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.

Biogenesis of lysosome-related organelles complex-2 is an evolutionarily ancient proto-coatomer complex.

Hermansky-Pudlak syndrome (HPS) is an inherited disorder of intracellular vesicle trafficking affecting the function of lysosome-related organelles (LROs). At least 11 genes underlie the disease, encoding four protein complexes, of which biogenesis of lysosome-related organelles complex-2 (BLOC-2) is the last whose molecular action is unknown. We find that the unicellular eukaryote Dictyostelium unexpectedly contains a complete BLOC-2, comprising orthologs of the mammalian subunits HPS3, -5, and -6, and a fourth subunit, an ortholog of the Drosophila LRO-biogenesis gene, Claret. Lysosomes from Dictyostelium BLOC-2 mutants fail to mature, similar to LROs from HPS patients, but for all endolysosomes rather than a specialized subset. They also strongly resemble lysosomes from WASH mutants. Dictyostelium BLOC-2 localizes to the same compartments as WASH, and in BLOC-2 mutants, WASH is inefficiently recruited, accounting for their impaired lysosomal maturation. BLOC-2 is recruited to endolysosomes via its HPS3 subunit. Structural modeling suggests that all four subunits are proto-coatomer proteins, with important implications for BLOC-2's molecular function. The discovery of Dictyostelium BLOC-2 permits identification of orthologs throughout eukaryotes. BLOC-2 and lysosome-related organelles, therefore, pre-date the evolution of Metazoa and have broader and more conserved functions than previously thought.

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.

Extracellular Signalling Modulates Scar/WAVE Complex Activity through Abi Phosphorylation.

The lamellipodia and pseudopodia of migrating cells are produced and maintained by the Scar/WAVE complex. Thus, actin-based cell migration is largely controlled through regulation of Scar/WAVE. Here, we report that the Abi subunit-but not Scar-is phosphorylated in response to extracellular signalling in Dictyostelium cells. Like Scar, Abi is phosphorylated after the complex has been activated, implying that Abi phosphorylation modulates pseudopodia, rather than causing new ones to be made. Consistent with this, Scar complex mutants that cannot bind Rac are also not phosphorylated. Several environmental cues also affect Abi phosphorylation-cell-substrate adhesion promotes it and increased extracellular osmolarity diminishes it. Both unphosphorylatable and phosphomimetic Abi efficiently rescue the chemotaxis of Abi KO cells and pseudopodia formation, confirming that Abi phosphorylation is not required for activation or inactivation of the Scar/WAVE complex. However, pseudopodia and Scar patches in the cells with unphosphorylatable Abi protrude for longer, altering pseudopod dynamics and cell speed. Dictyostelium, in which Scar and Abi are both unphosphorylatable, can still form pseudopods, but migrate substantially faster. We conclude that extracellular signals and environmental responses modulate cell migration by tuning the behaviour of the Scar/WAVE complex after it has been activated.

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-substrate adhesion drives Scar/WAVE activation and phosphorylation by a Ste20-family kinase, which controls pseudopod lifetime.

The Scar/WAVE complex is the principal catalyst of pseudopod and lamellipod formation. Here we show that Scar/WAVE's proline-rich domain is polyphosphorylated after the complex is activated. Blocking Scar/WAVE activation stops phosphorylation in both Dictyostelium and mammalian cells, implying that phosphorylation modulates pseudopods after they have been formed, rather than controlling whether they are initiated. Unexpectedly, phosphorylation is not promoted by chemotactic signaling but is greatly stimulated by cell:substrate adhesion and diminished when cells deadhere. Phosphorylation-deficient or phosphomimetic Scar/WAVE mutants are both normally functional and rescue the phenotype of knockout cells, demonstrating that phosphorylation is dispensable for activation and actin regulation. However, pseudopods and patches of phosphorylation-deficient Scar/WAVE last substantially longer in mutants, altering the dynamics and size of pseudopods and lamellipods and thus changing migration speed. Scar/WAVE phosphorylation does not require ERK2 in Dictyostelium or mammalian cells. However, the MAPKKK homologue SepA contributes substantially-sepA mutants have less steady-state phosphorylation, which does not increase in response to adhesion. The mutants also behave similarly to cells expressing phosphorylation-deficient Scar, with longer-lived pseudopods and patches of Scar recruitment. We conclude that pseudopod engagement with substratum is more important than extracellular signals at regulating Scar/WAVE's activity and that phosphorylation acts as a pseudopod timer by promoting Scar/WAVE turnover.

WASP Restricts Active Rac to Maintain Cells' Front-Rear Polarization.

