Amoeboid Chemotaxis

Chemotaxis is the directed movement of a cell towards a gradient of chemicals such as chemokines or growth factors. This phenomenon can be studied in organisms ranging from bacteria to mammalian cells and here we will focus on eukaryotic amoeboid chemotaxis. Chemotactic responses are mediated by two major classes of receptors: GPCR’s and RTK’s, with multiple pathways signaling downstream of them, certain ones functioning in parallel. In this review we address two important features of amoeboid chemotaxis that will be important for further advances in the field. First, the application of in vivo imaging will be critical for providing insight into the functional requirements for chemotactic responses. We will briefly cover a number of systems in which in vivo imaging is providing new insights. Second, due to the network-type design of signaling pathways of eukaryotic chemotaxis, more refined phenotypic analysis will be necessary, and we will discuss recent analyses of the role of the phosphoinositide 3-kinase pathway in this light. We will close with some speculations regarding future applications of more detailed in vivo analysis and mechanistic understanding of eukaryotic amoeboid chemotaxis.     

Migrating fibroblasts reorient directionality by a metastable, PI3K-dependent mechanism

Mesenchymal cell migration as exhibited by fibroblasts is distinct from amoeboid cell migration and is characterized by dynamic competition among multiple protrusions, which determines directional persistence and responses to spatial cues. Localization of phosphoinositide 3-kinase (PI3K) signaling is thought to play a broadly important role in cell motility, yet the context-dependent functions of this pathway have not been adequately elucidated. By mapping the spatiotemporal dynamics of cell protrusion/retraction and PI3K signaling monitored by total internal reflection fluorescence microscopy, we show that randomly migrating fibroblasts reorient polarity through PI3K-dependent branching and pivoting of protrusions. PI3K inhibition did not affect the initiation of newly branched protrusions, nor did it prevent protrusion induced by photoactivation of Rac. Rather, PI3K signaling increased after, not before, the onset of local protrusion and was required for the lateral spreading and stabilization of nascent branches. During chemotaxis, the branch experiencing the higher chemoattractant concentration was favored, and, thus, the cell reoriented so as to align with the external gradient.     

Four key signaling pathways mediating chemotaxis in Dictyostelium discoideum

Chemotaxis is the ability of cells to move in the direction of an external gradient of signaling molecules. Cells are guided by actin-filled protrusions in the front, whereas myosin filaments retract the rear of the cell. Previous work demonstrated that chemotaxis of unpolarized amoeboid Dictyostelium discoideum cells is mediated by two parallel pathways, phosphoinositide-3-kinase (PI3K) and phospholipase A2 (PLA2). Here, we show that polarized cells exhibit very good chemotaxis with inhibited PI3K and PLA2 activity. Using genetic screens, we demonstrate that this activity is mediated by a soluble guanylyl cyclase, providing two signals. The protein localizes to the leading edge where it interacts with actin filaments, whereas the cyclic guanosine monophosphate product induces myosin filaments in the rear of the cell. We conclude that chemotaxis is mediated by multiple signaling pathways regulating protrusions at the front and rear of the cell. Cells that express only rear activity are polarized but do not exhibit chemotaxis, whereas cells with only front signaling are unpolarized but undergo chemotaxis.     

Effects of CapG overexpression on agonist-induced motility and second messenger generation

