GRminusRD: A Sensitive Assay to Detect Activation Processes at the Plasma Membrane in Living Cells.

Activation processes at the plasma membrane have been studied with life-cell imaging using GFP fused to a protein that binds to a component of the activation process. In this way, PIP3 formation has been monitored with CRAC-GFP, Ras-GTP with RBD-Raf-GFP, and Rap-GTP with Ral-GDS-GFP. The fluorescent sensors translocate from the cytoplasm to the plasma membrane upon activation of the process. Although this translocation assay can provide very impressive images and movies, the method is not very sensitive, and amount of GFP-sensor at the plasma membrane is not linear with the amount of activator. The fluorescence in pixels at the cell boundary is partly coming from the GFP-sensor that is bound to the activated membrane and partly from unbound GFP-sensor in the cytosolic volume of that boundary pixel. The variable and unknown amount of cytosol in boundary pixels causes the low sensitivity and nonlinearity of the GFP-translocation assay. Here we describe a method in which the GFP-sensor is co-expressed with cytosolic-RFP. For each boundary pixels, the RFP fluorescence is used to determine the amount of cytosol of that pixel and is subtracted from the GFP fluorescence of that pixel yielding the amount of GFP-sensor that is specifically associated with the plasma membrane in that pixel. This GRminusRD method using GFP-sensor/RFP is at least tenfold more sensitive, more reproducible, and linear with activator compared to GFP-sensor alone.

Analysis of cGMP Signaling in Dictyostelium.

Biochemical assays are described to analyze signal transduction by the second messenger cGMP in Dictyostelium. The methods include enzyme assays to measure the activity and regulation of cGMP synthesizing guanylyl cyclases and cGMP-degrading phosphodiesterases. In addition, several methods are described to quantify cGMP levels. The target of cGMP in Dictyostelium is the large protein GbpC that has multiple domains including a Roc domain, a kinase domain, and a cGMP-stimulated Ras-GEF domain. A cGMP-binding assay is described to detect and quantify GbpC.

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.

Combined FCS and PCH Analysis to Quantify Protein Dimerization in Living Cells.

Protein dimerization plays a crucial role in the regulation of numerous biological processes. However, detecting protein dimers in a cellular environment is still a challenge. Here we present a methodology to measure the extent of dimerization of GFP-tagged proteins in living cells, using a combination of fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis of single-color fluorescence fluctuation data. We named this analysis method brightness and diffusion global analysis (BDGA) and adapted it for biological purposes. Using cell lysates containing different ratios of GFP and tandem-dimer GFP (diGFP), we show that the average brightness per particle is proportional to the fraction of dimer present. We further adapted this methodology for its application in living cells, and we were able to distinguish GFP, diGFP, as well as ligand-induced dimerization of FKBP12 (FK506 binding protein 12)-GFP. While other analysis methods have only sporadically been used to study dimerization in living cells and may be prone to errors, this paper provides a robust approach for the investigation of any cytosolic protein using single-color fluorescence fluctuation spectroscopy.

Short- and long-term memory of moving amoeboid cells.

Amoeboid cells constantly change shape and extend protrusions. The direction of movement is not random, but is correlated with the direction of movement in the preceding minutes. The basis of this correlation is an underlying memory of direction. The presence of memory in movement is known for many decades, but its molecular mechanism is still largely unknown. This study reports in detail on the information content of directional memory, the kinetics of learning and forgetting this information, and the molecular basis for memory using Dictyostelium mutants. Two types of memory were characterized. A short-term memory stores for ~20 seconds the position of the last pseudopod using a local modification of the branched F-actin inducer SCAR/WAVE, which enhances one new pseudopod to be formed at the position of the previous pseudopod. A long term memory stores for ~2 minutes the activity of the last ~10 pseudopods using a cGMP-binding protein that induces myosin filaments in the rear of the cell; this inhibits pseudopods in the rear and thereby enhances pseudopods in the global front. Similar types of memory were identified in human neutrophils and mesenchymal stem cells, the protist Dictyostelium and the fungus B.d. chytrid. The synergy of short- and long-term memory explains their role in persistent movement for enhanced cell dispersal, food seeking and chemotaxis.

Unified control of amoeboid pseudopod extension in multiple organisms by branched F-actin in the front and parallel F-actin/myosin in the cortex.

