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Normal-sized cells of Dictyostelium build up a front-tail polarity when they respond to a gradient of chemoattractant. To challenge the polarity-generating system, cells are fused to study the chemotactic response of oversized cells that extend multiple fronts toward the source of attractant. An aspect that can be explored in these cells is the relationship of spontaneously generated actin waves to actin reorganization in response to chemoattractant., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The extracellular environment plays a crucial role in many physiological and pathological processes involving cell motility, such as metastatic invasion in cancer development, by heavily impacting the migration strategies adopted by the cells. The study of how mechanical constraints affect the dynamics of cell migration may be relevant to gain more insight into such processes, and it may prove to be a powerful tool in the hands of biologists. In this chapter, we describe the methods used to investigate the ability of neoplastic cells to migrate through narrowing, rigid microstructures upon chemoattractant stimulation., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

GPCR signaling is the most prevailing molecular mechanism for detecting ambient signals in eukaryotes. Chemotactic cells use GPCR signaling to process chemical cues for directional migration over a broad concentration range and with high sensitivity. Dictyostelium discoideum is a classical model, in which the molecular mechanism underlying eukaryotic chemotaxis has been well studied. Here, we describe protocols to evaluate the spatiotemporal chemotactic responses of Dictyostelium discoideum by different microscopic observations combined with biochemical assays. First, two different chemotaxis assays are presented to measure the dynamic concentration ranges for different cell strains or chemotactic parameters. Next, live-cell imaging and biochemical assays are provided to detect the activities of GPCR and its partner heterotrimeric G proteins upon chemoattractant stimulation. Finally, a method for detecting how a cell deciphers chemical gradients is described.

Receptor activation upon ligand binding induces activation of multiple signaling pathways. To fully understand how these signaling pathways coordinate, it is essential to determine the dynamic nature of the spatiotemporal activation profile of signaling components at the level of single living cells. Here, we outline a detailed methodology for visualizing and quantitatively measuring the spatiotemporal activation of Ras and PKD1 by applying advanced fluorescence imaging techniques, including multichannel, simultaneous imaging and Förster resonance energy transfer (FRET).

Dictyostelium discoideum has proven an excellent model for the study of eukaryotic chemotaxis. During growth in its native environment, Dictyostelium phagocytose bacteria and fungi for primary nutrient capture. Growing Dictyostelium can detect these nutrient sources through chemotaxis toward the metabolic by-product folate. Although Dictyostelium grow as individual cells, nutrient depletion induces a multicellular development program and a separate chemotactic response pathway. During development, Dictyostelium synthesize and secrete cAMP, which serves as a chemoattractant to mobilize and coordinate cells for multicellular formation and development. Separate classes of GPCRs and Gα proteins mediate chemotactic signaling to the chemically distinct ligands. We discuss common and separate component responses of Dictyostelium to folate and cAMP during growth and development, and the advantages and disadvantages for each. As examples, we present biochemical assays to characterize the chemoattractant-induced kinase activations of mTORC2 and the ERKs.

A better understanding of the molecular mechanisms leading to mast cell migration and chemotaxis is the long-term goal in mast cell research and is essential for comprehension of mast cell function in health and disease. Various techniques have been developed in recent decades for in vitro and in vivo assessment of mast cell motility and chemotaxis. In this chapter three microscopy assays facilitating real-time quantification of mast cell chemotaxis and migration are described, focusing on individual cell tracking and data analysis.

Macrophages, which are organized throughout every tissue, represent a key component of the immune system and the recruitment of macrophages to specific sites is important in normal host defense. However, when inappropriately recruited macrophages may damage or destroy healthy tissue; this is seen in several autoimmune diseases such as arthritis. Many cytokines, including CSF-1 and chemokines, are often upregulated in inflamed tissues and can induce the directional migration of macrophages towards the highest concentration of the cytokine in a process called chemotaxis. Chemokines were first described as chemoattractant cytokines synthesized at sites of inflammation that stimulate the directional migration of leukocytes and mediate inflammation. Whereas specific receptors for chemoattractants reside over the entire cell surface, macrophages can detect very shallow chemotactic gradients leading to spatially defined responses to the chemoattractant such as the extension of directed protrusions leading to cell migration. In this chapter we describe a method for the localized delivery of chemoattractants via a micropipette needle to macrophages in culture followed by methods for imaging and an outline of quantifying macrophage responses.

Trichoplax adhaerens is an enigmatic basal animal with an extraordinarily simple morphological organization and surprisingly complex behaviors. Basic morphological, molecular and behavioral work is essential to better understand the unique and curious life style of these organisms. We provide basic instructions on how Trichoplax can be cultured and studied in the laboratory emphasizing behavioral and cellular aspects.

