ras and PIP3 self-amplify within macropinocytic cups, in a way that depends on the structural integrity of the cup. Signalling that triggers macropinocytosis may therefore be amplified downstream in a way that depends on macropinocytosis. This argument provides a context for recent findings that signalling to Akt (an effector of PIP3) is sensitive to cytoskeletal and macropinocytic inhibitors.

The role of ADP-ribosylation in regulating DNA interstrand crosslink repair.

ADP-ribosylation by ADP-ribosyltransferases (ARTs) has a well-established role in DNA strand break repair by promoting enrichment of repair factors at damage sites through ADP-ribose interaction domains. Here, we exploit the simple eukaryote Dictyostelium to uncover a role for ADP-ribosylation in regulating DNA interstrand crosslink repair and redundancy of this pathway with non-homologous end-joining (NHEJ). In silico searches were used to identify a protein that contains a permutated macrodomain (which we call aprataxin/APLF-and-PNKP-like protein; APL). Structural analysis reveals that this permutated macrodomain retains features associated with ADP-ribose interactions and that APL is capable of binding poly(ADP-ribose) through this macrodomain. APL is enriched in chromatin in response to cisplatin treatment, an agent that induces DNA interstrand crosslinks (ICLs). This is dependent on the macrodomain of APL and the ART Adprt2, indicating a role for ADP-ribosylation in the cellular response to cisplatin. Although adprt2- cells are sensitive to cisplatin, ADP-ribosylation is evident in these cells owing to redundant signalling by the double-strand break (DSB)-responsive ART Adprt1a, promoting NHEJ-mediated repair. These data implicate ADP-ribosylation in DNA ICL repair and identify that NHEJ can function to resolve this form of DNA damage in the absence of Adprt2.

Lipid droplet dynamics at early stages of Mycobacterium marinum infection in Dictyostelium.

Lipid droplets exist in virtually every cell type, ranging not only from mammals to plants, but also to eukaryotic and prokaryotic unicellular organisms such as Dictyostelium and bacteria. They serve among other roles as energy reservoir that cells consume in times of starvation. Mycobacteria and some other intracellular pathogens hijack these organelles as a nutrient source and to build up their own lipid inclusions. The mechanisms by which host lipid droplets are captured by the pathogenic bacteria are extremely poorly understood. Using the powerful Dictyostelium discoideum/Mycobacterium marinum infection model, we observed that, immediately after their uptake, lipid droplets translocate to the vicinity of the vacuole containing live but not dead mycobacteria. Induction of lipid droplets in Dictyostelium prior to infection resulted in a vast accumulation of neutral lipids and sterols inside the bacterium-containing compartment. Subsequently, under these conditions, mycobacteria accumulated much larger lipid inclusions. Strikingly, the Dictyostelium homologue of perilipin and the murine perilipin 2 surrounded bacteria that had escaped to the cytosol of Dictyostelium or microglial BV-2 cells respectively. Moreover, bacterial growth was inhibited in Dictyostelium plnA knockout cells. In summary, our results provide evidence that mycobacteria actively manipulate the lipid metabolism of the host from very early infection stages.

Dictyostelium lipid droplets host novel proteins.

Across all kingdoms of life, cells store energy in a specialized organelle, the lipid droplet. In general, it consists of a hydrophobic core of triglycerides and steryl esters surrounded by only one leaflet derived from the endoplasmic reticulum membrane to which a specific set of proteins is bound. We have chosen the unicellular organism Dictyostelium discoideum to establish kinetics of lipid droplet formation and degradation and to further identify the lipid constituents and proteins of lipid droplets. Here, we show that the lipid composition is similar to what is found in mammalian lipid droplets. In addition, phospholipids preferentially consist of mainly saturated fatty acids, whereas neutral lipids are enriched in unsaturated fatty acids. Among the novel protein components are LdpA, a protein specific to Dictyostelium, and Net4, which has strong homologies to mammalian DUF829/Tmem53/NET4 that was previously only known as a constituent of the mammalian nuclear envelope. The proteins analyzed so far appear to move from the endoplasmic reticulum to the lipid droplets, supporting the concept that lipid droplets are formed on this membrane.