The trajectory of moving eukaryotic cells depends on the kinetics and direction of extending pseudopods. The direction of pseudopods has been well studied to unravel mechanisms for chemotaxis, wound healing and inflammation. However, the kinetics of pseudopod extension-when and why do pseudopods start and stop- is equally important, but is largely unknown. Here the START and STOP of about 4000 pseudopods was determined in four different species, at four conditions and in nine mutants (fast amoeboids Dictyostelium and neutrophils, slow mesenchymal stem cells, and fungus B.d. chytrid with pseudopod and a flagellum). The START of a first pseudopod is a random event with a probability that is species-specific (23%/s for neutrophils). In all species and conditions, the START of a second pseudopod is strongly inhibited by the extending first pseudopod, which depends on parallel filamentous actin/myosin in the cell cortex. Pseudopods extend at a constant rate by polymerization of branched F-actin at the pseudopod tip, which requires the Scar complex. The STOP of pseudopod extension is induced by multiple inhibitory processes that evolve during pseudopod extension and mainly depend on the increasing size of the pseudopod. Surprisingly, no differences in pseudopod kinetics are detectable between polarized, unpolarized or chemotactic cells, and also not between different species except for small differences in numerical values. This suggests that the analysis has uncovered the fundament of cell movement with distinct roles for stimulatory branched F-actin in the protrusion and inhibitory parallel F-actin in the contractile cortex.

Symmetry Breaking during Cell Movement in the Context of Excitability, Kinetic Fine-Tuning and Memory of Pseudopod Formation.

The path of moving eukaryotic cells depends on the kinetics and direction of extending pseudopods. Amoeboid cells constantly change their shape with pseudopods extending in different directions. Detailed analysis has revealed that time, place and direction of pseudopod extension are not random, but highly ordered with strong prevalence for only one extending pseudopod, with defined life-times, and with reoccurring events in time and space indicative of memory. Important components are Ras activation and the formation of branched F-actin in the extending pseudopod and inhibition of pseudopod formation in the contractile cortex of parallel F-actin/myosin. In biology, order very often comes with symmetry. In this essay, I discuss cell movement and the dynamics of pseudopod extension from the perspective of symmetry and symmetry changes of Ras activation and the formation of branched F-actin in the extending pseudopod. Combining symmetry of Ras activation with kinetics and memory of pseudopod extension results in a refined model of amoeboid movement that appears to be largely conserved in the fast moving Dictyostelium and neutrophils, the slow moving mesenchymal stem cells and the fungus B.d. chytrid.

The cytoskeleton regulates symmetry transitions in moving amoeboid cells.

Symmetry and symmetry breaking are essential in biology. Symmetry comes in different forms: rotational symmetry, mirror symmetry and alternating right-left symmetry (for example, gliding reflection symmetry). Especially the transitions between the different symmetry forms are important because they specify crucial points in cell biology, including gastrulation in development, formation of the cleavage furrow in cell division, or the front in cell polarity. However, the mechanisms of these symmetry transitions are not well understood. Here, we have investigated the fundamental properties of symmetry and symmetry transitions of the cytoskeleton during cell movement. Our data show that the dynamic shape changes of amoeboid cells are far from random, but are the consequence of refined symmetries and symmetry changes that are orchestrated by small G-proteins and the cytoskeleton, with local stimulation by F-actin and Scar, and local inhibition by IQGAP2 and myosin.

A homologue of the Parkinson's disease-associated protein LRRK2 undergoes a monomer-dimer transition during GTP turnover.

Mutations in LRRK2 are a common cause of genetic Parkinson's disease (PD). LRRK2 is a multi-domain Roco protein, harbouring kinase and GTPase activity. In analogy with a bacterial homologue, LRRK2 was proposed to act as a GTPase activated by dimerization (GAD), while recent reports suggest LRRK2 to exist under a monomeric and dimeric form in vivo. It is however unknown how LRRK2 oligomerization is regulated. Here, we show that oligomerization of a homologous bacterial Roco protein depends on the nucleotide load. The protein is mainly dimeric in the nucleotide-free and GDP-bound states, while it forms monomers upon GTP binding, leading to a monomer-dimer cycle during GTP hydrolysis. An analogue of a PD-associated mutation stabilizes the dimer and decreases the GTPase activity. This work thus provides insights into the conformational cycle of Roco proteins and suggests a link between oligomerization and disease-associated mutations in LRRK2.

Coupled excitable Ras and F-actin activation mediates spontaneous pseudopod formation and directed cell movement.