Actin modulating proteins that bind polyphosphoinositides, such as phosphatidylinositol 4, 5-bisphosphate (PIP2), can potentially participate in receptor signaling by restructuring the membrane cytoskeleton and modulating second messenger generation through the phosphoinositide cycle. We examined these possibilities by overexpressing CapG, an actin filament end capping, Ca(2+)- and polyphosphoinositide-binding protein of the gelsolin family. High level transient overexpression decreased actin filament staining in the center of the cells but not in the cell periphery. Moderate overexpression in clonally selected cell lines did not have a detectible effect on actin filament content or organization. Nevertheless, it promoted a dose-dependent increase in rates of wound healing and chemotaxis. The motile phenotype was similar to that observed with gelsolin overexpression, which in addition to capping, also severs and nucleates actin filaments. CapG overexpressing clones are more responsive to platelet-derived growth factor than control- transfected clones. They form more circular dorsal membrane ruffles, have higher phosphoinositide turnover, inositol 1,4,5-trisphosphate generation and Ca2+ signaling. These responses are consistent with enhanced PLC gamma activity. Direct measurements of PIP2 mass showed that the CapG effect on PLC gamma was not due primarily to an increase in the PIP2 substrate concentration. The observed changes in cell motility and membrane signaling are consistent with the hypothesis that PIP(2)-binding actin regulatory proteins modulate phosphoinositide turnover and second messenger generation in vivo. We infer that CapG and related proteins are poised to coordinate membrane signaling with actin filament dynamics following cell stimulation.     

Naringenin is a novel inhibitor of Dictyostelium cell proliferation and cell migration

Naringenin is a flavanone compound that alters critical cellular processes such as cell multiplication, glucose uptake, and mitochondrial activity. In this study, we used the social amoeba, Dictyostelium discoideum, as a model system for examining the cellular processes and signaling pathways affected by naringenin. We found that naringenin inhibited Dictyostelium cell division in a dose-dependent manner (IC(50) approximately 20 microM). Assays of Dictyostelium chemotaxis and multicellular development revealed that naringenin possesses a previously unrecognized ability to suppress amoeboid cell motility. We also found that naringenin, which is known to inhibit phosphatidylinositol 3-kinase activity, had no apparent effect on phosphatidylinositol 3,4,5-trisphosphate synthesis in live Dictyostelium cells; suggesting that this compound suppresses cell growth and migration via alternative signaling pathways. In another context, the discoveries described here highlight the value of using the Dictyostelium model system for identifying and characterizing the mechanisms by which naringenin, and related compounds, exert their effects on eukaryotic cells.     

Differential activation of formyl peptide receptor-like 1 by peptide ligands

Formyl peptide receptor-like 1 (FPRL1) plays a key role in the regulation of immune responses. The activation of FPRL1 induces a complicated pattern of cellular signaling, which results in the regulation of several immune responses, such as chemotactic migration and the production of reactive oxygen species (ROS). Because some of these cellular responses are not beneficial to the host, ligands that selectively modulate these cellular responses are useful. His-Phe-Tyr-Leu-Pro-Met (HFYLPM) is a synthetic peptide that binds to FPRL1. In this study, we generated various HFYLPM analogues and examined their effects on cellular responses via FPRL1 in FPRL1-expressing rat basophilic leukemia-2H3 cells or in primary human neutrophils. Among the HXYLPM analogues, His-Arg-Tyr-Leu-Pro-Met (HRYLPM) activated a broad spectrum of cellular signaling events, including an intracellular Ca(2+) concentration increase, phosphoinositide 3-kinase, extracellular signal-regulated kinase, and Akt activation, however, His-Glu-Tyr-Leu-Pro-Met (HEYLPM) activated only intracellular Ca(2+) concentration and Akt but did not increase Ca(2+). In addition, HRYLPM was found to stimulate chemotaxis and ROS generation via phosphoinositide 3-kinase and an intracellular Ca(2+) concentration increase, respectively, whereas HEYLPM stimulated chemotaxis but not ROS generation. With respect to the molecular mechanisms involved in the differential action of HRYLPM and HEYLPM, we found that HRYLPM but not HEYLPM competitively inhibited the binding of (125)I-labeled Trp-Lys-Tyr-Met-Val-D-Met-NH(2) (WKYMVm, a FPRL1 ligand) to FPRL1. This study demonstrates that the important chemoattractant receptor, FPRL1, may be differentially modulated by distinct peptide ligands. We also suggest that HRYLPM and HEYLPM may be used to selectively modulate FPRL1.     

