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.     

Arp2/3 is critical for lamellipodia and response to extracellular matrix cues but is dispensable for chemotaxis

Lamellipodia are sheet-like, leading edge protrusions in firmly adherent cells that contain Arp2/3-generated dendritic actin networks. Although lamellipodia are widely believed to be critical for directional cell motility, this notion has not been rigorously tested. Using fibroblasts derived from Ink4a/Arf-deficient mice, we generated a stable line depleted of Arp2/3 complex that lacks lamellipodia. This line shows defective random cell motility and relies on a filopodia-based protrusion system. Utilizing a microfluidic gradient generation system, we tested the role of Arp2/3 complex and lamellipodia in directional cell migration. Surprisingly, Arp2/3-depleted cells respond normally to shallow gradients of PDGF, indicating that lamellipodia are not required for fibroblast chemotaxis. Conversely, these cells cannot respond to a surface-bound gradient of extracellular matrix (haptotaxis). Consistent with this finding, cells depleted of Arp2/3 fail to globally align focal adhesions, suggesting that one principle function of lamellipodia is to organize cell-matrix adhesions in a spatially coherent manner.     

Differences in signalling by directly and indirectly binding ligands in bacterial chemotaxis

In chemotaxis of Escherichia coli and other bacteria, extracellular stimuli are perceived by transmembrane receptors that bind their ligands either directly, or indirectly through periplasmic‐binding proteins (BPs). As BPs are also involved in ligand uptake, they provide a link between chemotaxis and nutrient utilization by cells. However, signalling by indirectly binding ligands remains much less understood than signalling by directly binding ligands. Here, we compared intracellular responses mediated by both types of ligands and developed a new mathematical model for signalling by indirectly binding ligands. We show that indirect binding allows cells to better control sensitivity to specific ligands in response to their nutrient environment and to coordinate chemotaxis with ligand transport, but at the cost of the dynamic range being much narrower than for directly binding ligands. We further demonstrate that signal integration by the chemosensory complexes does not depend on the type of ligand. Overall, our data suggest that the distinction between signalling by directly and indirectly binding ligands is more physiologically important than the traditional distinction between high‐ and low‐abundance receptors.     

Mechanotransduction of mesenchymal melanoma cell invasion into 3D collagen lattices: Filopod-mediated extension–relaxation cycles and force anisotropy

Mesenchymal cell migration in interstitial tissue is a cyclic process of coordinated leading edge protrusion, adhesive interaction with extracellular matrix (ECM) ligands, cell contraction followed by retraction and movement of the cell rear. During migration through 3D tissue, the force fields generated by moving cells are non-isotropic and polarized between leading and trailing edge, however the integration of protrusion formation, cell–substrate adhesion, traction force generation and cell translocation in time and space remain unclear. Using high-resolution 3D confocal reflectance and fluorescence microscopy in GFP/actin expressing melanoma cells, we here employ time-resolved subcellular coregistration of cell morphology, interaction and alignment of actin-rich protrusions engaged with individual collagen fibrils. Using single fibril displacement as sensitive measure for force generated by the leading edge, we show how a dominant protrusion generates extension–retraction cycles transmitted through multiple actin-rich filopods that move along the scaffold in a hand-over-hand manner. The resulting traction force is oscillatory, occurs in parallel to cell elongation and, with maximum elongation reached, is followed by rear retraction and movement of the cell body. Combined live-cell fluorescence and reflection microscopy of the leading edge thus reveals step-wise caterpillar-like extension–retraction cycles that underlie mesenchymal migration in 3D tissue.     

Cytomechanics of cell deformations and migration: from models to experiments

A cytomechanical model bas been proposed to analyse cell-cell interactions and cell migration through chemotaxis. We consider as the leading assumption that the cell cortical tension is locally modified by the protrusive activity of neighbour cells and binding of chemoattractant molecules to membrane receptors respectively. The model derives from the one initially proposed by Alt and Tranquillo (1995), which successfully describes experimentally observed cyclic autonomous cell shape changes. It is based on force balance equations coupling intracellular hydrostatic pressure and cell cortex contraction. Considering the protrusive dynamics of L929 fibroblats observed by videomicroscopy, we simulated the influence of neighbouring protrusions on a cell spontaneous pulsating behaviour. We further investigated the role of an extracellular gradient as another kind of external stimulus. The model illustrates how binding of chemoattractant molecules can induce a cell morphological instability that, above an intracellular stress threshold, will break the cell-substratum attachment. As a result, realistic cell chemotaxis can be simulated.     