A Reliable System for Quantitative G-Protein Activation Imaging in Cancer Cells.

Fluorescence resonance energy transfer (FRET) biosensors have proven to be an indispensable tool in cell biology and, more specifically, in the study of G-protein signalling. The best method of measuring the activation status or FRET state of a biosensor is often fluorescence lifetime imaging microscopy (FLIM), as it does away with many disadvantages inherent to fluorescence intensity-based methods and is easily quantitated. Despite the significant potential, there is a lack of reliable FLIM-FRET biosensors, and the data processing and analysis workflows reported previously face reproducibility challenges. Here, we established a system in live primary mouse pancreatic ductal adenocarcinoma cells, where we can detect the activation of an mNeonGreen-Gαi3-mCherry-Gγ2 biosensor through the lysophosphatidic acid receptor (LPAR) with 2-photon time-correlated single-photon counting (TCSPC) FLIM. This combination gave a superior signal to the commonly used mTurquoise2-mVenus G-protein biosensor. This system has potential as a platform for drug screening, or to answer basic cell biology questions in the field of G-protein signalling.

Competition between chemoattractants causes unexpected complexity and can explain negative chemotaxis.

Negative chemotaxis, where eukaryotic cells migrate away from repellents, is important throughout biology, for example, in nervous system patterning and resolution of inflammation. However, the mechanisms by which molecules repel migrating cells are unknown. Here, we use predictive modeling and experiments with Dictyostelium cells to show that competition between different ligands that bind to the same receptor leads to effective chemorepulsion. 8-CPT-cAMP, widely described as a simple chemorepellent, is inactive on its own and only repels cells when it acts in combination with the attractant cAMP. If cells degrade either competing ligand, the pattern of migration becomes more complex; cells may be repelled in one part of a gradient but attracted elsewhere, leading to populations moving in different directions in the same assay or converging in an arbitrary place. More counterintuitively still, two chemicals that normally attract cells can become repellent when combined. Computational models of chemotaxis are now accurate enough to predict phenomena that have not been anticipated by experiments. We have used them to identify new mechanisms that drive reverse chemotaxis, which we have confirmed through experiments with real cells. These findings are important whenever multiple ligands compete for the same receptors.

Formation and closure of macropinocytic cups in Dictyostelium.

Macropinocytosis is a conserved endocytic process by which cells engulf droplets of medium into micron-sized vesicles. We use light-sheet microscopy to define an underlying set of principles by which macropinocytic cups are shaped and closed in Dictyostelium amoebae. Cups form around domains of PIP3 stretching almost to their lip and are supported by a specialized F-actin scaffold from lip to base. They are shaped by a ring of actin polymerization created by recruiting Scar/WAVE and Arp2/3 around PIP3 domains, but how cups evolve over time to close and form a vesicle is unknown. Custom 3D analysis shows that PIP3 domains expand from small origins, capturing new membrane into the cup, and crucially, that cups close when domain expansion stalls. We show that cups can close in two ways: either at the lip, by inwardly directed actin polymerization, or the base, by stretching and delamination of the membrane. This provides the basis for a conceptual mechanism whereby closure is brought about by a combination of stalled cup expansion, continued actin polymerization at the lip, and membrane tension. We test this through the use of a biophysical model, which can recapitulate both forms of cup closure and explain how 3D cup structures evolve over time to mediate engulfment.

Glioblastoma extracellular vesicles influence glial cell hyaluronic acid deposition to promote invasiveness.