Three-dimensional simulation of obstacle-mediated chemotaxis

Amoeboid cells exhibit a highly dynamic motion that can be directed by external chemical signals, through the process of chemotaxis. Here, we propose a three-dimensional model for chemotactic motion of amoeboid cells. We account for the interactions between the extracellular substances, the membrane-bound proteins, and the cytosolic components involved in the signaling pathway that originates cell motility. We show two- and three-dimensional simulations of cell migration on planar substrates, flat surfaces with obstacles, and fibrous networks. The results show that our model reproduces the main features of chemotactic amoeboid motion. Our simulations unveil a complicated interplay between the geometry of the cell’s environment and the chemoattractant dynamics that tightly regulates cell motion. The model opens new opportunities to simulate the interactions between extra- and intra-cellular compounds mediated by the matrix geometry.     

Minimal Network Topologies for Signal Processing during Collective Cell Chemotaxis

Cell-cell communication plays an important role in collective cell migration. However, it remains unclear how cells in a group cooperatively process external signals to determine the group’s direction of motion. Although the topology of signaling pathways is vitally important in single-cell chemotaxis, the signaling topology for collective chemotaxis has not been systematically studied. Here, we combine mathematical analysis and simulations to find minimal network topologies for multicellular signal processing in collective chemotaxis. We focus on border cell cluster chemotaxis in the Drosophila egg chamber, in which responses to several experimental perturbations of the signaling network are known. Our minimal signaling network includes only four elements: a chemoattractant, the protein Rac (indicating cell activation), cell protrusion, and a hypothesized global factor responsible for cell-cell interaction. Experimental data on cell protrusion statistics allows us to systematically narrow the number of possible topologies from more than 40,000,000 to only six minimal topologies with six interactions between the four elements. This analysis does not require a specific functional form of the interactions, and only qualitative features are needed; it is thus robust to many modeling choices. Simulations of a stochastic biochemical model of border cell chemotaxis show that the qualitative selection procedure accurately determines which topologies are consistent with the experiment. We fit our model for all six proposed topologies; each produces results that are consistent with all experimentally available data. Finally, we suggest experiments to further discriminate possible pathway topologies.     

Stochastic signal inputs for chemotactic response in Dictyostelium cells revealed by single molecule imaging techniques

Chemotactic cells can exhibit extreme sensitivity to chemical gradients. Theoretical estimations of the signal inputs required for chemotaxis suggest that the response can be achieved under the strong influence of stochastic input noise generated by the receptors during the transmembrane signaling. This arises a fundamental question regarding the mechanisms for directional sensing: how do cells obtain reliable information regarding gradient direction by using stochastically operating receptors and the downstream molecules? To address this question, we have developed single molecule imaging techniques to visualize signaling molecules responsible for chemotaxis in living Dictyostelium cells, allowing us to monitor the stochastic signaling processes directly. Single molecule imaging of a chemoattractant bound to a receptor demonstrates that signal inputs fluctuate with time and space. Downstream signaling molecules, such as PTEN and a PH domain-containing protein that are constituent parts of chemotactic signaling system, can also be followed at single molecule level in living cells, illuminating the stochastic nature of chemotactic signaling processes. In this report, we start with a brief introduction of chemotactic response of the eukaryotic cells, followed by an explanation for single molecule imaging techniques, and finally discuss these applications to chemotactic signaling system of Dictyostelium cells.     

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (''APCs'') referred to as ''immunological synapses''. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of ''semi-professional APCs''. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic ''planar cellular APC model'' for improved imaging and interrogation of fundamental antigenic signaling processes will be described.     

Quantitative elucidation of a distinct spatial gradient-sensing mechanism in fibroblasts

Migration of eukaryotic cells toward a chemoattractant often relies on their ability to distinguish receptor-mediated signaling at different subcellular locations, a phenomenon known as spatial sensing. A prominent example that is seen during wound healing is fibroblast migration in platelet-derived growth factor (PDGF) gradients. As in the well-characterized chemotactic cells Dictyostelium discoideum and neutrophils, signaling to the cytoskeleton via the phosphoinositide 3-kinase pathway in fibroblasts is spatially polarized by a PDGF gradient; however, the sensitivity of this process and how it is regulated are unknown. Through a quantitative analysis of mathematical models and live cell total internal reflection fluorescence microscopy experiments, we demonstrate that PDGF detection is governed by mechanisms that are fundamentally different from those in D. discoideum and neutrophils. Robust PDGF sensing requires steeper gradients and a much narrower range of absolute chemoattractant concentration, which is consistent with a simpler system lacking the feedback loops that yield signal amplification and adaptation in amoeboid cells.     