Imaging sites of N-wasp activity in lamellipodia and invadopodia of carcinoma cells

Cell migration is crucial for many biological and pathological processes such as chemotaxis of immune cells, fibroblast migration during wound healing, and tumor cell invasion and metastasis. Cells migrate forward by extending membrane protrusions. The formation of these protrusions is driven by assembly of actin filaments at the leading edge. Neural Wiskott-Aldrich syndrome protein (N-WASP), a ubiquitous member of the WASP family, induces actin polymerization by activating Arp2/3 complex and is thought to regulate the formation of membrane protrusions. However, it is totally unclear how N-WASP activity is spatially and temporally regulated inside migrating cells. To detect and image sites of N-WASP activity during cell motility and invasion in carcinoma cells, we designed an N-WASP fluorescence resonance energy transfer (FRET) biosensor that distinguishes between the active and inactive conformations and mimics the function of endogenous N-WASP. Our data show that N-WASP is involved in lamellipodia extension, where it is activated at the leading edge, as well as in invadopodia formation of invasive carcinoma cells, where it is activated at the base. This is the first time that the activity of full-length N-WASP has been visualized in vivo, and this has lead to new insights for N-WASP function.     

Chemotaxis and calcium responses of phagocytes to formyl peptide receptor ligands is differentially regulated by cyclic ADP ribose

Cyclic ADP ribose (cADPR) is a calcium-mobilizing metabolite that regulates intracellular calcium release and extracellular calcium influx. Although the role of cADPR in modulating calcium mobilization has been extensively examined, its potential role in regulating immunologic responses is less well understood. We previously reported that cADPR, produced by the ADP-ribosyl cyclase, CD38, controls calcium influx and chemotaxis of murine neutrophils responding to fMLF, a peptide agonist for two chemoattractant receptor subtypes, formyl peptide receptor and formyl peptide receptor-like 1. In this study, we examine whether cADPR is required for chemotaxis of human monocytes and neutrophils to a diverse array of chemoattractants. We found that a cADPR antagonist and a CD38 substrate analogue inhibited the chemotaxis of human phagocytic cells to a number of formyl peptide receptor-like 1-specific ligands but had no effect on the chemotactic response of these cells to ligands selective for formyl peptide receptor. In addition, we show that the cADPR antagonist blocks the chemotaxis of human monocytes to CXCR4, CCR1, and CCR5 ligands. In all cases, we found that cADPR modulates intracellular free calcium levels in cells activated by chemokines that induce extracellular calcium influx in the apparent absence of significant intracellular calcium release. Thus, cADPR regulates calcium signaling of a discrete subset of chemoattractant receptors expressed by human leukocytes. Since many of the chemoattractant receptors regulated by cADPR bind to ligands that are associated with clinical pathology, cADPR and CD38 represent novel drug targets with potential application in chronic inflammatory and neurodegenerative disease.     

The chemotactic defect in wiskott-Aldrich syndrome macrophages is due to the reduced persistence of directional protrusions