Background: Infiltration of glioblastoma (GBM) throughout the brain leads to its inevitable recurrence following standard-of-care treatments, such as surgical resection, chemo-, and radiotherapy. A deeper understanding of the mechanisms invoked by GBM to infiltrate the brain is needed to develop approaches to contain the disease and reduce recurrence. The aim of this study was to discover mechanisms through which extracellular vesicles (EVs) released by GBM influence the brain microenvironment to facilitate infiltration, and to determine how altered extracellular matrix (ECM) deposition by glial cells might contribute to this. Methods: CRISPR was used to delete genes, previously established to drive carcinoma invasiveness and EV production, from patient-derived primary and GBM cell lines. We purified and characterized EVs released by these cells, assessed their capacity to foster pro-migratory microenvironments in mouse brain slices, and evaluated the contribution made by astrocyte-derived ECM to this. Finally, we determined how CRISPR-mediated deletion of genes, which we had found to control EV-mediated communication between GBM cells and astrocytes, influenced GBM infiltration when orthotopically injected into CD1-nude mice. Results: GBM cells expressing a p53 mutant (p53R273H) with established pro-invasive gain-of-function release EVs containing a sialomucin, podocalyxin (PODXL), which encourages astrocytes to deposit ECM with increased levels of hyaluronic acid (HA). This HA-rich ECM, in turn, promotes migration of GBM cells. Consistently, CRISPR-mediated deletion of PODXL opposes infiltration of GBM in vivo. Conclusions: This work describes several key components of an EV-mediated mechanism though which GBM cells educate astrocytes to support infiltration of the surrounding healthy brain tissue.

Steering yourself by the bootstraps: how cells create their own gradients for chemotaxis.

Chemotaxis, where cell movement is steered by chemical gradients, is a widespread and essential way of organising cell behaviour. But where do the instructions come from - who makes gradients, and how are they controlled? We discuss the emerging concept that chemotactic cells often create attractant gradients at the same time as responding to them. This self-guidance is more robust, works across greater distances, and is more informative about the local environment than passive responses. Several mechanisms can establish autonomous gradients. Best known are self-generated gradients, in which the cells degrade a widespread attractant, but cells also produce repellents and 'relay' by secreting fresh attractant after stimulation. Understanding how cells make and interpret their own chemoattractant gradients is fundamental to understanding the spatial patterns seen in all organisms.

AKT and SGK kinases regulate cell migration by altering Scar/WAVE complex activation and Arp2/3 complex recruitment.

Cell polarity and cell migration both depend on pseudopodia and lamellipodia formation. These are regulated by coordinated signaling acting through G-protein coupled receptors and kinases such as PKB/AKT and SGK, as well as the actin cytoskeletal machinery. Here we show that both Dictyostelium PKB and SGK kinases (encoded by pkbA and pkgB) are dispensable for chemotaxis towards folate. However, both are involved in the regulation of pseudopod formation and thus cell motility. Cells lacking pkbA and pkgB showed a substantial drop in cell speed. Actin polymerization is perturbed in pkbA- and reduced in pkgB- and pkbA-/pkgB- mutants. The Scar/WAVE complex, key catalyst of pseudopod formation, is recruited normally to the fronts of all mutant cells (pkbA-, pkgB- and pkbA-/pkgB-), but is unexpectedly unable to recruit the Arp2/3 complex in cells lacking SGK. Consequently, loss of SGK causes a near-complete loss of normal actin pseudopodia, though this can be rescued by overexpression of PKB. Hence both PKB and SGK are required for correct assembly of F-actin and recruitment of the Arp2/3 complex by the Scar/WAVE complex during pseudopodia formation.

Seeing around corners: Cells solve mazes and respond at a distance using attractant breakdown.

During development and metastasis, cells migrate large distances through complex environments. Migration is often guided by chemotaxis, but simple chemoattractant gradients between a source and sink cannot direct cells over such ranges. We describe how self-generated gradients, created by cells locally degrading attractant, allow single cells to navigate long, tortuous paths and make accurate choices between live channels and dead ends. This allows cells to solve complex mazes efficiently. Cells' accuracy at finding live channels was determined by attractant diffusivity, cell speed, and path complexity. Manipulating these parameters directed cells in mathematically predictable ways; specific combinations can even actively misdirect them. We propose that the length and complexity of many long-range migratory processes, including inflammation and germ cell migration, means that self-generated gradients are needed for successful navigation.

Cell and molecular transitions during efficient dedifferentiation.