The endogenous chemotaxis of cells toward sites of tissue injury and/or biomaterial implantation is an important component of the host response. Implanted biomaterials capable of recruiting host stem/progenitor cells to a site of interest may obviate challenges associated with cell transplantation. An assay for the identification and quantification of chemotaxis induced by surgically placed biologic scaffolds composed of extracellular matrix is described herein.

Chemotaxis is the phenomenon in which cells direct their movements in the presence of certain chemicals (chemoattractants or chemorepellents). Leukocyte recruitment (via chemotaxis) is an important component of the inflammatory response, both in physiological host defense and in a range of prevalent disorders that include an inflammatory component. Circulating leukocytes in the bloodstream migrate towards the site of inflammation in response to a complex network of proinflammatory signaling molecules (including cytokines, chemokines and prostaglandins). This chapter describes a method for rapid measure of the chemoattractant capacity of nanoparticulate materials. This method is an in vitro model for chemotaxis, in which promyelocytic leukemia cell migration through a filter is monitored using a fluorescent dye.

Dictyostelium discoideum is one of the most facile eukaryotic systems for the study of chemotactic response to secreted chemical ligands. Dictyostelium grow as individual cells, using bacteria and fungi as primary nutrient sources; during growth, Dictyostelium moves directionally toward folate, a bacterial byproduct. Upon nutrient depletion Dictyostelium initiates a multicellular development program characterized by the production and secretion of cAMP. Cell surface receptors specifically recognize extracellular cAMP, which serves as both a morphogen to promote development and a chemoattractant to organize multicellularity. We discuss several approaches for the study of ligand-receptor interaction, with focus on affinity class determination and quantification of ligand binding sites (i.e., receptors) per cell. We further present examples for the application of biochemical assays to characterize the ligand-induced kinase activation of PI3K, GSK3, and ERK2.

The involvement of calcium signalling during chemotaxis in Dictyostelium discoideum is well documented. Spatiotemporal increases of intracellular calcium ([Ca(2+)](i)) have been observed within seconds of stimulation with the chemoattractants folic acid and cAMP. This rise in [Ca(2+)](i) localises to the rear cortex of the cell (J. Cell Sci. 109:2673-2678, 1996) and has been found to be not essential for chemotaxis, but likely to be involved in fine tuning of chemotactic responses (EMBO J. 19:4846-4854, 2000). Measurements of cytosolic Ca(2+) ([Ca(2+)](c)) responses have involved the use of different Ca(2+) probes including ectopically expressed aequorin (a Ca(2+)-sensitive photoprotein), the fluorescent dye fura-2-dextran and the radioisotope (45)Ca(2+). The aequorin method (J. Cell Sci. 110:2845-2853, 1997) offers nonperturbing, real-time measurement of cytosolic free Ca(2+) in suspensions of cells, but the low levels of light emission preclude measurements on individual cells. Fura-2 imaging (Cell Calcium 22:65-74, 1997; Eur. J. Cell Biol. 58:172-181, 1992; Biochem. J. 332:541-548, 1998; BMC Cell Biol. 6:13, 2005) has the advantage of allowing Ca(2+) responses to be observed in individual cells so that the subcellular localisation of the response and differences amongst individual cells can be observed. However data collection is more labour intensive, much smaller numbers of cells are sampled, the cells are unavoidably damaged physically during loading and the time resolution (s) is much less than that provided by the aequorin method (ms). (45)Ca(2+) uptake assays (Cell Biol. Int. Rep. 2:71-79, 1978; J. Cell Biol. 112:103-110, 1991) allow measurement of Ca(2+) influx from the medium by cell suspensions with a time resolution of the order of seconds. Radioactive Ca(2+) uptake measurements are unsullied by but equally do not provide information about Ca(2+) efflux, intracellular Ca(2+) release or sequestration or changes in cytosolic free Ca(2+) levels.

We describe an in vivo invasion assay that enables the collection of invasive cells from the primary tumor. In addition to determination of the endogenous, unstimulated invasive properties of cells in vivo, the assay can take advantage of the chemotactic properties of cancer cells. Microneedles are filled with a mixture of extracellular matrix components such as Matrigel with or without a chemoattractant such as EGF, and then introduced into the primary tumor of a rat or mouse that is generated either by orthotopic injection of carcinoma cell lines or by a transgene such as polyoma Middle T. Over the course of 4 h the invasive cell population enters the needles while the animal is kept under anesthesia. At the end of the collection time, the invasive cells are extruded from the microneedles and can be analyzed in terms of the number and type of cells that invade in response to defined stimuli. By including pharmacological inhibitors in the needle, signaling pathways contributing to in vivo invasion can also be identified. This assay leads to a better understanding of the cell types and signaling involved in the tumor microenvironment, and has the potential to be applied to a variety of in vivo models.