Wiskott-Aldrich syndrome protein (WASp) is an actin nucleation promoting factor that is required for macrophages to directionally migrate towards various chemoattractants. The chemotaxis defect of WASp-deficient cells and its activation by Cdc42 in vivo suggest that WASp plays a role in directional sensing, however, its precise role in macrophage chemotaxis is still unclear. Using shRNA-mediated downregulation of WASp in the murine monocyte/macrophage cell line RAW/LR5 (shWASp), we found that WASp was responsible for the initial wave of actin polymerization in response to global stimulation with CSF-1, which in Dictyostelium discoideum amoebae and carcinoma cells has been correlated with the ability to migrate towards chemoattractants. Real-time monitoring of shWASp cells, as well as WASp^−/− bone marrow-derived macrophages (BMMs), in response to a CSF-1 gradient revealed that the protrusions from WASp-deficient cells were directional, showing intact directional sensing. However, the protrusions from WASp-deficient cells demonstrated reduced persistence compared to their respective control shRNA and wild-type cells. Further examination showed that tyrosine phosphorylation of WASp was required for both the first wave of actin polymerization following global CSF-1 stimulation and proper directional responses towards CSF-1. Importantly, the PI3K, Rac1 and WAVE2 proteins were incorporated normally in CSF-1 – elicited protrusions in the absence of WASp, suggesting that membrane protrusion driven by the WAVE2 complex signaling is intact. Collectively, these results suggest that WASp and its phosphorylation play critical roles in coordinating the actin cytoskeleton rearrangements necessary for the persistence of protrusions required for directional migration of macrophages towards CSF-1.     

Lateral density of receptor arrays in the membrane plane influences sensitivity of the E. coli chemotaxis response

In chemotactic bacteria, transmembrane chemoreceptors, CheA and CheW form the core signalling complex of the chemotaxis sensory apparatus. These complexes are organized in extended arrays in the cytoplasmic membrane that allow bacteria to respond to changes in concentration of extracellular ligands via a cooperative, allosteric response that leads to substantial amplification of the signal induced by ligand binding. Here, we have combined cryo-electron tomographic studies of the 3D spatial architecture of chemoreceptor arrays in intact E. coli cells with computational modelling to develop a predictive model for the cooperativity and sensitivity of the chemotaxis response. The predictions were tested experimentally using fluorescence resonance energy transfer (FRET) microscopy. Our results demonstrate that changes in lateral packing densities of the partially ordered, spatially extended chemoreceptor arrays can modulate the bacterial chemotaxis response, and that information about the molecular organization of the arrays derived by cryo-electron tomography of intact cells can be translated into testable, predictive computational models of the chemotaxis response.     

A Family of Heptahelical Receptors With Adhesion-Like Domains: A Marriage Between Two Super Families

Abstract

Some G protein-coupled receptors (GPCRs) are regulators of cell adhesion via inside-out effector signaling pathways. Such is the case with leukocyte chemokine receptors which stimulate intracellular second messenger pathways resulting in upregulation of integrin adhesion to ligands present in the extracellular matrix or on opposing cells resulting in chemotaxis and extravasation during immune surveillance. Remarkably, a family of GPCRs has recently been discovered that may themselves be triggered by cell-cell or cell-matrix interactions. Along with a canonical heptahelical membrane-spanning region, these intriguing proteins contain putative cell adhesionlike modules. The evidence to date suggests that they are involved in lymphocyte activation, macrophage biology, synaptic exocytosis and planar polarization during organogenesis.     

Cell shape dynamics: from waves to migration

We observe and quantify wave-like characteristics of amoeboid migration. Using the amoeba Dictyostelium discoideum, a model system for the study of chemotaxis, we demonstrate that cell shape changes in a wave-like manner. Cells have regions of high boundary curvature that propagate from the leading edge toward the back, usually along alternating sides of the cell. Curvature waves are easily seen in cells that do not adhere to a surface, such as cells that are electrostatically repelled from surfaces or cells that extend over the edge of micro-fabricated cliffs. Without surface contact, curvature waves travel from the leading edge to the back of a cell at -35 μm/min. Non-adherent myosin II null cells do not exhibit these curvature waves. At the leading edge of adherent cells, curvature waves are associated with protrusive activity. Like regions of high curvature, protrusive activity travels along the boundary in a wave-like manner. Upon contact with a surface, the protrusions stop moving relative to the surface, and the boundary shape thus reflects the history of protrusive motion. The wave-like character of protrusions provides a plausible mechanism for the zig-zagging of pseudopods and for the ability of cells both to swim in viscous fluids and to navigate complex three dimensional topography.     

