Pseudopod Tracking and Statistics During Cell Movement in Buffer and Chemotaxis.

Amoeboid cells such as the protist Dictyostelium, human neutrophils, and the fungus B.d. chytrid move by extending pseudopods.

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.

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.

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

In , 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 Here we combine published experiments with many unpublished experiments performed in the past 45 years on the regulation and function of the cGMP signaling pathway.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 Gβγ 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.

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.

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.

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.

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).

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 Gβγ dimer. In Dictyostelium, a basal chemotaxis pathway consisting of heterotrimeric and monomeric G proteins is sufficient for chemotaxis.

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.

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.

Roco kinase structures give insights into the mechanism of Parkinson disease-related leucine-rich-repeat kinase 2 mutations.

Mutations in human leucine-rich-repeat kinase 2 (LRRK2) have been found to be the most frequent cause of late-onset Parkinson disease. Here we show that Dictyostelium discoideum Roco4 is a suitable model to study the structural and biochemical characteristics of the LRRK2 kinase and can be used for optimization of current and identification of new LRRK2 inhibitors. We have solved the structure of Roco4 kinase wild-type, Parkinson disease-related mutants G1179S and L1180T (G2019S and I2020T in LRRK2) and the structure of Roco4 kinase in complex with the LRRK2 inhibitor H1152.

Multiple regulatory mechanisms for the Dictyostelium Roco protein GbpC.

GbpC is a multidomain Roco protein in Dictyostelium, involved in transduction of intracellular cGMP that is produced by chemotactic signals. We have shown previously that cGMP binding to GbpC induces an intramolecular signaling cascade by activating subsequently the GEF, Ras, and kinase domains. In this study, we report on the cellular localization of GbpC.

Amoeboid cells use protrusions for walking, gliding and swimming.

Amoeboid cells crawl using pseudopods, which are convex extensions of the cell surface. In many laboratory experiments, cells move on a smooth substrate, but in the wild cells may experience obstacles of other cells or dead material, or may even move in liquid. To understand how cells cope with heterogeneous environments we have investigated the pseudopod life cycle of wild type and mutant cells moving on a substrate and when suspended in liquid.