Chemotactic Cell Movement Research Articles

Spatial regulation of the cAMP-dependent protein kinase during chemotactic cell migration

Historically, the cAMP-dependent protein kinase (PKA) has a paradoxical role in cell motility, having been shown to both facilitate and inhibit actin cytoskeletal dynamics and cell migration. In an effort to understand this dichotomy, we show here that PKA is regulated in subcellular space during cell migration. Immunofluorescence microscopy and biochemical enrichment of pseudopodia showed that type II regulatory subunits of PKA and PKA activity are enriched in protrusive cellular structures formed during chemotaxis. This enrichment correlates with increased phosphorylation of key cytoskeletal substrates for PKA, including the vasodilator-stimulated phosphoprotein (VASP) and the protein tyrosine phosphatase containing a PEST motif. Importantly, inhibition of PKA activity or its ability to interact with A kinase anchoring proteins inhibited the activity of the Rac GTPase within pseudopodia. This effect correlated with both decreased guanine nucleotide exchange factor activity and increased GTPase activating protein activity. Finally, inhibition of PKA anchoring, like inhibition of total PKA activity, inhibited pseudopod formation and chemotactic cell migration. These data demonstrate that spatial regulation of PKA via anchoring is an important facet of normal chemotactic cell movement.

Autocrine/paracrine role of the angiopoietin-1 and -2/Tie2 system in cell proliferation and chemotaxis of cultured fibroblastic synoviocytes in rheumatoid arthritis

Hypervascularity is a characteristic synovial feature of rheumatoid arthritis (RA). We previously reported that Tie1 and Tie2, endothelium-specific tyrosine kinase receptors essential for angiogenesis, are expressed not only by vascular cells, but also by a subpopulation of synovial lining and stromal cells in the inflamed RA synovium. The present study used immunohistochemistry and in situ hybridization to examine whether angiopoietin-1 and -2 (Ang1 and Ang2), ligands for Tie2, are also expressed in the RA synovium. Ang1 and Ang2 were expressed in all of 15 RA synovial samples, and their distribution pattern was similar to that of Tie2. Intense staining was noted in synovial lining and stromal cells, as well as in small vessels in areas of papillary projection and high cell density. Double immunohistochemistry revealed coexpression of Ang1, Ang2, and Tie2 in synovial components exhibiting proliferating cell nuclear antigen immunoreactivity. In addition, Ang1 and Ang2 were preferentially expressed in vimentin-positive fibroblastic cells. To address the functional role of Ang/Tie signaling in RA pathophysiology, we carried out [ 3H]thymidine incorporation and transwell chemotaxis assays using cultured fibroblastic synoviocytes obtained from 2 RA patients. Incubation with various concentrations of recombinant Ang1 or Ang2 did not alter DNA synthesis, but the ligands enhanced chemotactic migration of RA fibroblastic synoviocytes. Our results suggest that the autocrine/paracrine signaling of the Ang/Tie2 system is important for the up-regulated angiogenesis in the RA synovium, as well as for synoviocyte behavior, by regulating chemotactic cell movement.

Differential inhibition of receptor activation by two mouse monoclonal antibodies specific for the human leukotriene B 4 receptor, BLT 1

The inflammatory mediator leukotriene B 4 (LTB 4) binds to and activates a G-protein-coupled receptor named BLT 1. We have previously produced two monoclonal antibodies, named 7B1 and 14F11, that bind specifically to this receptor. Using a HeLa cell line expressing human BLT 1, we find that both antibodies inhibit LTB 4-induced calcium release, and activation of a MAP-kinase-sensitive luciferase reporter system. The normal chemotactic movement of polymorphonuclear cells towards higher LTB 4 concentrations was also strongly inhibited by both antibodies. Neither antibody was found to activate BLT 1, and experiments using cyclic peptide fragments of the BLT 1 n-terminal and extracellular loops showed that these antibodies bind only to complex epitopes in the tertiary, membrane bound, conformation of the receptor protein. In ligand binding experiments, 7B1 was found to be a competitive antagonist, while 14F11 was a noncompetitive antagonist that inhibited receptor activation, but not agonist (LTB 4) binding. 14F11 will be a useful tool for studying the mechanisms of receptor activation.