STIM1-directed reorganization of microtubules in activated mast cells

Activation of mast cells by aggregation of the high-affinity IgE receptors (FcεRI) initiates signaling events leading to the release of inflammatory and allergic mediators stored in cytoplasmic granules. A key role in this process play changes in concentrations of intracellular Ca(2+) controlled by store-operated Ca(2+) entry (SOCE). Although microtubules are also involved in the process leading to degranulation, the molecular mechanisms that control microtubule rearrangement during activation are largely unknown. In this study, we report that activation of bone marrow-derived mast cells (BMMCs) induced by FcεRI aggregation or treatment with pervanadate or thapsigargin results in generation of protrusions containing microtubules (microtubule protrusions). Formation of these protrusions depended on the influx of extracellular Ca(2+). Changes in cytosolic Ca(2+)concentration also affected microtubule plus-end dynamics detected by microtubule plus-end tracking protein EB1. Experiments with knockdown or reexpression of STIM1, the key regulator of SOCE, confirmed the important role of STIM1 in the formation of microtubule protrusions. Although STIM1 in activated cells formed puncta associated with microtubules in protrusions, relocation of STIM1 to a close proximity of cell membrane was independent of growing microtubules. In accordance with the inhibition of Ag-induced Ca(2+) response and decreased formation of microtubule protrusions in BMMCs with reduced STIM1, the cells also exhibited impaired chemotactic response to Ag. We propose that rearrangement of microtubules in activated mast cells depends on STIM1-induced SOCE, and that Ca(2+) plays an important role in the formation of microtubule protrusions in BMMCs.     

Proteolytic activity of specialized surface protrusions formed at rosette contact sites of transformed cells

Surface protrusions at the leading edge of a moving cell that make contact with the surrounding extracellular matrix (ECM) are its main motor for locomotion and invasion. Chicken embryonic fibroblasts transformed by Rous sarcoma virus (RSV-CEF) form specialized membrane rosette-shaped contact sites on planar substrata as shown by interference reflection microscopy (IRM). Such activity is lacking in normal cells. These rosette contacts are more labile than other adhesion sites, such as focal and close contacts. Ultrastructural studies demonstrate that rosettes are sites at which membrane protrusions from the ventral cell surface contact the substratum. These protrusions are filled with meshworks of microfilaments and contain the pp60src oncogene product, actin, vinculin, and alpha-actinin. However, unlike focal contacts, at the rosettes these proteins interact to extend a highly motile membrane. Rosettes have the biological activity of degrading ECM components, as demonstrated by (1) local degradation of fibronectin substrata at sites of rosette contacts, but not focal and close contacts; (2) localization of putative antiprotease antibody at sites of rosette contacts, but not at focal an close contacts; and (3) local disruption of fibronectin matrix at sites of protrusive activity seen by transmission electron microscopy (TEM). In addition, formation of the rosette contact is insensitive to the ionophore monensin, and to inhibitors of proteolytic enzymes, while local fibronectin degradation at rosette contacts is inhibited by inhibitors of metalloproteases, 1,10-phenanthroline and NP-20. I consider these membrane protrusions of the rosette contacts in RSV-transformed cells specialized structural entities--invadopodia--that are involved in the local degradation of the ECM.     

The orientation of nucleus, nucleus-associated body and protruding nucleolus in aggregating Dictyostelium discoideum

Dictyostelium discoideum growing or developing on cellulose dialysis membranes were fixed with acrolein vapour for electron microscopy. In interphase amoebae, nucleoli began to protrude from the nuclei. The percentage of cells with protruding nucleoli increased during aggregation by a value approximately twice as high in aggregation streams as in centers. Cells in pseudoplasmodia showed only a low percentage and protrusions disappeared at early culmination stage. The protrusions did not reappear when cells from dissociated pseudoplasmodia migrated toward cAMP. Thus the formation of the protrusions did not depend solely on chemotaxis; rather, it was specific to the aggregation stage. In aggregation streams, the nucleus was anterior in the cell, with the protrusion at its anterior periphery. In contrast, the nucleus associated body (NAB) was evident at the cell's mid-point. This orientation of nucleus and NAB in the aggregating slime mould amoeba is contrary to that seen in human neutrophils or cultured mouse 3T3 cells.     