Efficient motility requires polarized cells, with pseudopods at the front and a retracting rear. Polarization is maintained by restricting the pseudopod catalyst, active Rac, to the front. Here, we show that the actin nucleation-promoting factor Wiskott-Aldrich syndrome protein (WASP) contributes to maintenance of front-rear polarity by controlling localization and cellular levels of active Rac. Dictyostelium cells lacking WASP inappropriately activate Rac at the rear, which affects their polarity and speed. WASP's Cdc42 and Rac interacting binding ("CRIB") motif has been thought to be essential for its activation. However, we show that the CRIB motif's biological role is unexpectedly complex. WASP CRIB mutants are no longer able to restrict Rac activity to the front, and cannot generate new pseudopods when SCAR/WAVE is absent. Overall levels of Rac activity also increase when WASP is unable to bind to Rac. However, WASP without a functional CRIB domain localizes normally at clathrin pits during endocytosis, and activates Arp2/3 complex. Similarly, chemical inhibition of Rac does not affect WASP localization or activation at sites of endocytosis. Thus, the interaction between small GTPases and WASP is more complex than previously thought-Rac regulates a subset of WASP functions, but WASP reciprocally restricts active Rac through its CRIB motif.

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.

Fam49/CYRI interacts with Rac1 and locally suppresses protrusions.

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.

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.

The trimeric coiled-coil HSBP1 protein promotes WASH complex assembly at centrosomes.

The Arp2/3 complex generates branched actin networks that exert pushing forces onto different cellular membranes. WASH complexes activate Arp2/3 complexes at the surface of endosomes and thereby fission transport intermediates containing endocytosed receptors, such as α5ß1 integrins. How WASH complexes are assembled in the cell is unknown. Here, we identify the small coiled-coil protein HSBP1 as a factor that specifically promotes the assembly of a ternary complex composed of CCDC53, WASH, and FAM21 by dissociating the CCDC53 homotrimeric precursor. HSBP1 operates at the centrosome, which concentrates the building blocks. HSBP1 depletion in human cancer cell lines and in Dictyostelium amoebae phenocopies WASH depletion, suggesting a critical role of the ternary WASH complex for WASH functions. HSBP1 is required for the development of focal adhesions and of cell polarity. These defects impair the migration and invasion of tumor cells. Overexpression of HSBP1 in breast tumors is associated with increased levels of WASH complexes and with poor prognosis for patients.

WASP family proteins and formins compete in pseudopod- and bleb-based migration.

Actin pseudopods induced by SCAR/WAVE drive normal migration and chemotaxis in eukaryotic cells. Cells can also migrate using blebs, in which the edge is driven forward by hydrostatic pressure instead of actin. In Dictyostelium discoideum, loss of SCAR is compensated by WASP moving to the leading edge to generate morphologically normal pseudopods. Here we use an inducible double knockout to show that cells lacking both SCAR and WASP are unable to grow, make pseudopods or, unexpectedly, migrate using blebs. Remarkably, amounts and dynamics of actin polymerization are normal. Pseudopods are replaced in double SCAR/WASP mutants by aberrant filopods, induced by the formin dDia2. Further disruption of the gene for dDia2 restores cells' ability to initiate blebs and thus migrate, though pseudopods are still lost. Triple knockout cells still contain near-normal F-actin levels. This work shows that SCAR, WASP, and dDia2 compete for actin. Loss of SCAR and WASP causes excessive dDia2 activity, maintaining F-actin levels but blocking pseudopod and bleb formation and migration.

LPP3 mediates self-generation of chemotactic LPA gradients by melanoma cells.

Melanoma cells steer out of tumours using self-generated lysophosphatidic acid (LPA) gradients. The cells break down LPA, which is present at high levels around the tumours, creating a dynamic gradient that is low in the tumour and high outside. They then migrate up this gradient, creating a complex and evolving outward chemotactic stimulus. Here, we introduce a new assay for self-generated chemotaxis, and show that raising LPA levels causes a delay in migration rather than loss of chemotactic efficiency. Knockdown of the lipid phosphatase LPP3 - but not of its homologues LPP1 or LPP2 - diminishes the cell's ability to break down LPA. This is specific for chemotactically active LPAs, such as the 18:1 and 20:4 species. Inhibition of autotaxin-mediated LPA production does not diminish outward chemotaxis, but loss of LPP3-mediated LPA breakdown blocks it. Similarly, in both 2D and 3D invasion assays, knockdown of LPP3 diminishes the ability of melanoma cells to invade. Our results demonstrate that LPP3 is the key enzyme in the breakdown of LPA by melanoma cells, and confirm the importance of attractant breakdown in LPA-mediated cell steering.This article has an associated First Person interview with the first author of the paper.

Mroh1, a lysosomal regulator localized by WASH-generated actin.

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.

The Inositol-3-Phosphate Synthase Biosynthetic Enzyme Has Distinct Catalytic and Metabolic Roles.