A talin fragment as an actin trap visualizing actin flow in chemotaxis, endocytosis, and cytokinesis.

A C-terminal 63-kDa fragment of talin A from Dictyostelium discoideum forms a slowly dissociating complex with F-actin in vitro. This talin fragment (TalC63) has been tagged with GFP and used as a trap for actin filaments in chemotactic cell movement, endocytosis, and mitotic cell division. TalC63 efficiently sequesters actin filaments in vivo. Its translocation reflects the direction and efficiency of an actin flow. Along the body of a migrating Dictyostelium cell, this flow is directed from the front to the tail. If during chemotaxis one or two new fronts are induced, the flow is always directed away from these fronts. The flow thus reflects the re-programming of cell polarity in response to changing gradients of chemoattractant. In endocytosis, the fluorescent complexes are translocated to the base of a phagocytic or macropinocytic cup. During mitosis, the complexes of F-actin with TalC63 accumulate within the midzone of anaphase cells. If TalC63 is strongly expressed, the entire cleavage furrow is filled out by sequestered actin filaments, and cytokinesis is severely impaired. These cells are considered to mimic the phenotype of mutants deficient in the shredding of actin filaments that normally occurs in the mid-zone of a dividing cell.

HDAC6 is a microtubule-associated deacetylase.

Reversible acetylation of alpha-tubulin has been implicated in regulating microtubule stability and function. The distribution of acetylated alpha-tubulin is tightly controlled and stereotypic. Acetylated alpha-tubulin is most abundant in stable microtubules but is absent from dynamic cellular structures such as neuronal growth cones and the leading edges of fibroblasts. However, the enzymes responsible for regulating tubulin acetylation and deacetylation are not known. Here we report that a member of the histone deacetylase family, HDAC6, functions as a tubulin deacetylase. HDAC6 is localized exclusively in the cytoplasm, where it associates with microtubules and localizes with the microtubule motor complex containing p150(glued) (ref. 3). In vivo, the overexpression of HDAC6 leads to a global deacetylation of alpha-tubulin, whereas a decrease in HDAC6 increases alpha-tubulin acetylation. In vitro, purified HDAC6 potently deacetylates alpha-tubulin in assembled microtubules. Furthermore, overexpression of HDAC6 promotes chemotactic cell movement, supporting the idea that HDAC6-mediated deacetylation regulates microtubule-dependent cell motility. Our results show that HDAC6 is the tubulin deacetylase, and provide evidence that reversible acetylation regulates important biological processes beyond histone metabolism and gene transcription.

CAMP receptor affinity controls wave dynamics, geometry and morphogenesis in Dictyostelium.

Serpentine G-protein-coupled cAMP receptors are key components in the detection and relay of the extracellular cAMP waves that control chemotactic cell movement during Dictyostelium development. During development the cells sequentially express four closely related cAMP receptors of decreasing affinity. In this study, we investigated the effect of cAMP receptor type and affinity on the dynamics of cell-cell signalling in vivo, by measuring the dynamics of wave initiation and propagation in a variety of cAMP receptor mutants. We found that receptor affinity controls the frequency of wave initiation, but it does not determine wave propagation velocity, thus resulting in dramatic changes in wave geometry. In the limiting case, the affinity of the receptor is so low that waves can still be initiated but no stable centres form - thus, the cells cannot aggregate. In mounds, expression of low affinity receptors results in slow concentric waves instead of the normally observed multi-armed spiral waves. Under these conditions there is no rotational cell movement and the hemispherical mounds cannot transform into slugs. These results highlight the importance of receptor number and affinity in the proper control of cell-cell signalling dynamics required for the successful completion of development.