Real-time phase-contrast x-ray imaging: a new technique for the study of animal form and function

Background

Cell shape changes during cytokinesis and chemotaxis require regulation of the actin cytoskeletal network. Dynacortin, an actin cross-linking protein, localizes to the cell cortex and contributes to cortical resistance, thereby helping to define the cell shape changes of cytokinesis. Dynacortin also becomes highly enriched in cortical protrusions, which are sites of new actin assembly.

Results

We studied the effect of dynacortin on cell motility during chemotaxis and on actin dynamics in vivo and in vitro. Dynacortin enriches with the actin, particularly at the leading edge of chemotaxing cells. Cells devoid of dynacortin do not become as polarized as wild-type control cells but move with similar velocities as wild-type cells. In particular, they send out multiple pseudopods that radiate at a broader distribution of angles relative to the chemoattractant gradient. Wild-type cells typically only send out one pseudopod at a time that does not diverge much from 0° on average relative to the gradient. Though dynacortin-deficient cells show normal bulk (whole-cell) actin assembly upon chemoattractant stimulation, dynacortin can promote actin assembly in vitro. By fluorescence spectroscopy, co-sedimentation and transmission electron microscopy, dynacortin acts as an actin scaffolder in which it assembles actin monomers into polymers with a stoichiometry of 1 Dyn₂:1 actin under salt conditions that disfavor polymer assembly.

Conclusion

Dynacortin contributes to cell polarization during chemotaxis. By cross-linking and possibly stabilizing actin polymers, dynacortin also contributes to cortical viscoelasticity, which may be critical for establishing cell polarity. Though not essential for directional sensing or motility, dynacortin is required to establish cell polarity, the third core feature of chemotaxis.     

A family of heptahelical receptors with adhesion-like domains: a marriage between two super families

Some G protein-coupled receptors (GPCRs) are regulators of cell adhesion via inside-out effector signaling pathways. Such is the case with leukocyte chemokine receptors which stimulate intracellular second messenger pathways resulting in upregulation of integrin adhesion to ligands present in the extracellular matrix or on opposing cells resulting in chemotaxis and extravasation during immune surveillance. Remarkably, a family of GPCRs has recently been discovered that may themselves be triggered by cell-cell or cell-matrix interactions. Along with a canonical heptahelical membrane-spanning region, these intriguing proteins contain putative cell adhesion-like modules. The evidence to date suggests that they are involved in lymphocyte activation, macrophage biology, synaptic exocytosis and planar polarization during organogenesis.     

A mechanism of leading-edge protrusion in the absence of Arp2/3 complex

Cells employ protrusive leading edges to navigate and promote their migration in diverse physiological environments. Classical models of leading-edge protrusion rely on a treadmilling dendritic actin network that undergoes continuous assembly nucleated by the Arp2/3 complex, forming ruffling lamellipodia. Recent work demonstrated, however, that, in the absence of the Arp2/3 complex, fibroblast cells adopt a leading edge with filopodia-like protrusions (FLPs) and maintain an ability to move, albeit with altered responses to different environmental signals. We show that formin-family actin nucleators are required for the extension of FLPs but are insufficient to produce a continuous leading edge in fibroblasts lacking Arp2/3 complex. Myosin II is concentrated in arc-like regions of the leading edge in between FLPs, and its activity is required for coordinated advancement of these regions with formin-generated FLPs. We propose that actomyosin contraction acting against membrane tension advances the web of arcs between FLPs. Predictions of this model are verified experimentally. The dependence of myosin II in leading-edge advancement helps explain the previously reported defect in directional movement in the Arpc3-null fibroblasts. We provide further evidence that this defect is cell autonomous during chemotaxis.     