Many eukaryotic cells regulate their mobility by external cues. Genetic studies have identified >100 components that participate in chemotaxis, which hinders the identification of the conceptual framework of how cells sense and respond to shallow chemical gradients. The activation of Ras occurs during basal locomotion and is an essential connector between receptor and cytoskeleton during chemotaxis. Using a sensitive assay for activated Ras, we show here that activation of Ras and F-actin forms two excitable systems that are coupled through mutual positive feedback and memory. This coupled excitable system leads to short-lived patches of activated Ras and associated F-actin that precede the extension of protrusions. In buffer, excitability starts frequently with Ras activation in the back/side of the cell or with F-actin in the front of the cell. In a shallow gradient of chemoattractant, local Ras activation triggers full excitation of Ras and subsequently F-actin at the side of the cell facing the chemoattractant, leading to directed pseudopod extension and chemotaxis. A computational model shows that the coupled excitable Ras/F-actin system forms the driving heart for the ordered-stochastic extension of pseudopods in buffer and for efficient directional extension of pseudopods in chemotactic gradients.

A Worldwide Competition to Compare the Speed and Chemotactic Accuracy of Neutrophil-Like Cells.

Chemotaxis is the ability to migrate towards the source of chemical gradients. It underlies the ability of neutrophils and other immune cells to hone in on their targets and defend against invading pathogens. Given the importance of neutrophil migration to health and disease, it is crucial to understand the basic mechanisms controlling chemotaxis so that strategies can be developed to modulate cell migration in clinical settings. Because of the complexity of human genetics, Dictyostelium and HL60 cells have long served as models system for studying chemotaxis. Since many of our current insights into chemotaxis have been gained from these two model systems, we decided to compare them side by side in a set of winner-take-all races, the Dicty World Races. These worldwide competitions challenge researchers to genetically engineer and pharmacologically enhance the model systems to compete in microfluidic racecourses. These races bring together technological innovations in genetic engineering and precision measurement of cell motility. Fourteen teams participated in the inaugural Dicty World Race 2014 and contributed cell lines, which they tuned for enhanced speed and chemotactic accuracy. The race enabled large-scale analyses of chemotaxis in complex environments and revealed an intriguing balance of speed and accuracy of the model cell lines. The successes of the first race validated the concept of using fun-spirited competition to gain insights into the complex mechanisms controlling chemotaxis, while the challenges of the first race will guide further technological development and planning of future events.

Mathematics of Experimentally Generated Chemoattractant Gradients.

Many eukaryotic cells move in the direction of a chemical gradient. Several assays have been developed to measure this chemotactic response, but no complete mathematical models of the spatial and temporal gradients are available to describe the fundamental principles of chemotaxis. Here we provide analytical solutions for the gradients formed by release of chemoattractant from a point source by passive diffusion or forced flow (micropipettes) and gradients formed by laminar diffusion in a Zigmond chamber. The results show that gradients delivered with a micropipette are formed nearly instantaneously, are very steep close to the pipette, and have a steepness that is strongly dependent on the distance from the pipette. In contrast, gradients in a Zigmond chamber are formed more slowly, are nearly independent of the distance from the source, and resemble the temporal and spatial properties of the natural cAMP wave that Dictyostelium cells experience during cell aggregation.

The small GTPases Ras and Rap1 bind to and control TORC2 activity.

Target of Rapamycin Complex 2 (TORC2) has conserved roles in regulating cytoskeleton dynamics and cell migration and has been linked to cancer metastasis. However, little is known about the mechanisms regulating TORC2 activity and function in any system. In Dictyostelium, TORC2 functions at the front of migrating cells downstream of the Ras protein RasC, controlling F-actin dynamics and cAMP production. Here, we report the identification of the small GTPase Rap1 as a conserved binding partner of the TORC2 component RIP3/SIN1, and that Rap1 positively regulates the RasC-mediated activation of TORC2 in Dictyostelium. Moreover, we show that active RasC binds to the catalytic domain of TOR, suggesting a mechanism of TORC2 activation that is similar to Rheb activation of TOR complex 1. Dual Ras/Rap1 regulation of TORC2 may allow for integration of Ras and Rap1 signaling pathways in directed cell migration.

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.

Direct Interaction between TalinB and Rap1 is necessary for adhesion of Dictyostelium cells.

BACKGROUND: The small G-protein Rap1 is an important regulator of cellular adhesion in Dictyostelium, however so far the downstream signalling pathways for cell adhesion are not completely characterized. In mammalian cells talin is crucial for adhesion and Rap1 was shown to be a key regulator of talin signalling. RESULTS: In a proteomic screen we identified TalinB as a potential Rap1 effector in Dictyostelium. In subsequent pull-down experiments we demonstrate that the Ras association (RA) domain of TalinB interacts specifically with active Rap1. Studies with a mutated RA domain revealed that the RA domain is essential for TalinB-Rap1 interaction, and that this interaction contributes to cell-substrate adhesion during single-celled growth and is crucial for cell-cell adhesion during multicellular development. CONCLUSIONS: Dictyostelium Rap1 directly binds to TalinB via the conserved RA domain. This interaction is critical for adhesion, which becomes essential for high adhesive force demanding processes, like morphogenesis during multicellular development of Dictyostelium. In mammalian cells the established Rap1-talin interaction is indirect and acts through the scaffold protein - RIAM. Interestingly, direct binding of mouse Rap1 to the RA domain of Talin1 has recently been demonstrated.