The Amyloid Precursor Protein Controls PIKfyve Function

While the Amyloid Precursor Protein (APP) plays a central role in Alzheimer’s disease, its cellular function still remains largely unclear. It was our goal to establish APP function which will provide insights into APP''s implication in Alzheimer''s disease. Using our recently developed proteo-liposome assay we established the interactome of APP''s intracellular domain (known as AICD), thereby identifying novel APP interactors that provide mechanistic insights into APP function. By combining biochemical, cell biological and genetic approaches we validated the functional significance of one of these novel interactors. Here we show that APP binds the PIKfyve complex, an essential kinase for the synthesis of the endosomal phosphoinositide phosphatidylinositol-3,5-bisphosphate. This signalling lipid plays a crucial role in endosomal homeostasis and receptor sorting. Loss of PIKfyve function by mutation causes profound neurodegeneration in mammals. Using C. elegans genetics we demonstrate that APP functionally cooperates with PIKfyve in vivo. This regulation is required for maintaining endosomal and neuronal function. Our findings establish an unexpected role for APP in the regulation of endosomal phosphoinositide metabolism with dramatic consequences for endosomal biology and important implications for our understanding of Alzheimer''s disease.     

Signaling pathways mediating chemotaxis in the social amoeba, Dictyostelium discoideum

Chemotaxis, or cell migration guided by chemical cues, is critical for a multitude of biological processes in a diverse array of organisms. Dictyostelium discoideum amoebae rely on chemotaxis to find food and to survive starvation conditions, and we have taken advantage of this system to study the molecular regulation of this vital cell behavior. Previous work has identified phosphoinositide signaling as one mechanism which may contribute to directional sensing and actin polymerization during chemotaxis; a mechanism which is conserved in mammalian neutrophils. In this review, we will discuss recent data on genes and pathways governing directional sensing and actin polymerization, with a particular emphasis on contributions from our laboratory.     

Signaling pathways mediating chemotaxis in the social amoeba, Dictyostelium discoideum

Chemotaxis, or cell migration guided by chemical cues, is critical for a multitude of biological processes in a diverse array of organisms. Dictyostelium discoideum amoebae rely on chemotaxis to find food and to survive starvation conditions, and we have taken advantage of this system to study the molecular regulation of this vital cell behavior. Previous work has identified phosphoinositide signaling as one mechanism which may contribute to directional sensing and actin polymerization during chemotaxis; a mechanism which is conserved in mammalian neutrophils. In this review, we will discuss recent data on genes and pathways governing directional sensing and actin polymerization, with a particular emphasis on contributions from our laboratory.     

Self-Generated Chemoattractant Gradients: Attractant Depletion Extends the Range and Robustness of Chemotaxis

Chemotaxis is fundamentally important, but the sources of gradients in vivo are rarely well understood. Here, we analyse self-generated chemotaxis, in which cells respond to gradients they have made themselves by breaking down globally available attractants, using both computational simulations and experiments. We show that chemoattractant degradation creates steep local gradients. This leads to surprising results, in particular the existence of a leading population of cells that moves highly directionally, while cells behind this group are undirected. This leading cell population is denser than those following, especially at high attractant concentrations. The local gradient moves with the leading cells as they interact with their surroundings, giving directed movement that is unusually robust and can operate over long distances. Even when gradients are applied from external sources, attractant breakdown greatly changes cells'' responses and increases robustness. We also consider alternative mechanisms for directional decision-making and show that they do not predict the features of population migration we observe experimentally. Our findings provide useful diagnostics to allow identification of self-generated gradients and suggest that self-generated chemotaxis is unexpectedly universal in biology and medicine.     