Inositol levels, maintained by the biosynthetic enzyme inositol-3-phosphate synthase (Ino1), are altered in a range of disorders, including bipolar disorder and Alzheimer's disease. To date, most inositol studies have focused on the molecular and cellular effects of inositol depletion without considering Ino1 levels. Here we employ a simple eukaryote, Dictyostelium discoideum, to demonstrate distinct effects of loss of Ino1 and inositol depletion. We show that loss of Ino1 results in an inositol auxotrophy that can be rescued only partially by exogenous inositol. Removal of inositol supplementation from the ino1(-) mutant resulted in a rapid 56% reduction in inositol levels, triggering the induction of autophagy, reduced cytokinesis, and substrate adhesion. Inositol depletion also caused a dramatic generalized decrease in phosphoinositide levels that was rescued by inositol supplementation. However, loss of Ino1 triggered broad metabolic changes consistent with the induction of a catabolic state that was not rescued by inositol supplementation. These data suggest a metabolic role for Ino1 that is independent of inositol biosynthesis. To characterize this role, an Ino1 binding partner containing SEL1L1 domains (Q54IX5) and having homology to mammalian macromolecular complex adaptor proteins was identified. Our findings therefore identify a new role for Ino1, independent of inositol biosynthesis, with broad effects on cell metabolism.

Abi is required for modulation and stability but not localization or activation of the SCAR/WAVE complex.

The SCAR/WAVE complex drives actin-based protrusion, cell migration, and cell separation during cytokinesis. However, the contribution of the individual complex members to the activity of the whole remains a mystery. This is primarily because complex members depend on one another for stability, which limits the scope for experimental manipulation. Several studies suggest that Abi, a relatively small complex member, connects signaling to SCAR/WAVE complex localization and activation through its polyproline C-terminal tail. We generated a deletion series of the Dictyostelium discoideum Abi to investigate its exact role in regulation of the SCAR complex and identified a minimal fragment that would stabilize the complex. Surprisingly, loss of either the N terminus of Abi or the C-terminal polyproline tail conferred no detectable defect in complex recruitment to the leading edge or the formation of pseudopods. A fragment containing approximately 20% Abi--and none of the sites that couple to known signaling pathways--allowed the SCAR complex to function with normal localization and kinetics. However, expression of N-terminal Abi deletions exacerbated the cytokinesis defect of the Dictyostelium abi mutant, which was earlier shown to be caused by the inappropriate activation of SCAR. This demonstrates, unexpectedly, that Abi does not mediate the SCAR complex's ability to make pseudopods, beyond its role in complex stability. Instead, we propose that Abi has a modulatory role when the SCAR complex is activated through other mechanisms.

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.

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.

Phosphorylation of actin-related protein 2 (Arp2) is required for normal development and cAMP chemotaxis in Dictyostelium.

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.

Bio-informatics analysis of a gene co-expression module in adipose tissue containing the diet-responsive gene Nnat.

BACKGROUND: Obesity causes insulin resistance in target tissues - skeletal muscle, adipose tissue, liver and the brain. Insulin resistance predisposes to type-2 diabetes (T2D) and cardiovascular disease (CVD). Adipose tissue inflammation is an essential characteristic of obesity and insulin resistance. Neuronatin (Nnat) expression has been found to be altered in a number of conditions related to inflammatory or metabolic disturbance, but its physiological roles and regulatory mechanisms in adipose tissue, brain, pancreatic islets and other tissues are not understood. RESULTS: We identified transcription factor binding sites (TFBS) conserved in the Nnat promoter, and transcription factors (TF) abundantly expressed in adipose tissue. These include transcription factors concerned with the control of: adipogenesis (Pparγ, Klf15, Irf1, Creb1, Egr2, Gata3); lipogenesis (Mlxipl, Srebp1c); inflammation (Jun, Stat3); insulin signalling and diabetes susceptibility (Foxo1, Tcf7l2). We also identified NeuroD1 the only documented TF that controls Nnat expression. We identified KEGG pathways significantly associated with Nnat expression, including positive correlations with inflammation and negative correlations with metabolic pathways (most prominently oxidative phosphorylation, glycolysis and gluconeogenesis, pyruvate metabolism) and protein turnover. 27 genes, including; Gstt1 and Sod3, concerned with oxidative stress; Sncg and Cxcl9 concerned with inflammation; Ebf1, Lgals12 and Fzd4 involved in adipogenesis; whose expression co-varies with Nnat were identified, and conserved transcription factor binding sites identified on their promoters. Functional networks relating to each of these genes were identified. CONCLUSIONS: Our analysis shows that Nnat is an acute diet-responsive gene in white adipose tissue and hypothalamus; it may play an important role in metabolism, adipogenesis, and resolution of oxidative stress and inflammation in response to dietary excess.