Rap1-dependent pathways coordinate cytokinesis in Dictyostelium.

Cytokinesis is the final step of mitosis when a mother cell is separated into two daughter cells. Major cytoskeletal changes are essential for cytokinesis; it is, however, not well understood how the microtubules and actomyosin cytoskeleton are exactly regulated in time and space. In this paper, we show that during the early stages of cytokinesis, in rounded-up Dictyostelium discoideum cells, the small G-protein Rap1 is activated uniformly at the cell cortex. When cells begin to elongate, active Rap1 becomes restricted from the furrow region, where the myosin contractile ring is subsequently formed. In the final stages of cytokinesis, active Rap1 is only present at the cell poles. Mutant cells with decreased Rap1 activation at the poles showed strongly decreased growth rates. Hyperactivation of Rap1 results in severe growth delays and defective spindle formation in adherent cells and cell death in suspension. Furthermore, Rap mutants show aberrant regulation of the actomyosin cytoskeleton, resulting in extended furrow ingression times and asymmetrical cell division. We propose that Rap1 drives cytokinesis progression by coordinating the three major cytoskeletal components: microtubules, actin, and myosin II. Importantly, mutated forms of Rap also affect cytokinesis in other organisms, suggesting a conserved role for Rap in cell division.

Ras activation and symmetry breaking during Dictyostelium chemotaxis.

Central to chemotaxis is the molecular mechanism by which a shallow spatial gradient of chemoattractant induces symmetry breaking of activated signaling molecules. Previously, we have used Dictyostelium mutants to investigate the minimal requirements for chemotaxis, and identified a basal signaling module providing activation of Ras and F-actin at the leading edge. Here, we show that Ras activation after application of a pipette releasing the chemoattractant cAMP has three phases, each depending on specific guanine-nucleotide-exchange factors (GEFs). Initially a transient activation of Ras occurs at the entire cell boundary, which is proportional to the local cAMP concentrations and therefore slightly stronger at the front than in the rear of the cell. This transient Ras activation is present in gα2 (gpbB)-null cells but not in gß (gpbA)-null cells, suggesting that Gßγ mediates the initial activation of Ras. The second phase is symmetry breaking: Ras is activated only at the side of the cell closest to the pipette. Symmetry breaking absolutely requires Gα2 and Gßγ, but not the cytoskeleton or four cAMP-induced signaling pathways, those dependent on phosphatidylinositol (3,4,5)-triphosphate [PtdIns(3,4,5)P3], cGMP, TorC2 and PLA2. As cells move in the gradient, the crescent of activated Ras in the front half of the cell becomes confined to a small area at the utmost front of the cell. Confinement of Ras activation leads to cell polarization, and depends on cGMP formation, myosin and F-actin. The experiments show that activation, symmetry breaking and confinement of Ras during Dictyostelium chemotaxis uses different G-protein subunits and a multitude of Ras GEFs and GTPase-activating proteins (GAPs).

Dictyostelium Ric8 is a nonreceptor guanine exchange factor for heterotrimeric G proteins and is important for development and chemotaxis.

Heterotrimeric G proteins couple external signals to the activation of intracellular signal transduction pathways. Agonist-stimulated guanine nucleotide exchange activity of G-protein-coupled receptors results in the exchange of G-protein-bound GDP to GTP and the dissociation and activation of the complex into Gα-GTP and a Gßγ dimer. In Dictyostelium, a basal chemotaxis pathway consisting of heterotrimeric and monomeric G proteins is sufficient for chemotaxis. Symmetry breaking and amplification of chemoattractant sensing occurs between heterotrimeric G protein signaling and Ras activation. In a pull-down screen coupled to mass spectrometry, with Gα proteins as bait, we have identified resistant to inhibitors of cholinesterase 8 (Ric8) as a nonreceptor guanine nucleotide exchange factor for Gα-protein. Ric8 is not essential for the initial activation of heterotrimeric G proteins or Ras by uniform chemoattractant; however, it amplifies Gα signaling, which is essential for Ras-mediated symmetry breaking during chemotaxis and development.

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.