Imaging calcium in neurons

Calcium ions generate versatile intracellular signals that control key functions in all types of neurons. Imaging calcium in neurons is particularly important because calcium signals exert their highly specific functions in well-defined cellular subcompartments. In this Primer, we briefly review the general mechanisms of neuronal calcium signaling. We then introduce the calcium imaging devices, including confocal and two-photon microscopy as well as miniaturized devices that are used in freely moving animals. We provide an overview of the classical chemical fluorescent calcium indicators and of the protein-based genetically encoded calcium indicators. Using application examples, we introduce new developments in the field, such as calcium imaging in awake, behaving animals and the use of calcium imaging for mapping single spine sensory inputs in cortical neurons in vivo. We conclude by providing an outlook on the prospects of calcium imaging for the analysis of neuronal signaling and plasticity in various animal models.     

An information-theoretic characterization of the optimal gradient sensing response of cells

Many cellular systems rely on the ability to interpret spatial heterogeneities in chemoattractant concentration to direct cell migration. The accuracy of this process is limited by stochastic fluctuations in the concentration of the external signal and in the internal signaling components. Here we use information theory to determine the optimal scheme to detect the location of an external chemoattractant source in the presence of noise. We compute the minimum amount of mutual information needed between the chemoattractant gradient and the internal signal to achieve a prespecified chemotactic accuracy. We show that more accurate chemotaxis requires greater mutual information. We also demonstrate that a priori information can improve chemotaxis efficiency. We compare the optimal signaling schemes with existing experimental measurements and models of eukaryotic gradient sensing. Remarkably, there is good quantitative agreement between the optimal response when no a priori assumption is made about the location of the existing source, and the observed experimental response of unpolarized Dictyostelium discoideum cells. In contrast, the measured response of polarized D. discoideum cells matches closely the optimal scheme, assuming prior knowledge of the external gradient-for example, through prolonged chemotaxis in a given direction. Our results demonstrate that different observed classes of responses in cells (polarized and unpolarized) are optimal under varying information assumptions.     

Glutamate,Glutamate Receptors,and Downstream Signaling Pathways

Glutamate is a nonessential amino acid, a major bioenergetic substrate for proliferating normal and neoplastic cells, and an excitatory neurotransmitter that is actively involved in biosynthetic, bioenergetic, metabolic, and oncogenic signaling pathways. Glutamate signaling activates a family of receptors consisting of metabotropic glutamate receptors (mGluRs) and ionotropic glutamate receptors (iGluRs), both of which have been implicated in chronic disabling brain disorders such as Schizophrenia and neurodegenerative diseases like Alzheimer''s, Parkinson''s, and multiple sclerosis. In this review, we discuss the structural and functional relationship of mGluRs and iGluRs and their downstream signaling pathways. The three groups of mGluRs, the associated second messenger systems, and subsequent activation of PI3K/Akt, MAPK, NFkB, PLC, and Ca/CaM signaling systems will be discussed in detail. The current state of human mGluR1a as one of the most important isoforms of Group I-mGluRs will be highlighted. The lack of studies on the human orthologues of mGluRs family will be outlined. We conclude that upon further study, human glutamate-initiated signaling pathways may provide novel therapeutic opportunities for a variety of non-malignant and malignant human diseases.     

Moving toward understanding eukaryotic chemotaxis

The discovery in 1947 of directed cell movement in Dictyostelium discoideum quietly gave a birth to a new line of investigation into the molecular basis of chemotaxis. Some 60 years later, D. discoideum continues to be a key model system for the study of eukaryotic chemotaxis as well as an array of other important biological processes. As one of the most influential scientists, Guenther Gerisch has inspired several generations of researchers with his insightful and rigorous approaches applied to this model system. His studies have greatly contributed to current knowledge of many fundamental processes, such as cell-cell adhesion, phagocytosis, endocytosis, cytokinesis, cell signaling and chemotaxis. In this review, we wish to look back at the journey that has led to our current understanding of chemotaxis of eukaryotic cells.