Dedifferentiation is a critical response to tissue damage, yet is not well understood, even at a basic phenomenological level. Developing Dictyostelium cells undergo highly efficient dedifferentiation, completed by most cells within 24 hr. We use this rapid response to investigate the control features of dedifferentiation, combining single cell imaging with high temporal resolution transcriptomics. Gene expression during dedifferentiation was predominantly a simple reversal of developmental changes, with expression changes not following this pattern primarily associated with ribosome biogenesis. Mutation of genes induced early in dedifferentiation did not strongly perturb the reversal of development. This apparent robustness may arise from adaptability of cells: the relative temporal ordering of cell and molecular events was not absolute, suggesting cell programmes reach the same end using different mechanisms. In addition, although cells start from different fates, they rapidly converged on a single expression trajectory. These regulatory features may contribute to dedifferentiation responses during regeneration.

Function of small GTPases in Dictyostelium macropinocytosis.

Macropinocytosis-the large-scale, non-specific uptake of fluid by cells-is used by Dictyostelium discoideum amoebae to obtain nutrients. These cells form circular ruffles around regions of membrane defined by a patch of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and the activated forms of the small G-proteins Ras and Rac. When this ruffle closes, a vesicle of the medium is delivered to the cell interior for further processing. It is accepted that PIP3 is required for efficient macropinocytosis. Here, we assess the roles of Ras and Rac in Dictyostelium macropinocytosis. Gain-of-function experiments show that macropinocytosis is stimulated by persistent Ras activation and genetic analysis suggests that RasG and RasS are the key Ras proteins involved. Among the activating guanine exchange factors (GEFs), GefF is implicated in macropinocytosis by an insertional mutant. The individual roles of Rho family proteins are little understood but activation of at least some may be independent of PIP3. This article is part of the Theo Murphy meeting issue 'Macropinocytosis'.

Living on soup: macropinocytic feeding in amoebae.

Macropinocytosis is used by a variety of amoebae for feeding on liquid medium. The amoebae project cups and ruffles from their plasma membrane, driven by actin polymerization, and eventually fuse these back to the membrane, entrapping droplets of medium into internal vesicles. These vesicles are of up to several microns in diameter and are processed through the lysosomal digestive system to extract nutrients. Recognizably the same process is used in metazoan cells for a number of medically important purposes, including the pathological growth of cancer cells. We describe the discovery of macropinocytosis in Dictyostelium amoebae, its genetic regulation by the NF1 RasGAP, and the tools available for its investigation. Work on Dictyostelium over the last 30 years has identified many genes that may be important for macropinocytosis, which are listed at dictyBase, and give a basis for mechanistic studies. We argue that the actin cytoskeleton is organized for macropinocytosis by a signalling patch of PIP3 and active Ras and Rac, together with their regulatory proteins and effectors, including the protein kinases Akt and SGK. The Scar/WAVE complex is recruited to the periphery of this patch, triggering the formation of a hollow ring of protrusive actin polymerization, and eventually a macropinocytic cup. Major problems to be addressed include: the dynamics sustaining macropinocytic patches and the mechanism of Scar/WAVE recruitment; the mechanisms of cup closure and of membrane fusion; the ecological situations where amoebae feed by macropinocytosis; and the evolutionary relationship between macropinocytosis and growth factor signalling.

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.

The Atypical MAP Kinase ErkB Transmits Distinct Chemotactic Signals through a Core Signaling Module.

Signaling from chemoattractant receptors activates the cytoskeleton of crawling cells for chemotaxis. We show using phosphoproteomics that different chemoattractants cause phosphorylation of the same core set of around 80 proteins in Dictyostelium cells. Strikingly, the majority of these are phosphorylated at an [S/T]PR motif by the atypical MAP kinase ErkB. Unlike most chemotactic responses, ErkB phosphorylations are persistent and do not adapt to sustained stimulation with chemoattractant. ErkB integrates dynamic autophosphorylation with chemotactic signaling through G-protein-coupled receptors. Downstream, our phosphoproteomics data define a broad panel of regulators of chemotaxis. Surprisingly, targets are almost exclusively other signaling proteins, rather than cytoskeletal components, revealing ErkB as a regulator of regulators rather than acting directly on the motility machinery. ErkB null cells migrate slowly and orientate poorly over broad dynamic ranges of chemoattractant. Our data indicate a central role for ErkB and its substrates in directing chemotaxis.

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.