Many eukaryotic cells can elicit intracellular signaling relays to produce pseudopodia and move up to the chemoattractant gradient (chemotaxis) or move randomly in the absence of extracellular stimuli and nutrients (random movement). A precise spatiotemporal regulation of Ras-GTPases, such as Ras and Rap, is crucial to induce pseudopodia formation and cellular adhesion during the chemotaxis and random movement. Here, we describe biochemical and real-time imaging methods for using Dictyostelium to understand the signaling events important for chemotaxis and random cell movement. The chapter includes (1) a biochemical method to assess Ras and Rap1 activation in response to chemoattractant, (2) an imaging method to detect endogenous Ras and Rap1 activation in moving cells, and (3) a simultaneous imaging method to decipher the precise order and localization of these signaling events. With a combination of powerful Dictyostelium genetics, these methods will facilitate to elucidate a dynamic activation of Ras proteins and their inter relay with other signaling molecules during chemotaxis and random movement.

This chapter will focus on responses that the chemoattractant cyclic AMP elicits in the motility system of Dictyostelium. These cells can be permanently transfected to express cytoskeleton-associated proteins tagged with fluorescent proteins. Multiple proteins that are distinguishable by the excitation and emission spectra of their tags can be simultaneously expressed. This makes it possible to relate the spatial and temporal patterns of their chemoattractant-induced translocation to each other in one cell by a single recording. Since actin polymerization in live cells progresses with velocities of about 3 microm/s, high image frequencies and short acquisition times in the millisecond range are required. Techniques of total internal reflection fluorescence (TIRF) and spinning-disc confocal microscopy provide appropriate temporal and spatial resolution for the analysis of actin dynamics.

The ability of cells to migrate directionally in gradients of chemoattractant is a fundamental biological response that is essential for the survival of the social amoebae Dictyostelium discoideum. In Dictyostelium, cAMP is the most potent chemoattractant and the detection, synthesis, and degradation of cAMP is exquisitely regulated. Interestingly, as Dictyostelium cells migrate directionally, they do so in a head-to-tail fashion, forming characteristic streams. This group behavior is acquired through the relay of the cAMP signals to neighboring cells. This chapter describes experimental procedures used to obtain synchronized populations of chemotactically competent cells and to assess their streaming behavior. In addition, we provide a detailed account of the method used to measure the ability of chemoattractants to directly stimulate adenylyl cyclase activity. Together, these techniques provide a way to combine cell biological and biochemical approaches to the study of signal relay.

Chemotaxis is a highly coordinated biological system where chemoattractants trigger multiple signal transduction pathways which act in concert to bring about directed migration. A signaling pathway acting through PIP(3), which accumulates at the leading edge of the cell, has been extensively characterized. However, chemotaxis still remains in cells depleted of PIP(3), suggesting there are PIP(3)-independent pathways. We have identified a pathway involving TorC2-PKBR1 as well as another containing PLA2 activity that act in parallel with PIP(3). Activation of PKBR1, a myristoylated Protein Kinase B homolog, is dependent on TorC2 (Rapamycin-insensitive Tor complex 2) kinase but is completely independent of PIP(3). In response to chemoattractant, PKBs rapidly phosphorylate at least eight proteins, including Talin B, PI4P 5-kinase, two RasGefs, and a RhoGap. These studies help to link the signaling pathways to specific effectors and provide a more complete understanding of chemotaxis.

Polymorphonuclear neutrophil (PMN) recruitment from the blood stream into surrounding tissues, followed by migration through the tissue with triggered release of oxidative enzymes or eventual clearance from the epithelial surface, involves a regulated series of events central to acute responses in host defense. Accumulations of large numbers of neutrophils within mucosal tissues are pathognomonic features of both acute and chronic inflammatory conditions including sepsis and inflammatory bowel disease, but the precise signals governing neutrophil adhesion and transmigration remain to be fully characterized. Previous chapters examine methods employed for both neutrophil isolation and study of the mechanisms underlying regulation of PMN rolling behavior. Here, we describe in vitro experimental models for the examination of PMN adhesion to endothelial and epithelial monolayers as well as the characterization of signals influencing neutrophil migration, both along acellular matrices and across endothelial and epithelial monolayers, in the physiologically relevant directions. Studies employing these model systems allow further elucidation of the mechanisms governing PMN adhesion and transmigration.