Mouse Macrophages Completely Lacking Rho Subfamily GTPases (RhoA,RhoB, and RhoC) Have Severe Lamellipodial Retraction Defects,but Robust Chemotactic Navigation and Altered Motility

RhoA is thought to be essential for coordination of the membrane protrusions and retractions required for immune cell motility and directed migration. Whether the subfamily of Rho (Ras homolog) GTPases (RhoA, RhoB, and RhoC) is actually required for the directed migration of primary cells is difficult to predict. Macrophages isolated from myeloid-restricted RhoA/RhoB (conditional) double knock-out (dKO) mice did not express RhoC and were essentially “pan-Rho”-deficient. Using real-time chemotaxis assays, we found that retraction of the trailing edge was dissociated from the advance of the cell body in dKO cells, which developed extremely elongated tails. Surprisingly, velocity (of the cell body) was increased, whereas chemotactic efficiency was preserved, when compared with WT macrophages. Randomly migrating RhoA/RhoB dKO macrophages exhibited multiple small protrusions and developed large “branches” due to impaired lamellipodial retraction. A mouse model of peritonitis indicated that monocyte/macrophage recruitment was, surprisingly, more rapid in RhoA/RhoB dKO mice than in WT mice. In comparison with dKO cells, the phenotypes of single RhoA- or RhoB-deficient macrophages were mild due to mutual compensation. Furthermore, genetic deletion of RhoB partially reversed the motility defect of macrophages lacking the RhoGAP (Rho GTPase-activating protein) myosin IXb (Myo9b). In conclusion, the Rho subfamily is not required for “front end” functions (motility and chemotaxis), although both RhoA and RhoB are involved in pulling up the “back end” and resorbing lamellipodial membrane protrusions. Macrophages lacking Rho proteins migrate faster in vitro, which, in the case of the peritoneum, translates to more rapid in vivo monocyte/macrophage recruitment.     

G protein-coupled chemokine receptors induce both survival and apoptotic signaling pathways

Chemokine receptors are essential for triggering chemotaxis to immune cells; however, a number of them can also mediate death when engaged by nonchemokine ligands. When the chemokine receptor CXCR4 is engaged by stromal cell-derived factor (SDF1)alpha, it triggers cells to chemotax, and in some cell types such as neurons, causes cell death. To elucidate this dual and opposing receptor function, we have investigated whether CXCR4 activation by its chemokine SDF1alpha could lead to the simultaneous activation of both anti- and proapoptotic signaling pathways; the balance ultimately influencing cell survival. CXCR4 activation in CD4 T cells by SDF1alpha led to the activation of the prosurvival second messengers, Akt and extracellular signal-regulated protein kinase. Selective inhibition of each signal demonstrated that extracellular signal-regulated protein kinase is essential for mediating SDF1alpha-triggered chemotaxis but does not confer an antiapoptotic state. In contrast, Akt activation through CXCR4 by SDF1alpha interactions is necessary to confer resistance to apoptosis. The proapoptotic signaling pathway triggered by SDF1alpha-CXCR4 interaction involves the G(ialpha) protein-independent activation of the proapoptotic MAPK (p38). Furthermore, other chemokines and chemokine receptors also signal chemotaxis and proapoptotic effects via similar pathways. Thus, G(ialpha) protein-coupled chemokine receptors can function as death prone receptors and the balance between the above signaling pathways will ultimately mandate the fate of the activated cell.     

Travelling Waves in Hybrid Chemotaxis Models

Hybrid models of chemotaxis combine agent-based models of cells with partial differential equation models of extracellular chemical signals. In this paper, travelling wave properties of hybrid models of bacterial chemotaxis are investigated. Bacteria are modelled using an agent-based (individual-based) approach with internal dynamics describing signal transduction. In addition to the chemotactic behaviour of the bacteria, the individual-based model also includes cell proliferation and death. Cells consume the extracellular nutrient field (chemoattractant), which is modelled using a partial differential equation. Mesoscopic and macroscopic equations representing the behaviour of the hybrid model are derived and the existence of travelling wave solutions for these models is established. It is shown that cell proliferation is necessary for the existence of non-transient (stationary) travelling waves in hybrid models. Additionally, a numerical comparison between the wave speeds of the continuum models and the hybrid models shows good agreement in the case of weak chemotaxis and qualitative agreement for the strong chemotaxis case. In the case of slow cell adaptation, we detect oscillating behaviour of the wave, which cannot be explained by mean-field approximations.