Activated Gα Subunits Can Inhibit Multiple Signal Transduction Pathways during Dictyostelium Development

Mutations impairing the GTPase activity of G protein Gα subunits can result in activated Gα subunits that affect signal transduction and cellular responses and, in some cases, promote tumor formation. An analogous mutation in the Dictyostelium Gα4 subunit gene (Q200L substitution) was constructed and found to inhibit Gα4-mediated responses to folic acid, including the accumulation of cyclic nucleotides and chemotactic cell movement. The Gα4-Q200L subunit also severely inhibited responses to cAMP, including cyclic nucleotide accumulation, cAMP chemotaxis, and cellular aggregation. An analogous mutation in the Gα2 subunit (Q208L substitution), previously reported to inhibit cAMP responses (K. Okaichi et al., 1992, Mol. Biol. Cell 3, 735–747), was also found to partially inhibit folic acid chemotaxis. Chemotactic responses to folic acid and cAMP and developmental aggregation were also inhibited by a mutant Gα5 subunit with the analogous alteration (Q199L substitution). All aggregation-defective Gα mutants were capable of multicellular development after a temporary incubation at 4°C and this development was found to be dependent on wild-type Gα4 function. This study indicates that mutant Gα subunits can inhibit signal transduction pathways mediated by other Gα subunits.

Modeling Chemotactic Cell Sorting during Dictyostelium discoideum Mound Formation

Coordinated cell movement is a major mechanism of the multicellular development of most organisms. The multicellular morphogenesis of the slime mould Dictyostelium discoideum, from single cells into a multicellular fruiting body, results from differential chemotactic cell movement. During aggregation cells differentiate into prestalk and prespore cells that will form the stalk and spores in the fruiting body. These cell types arise in a salt and pepper pattern after what the prestalk cells chemotactically sort out to form a tip. The tip functions as an organizer because it directs the further development. It has been difficult to get a satisfactory formal description of the movement behavior of cells in tissues. Based on our experiments, we consider the aggregate as a drop of a viscous fluid and show that this consideration is very well suited to mathematically describe the motion of cells in the tissue. We show that the transformation of a hemispherical mound into an elongated slug can result from the coordinated chemotactic cell movement in response to scroll waves of the chemoattractant cAMP. The model calculations furthermore show that cell sorting can result from differences in chemotactic cell movement and cAMP relay kinetics between the two cell types. During this process, the faster moving and stronger signaling cells collect on the top of the mound to form a tip. The mound then extends into an elongated slug just as observed in experiments. The model is able to describe cell movement patterns in the complex multicellular morphogenesis of Dictyostelium rather well and we expect that this approach may be useful in the modeling of tissue transformations in other systems.

Propagating waves control Dictyostelium discoideum morphogenesis

The morphogenesis of Dictyostelium results from the coordinated movement of starving cells to form a multicellular aggregate (mound) which transforms into a motile slug and finally a fruiting body. Cells differentiate in the mound and sort out to form an organised pattern in the slug and fruiting body. During aggregation, cell movement is controlled by propagating waves of the chemo-attractant cAMP. We show that mounds are also organised by propagating waves. Their geometry changes from target or single armed spirals during aggregation to multi-armed spiral waves in the mound. Some mounds develop transiently into rings in which multiple propagating wave fronts can still be seen. We model cell sorting in the mound stage assuming cell type specific differences in cell movement speed and excitability. This sorting feeds back on the wave geometry to generate twisted scroll waves in the slug. Slime mould morphogenesis can be understood in terms of wave propagation directing chemotactic cell movement.

Analysis of Optical Density Wave Propagation and Cell Movement during Mound Formation inDictyostelium discoideum

Aggregation fields ofDictyosteliumamoebae are organized by propagating concentric or spiral waves of cAMP. These waves coordinate cell movement directed toward the aggregation center. We now systematically investigated dark-field wave propagation and chemotactic cell movement during late aggregation and mound formation. The period and the signal propagation velocity decreased continuously during aggregation leading to a 15-fold decrease of the chemical wavelength. By analyzing the behavior of single GFP-labeled cells in aggregates and mounds we measured cell movement velocity, changes in cell shape, periodicity of cell movement, and cell trajectories. In early mounds of strain AX-3 dark-field waves propagated frequently as multiarmed (high-frequency) spirals. During the high-frequency waves observed in the early mound stage, cell movement speed is low and cell movement rather undirected. During tip formation the wave period decreased again and the cells started to rotate in the mound at unusually high average speeds of 40 μm/min. The rotation was almost monotonic with no clear periodicity. Since at this time the majority of the cells had already differentiated into prespore cells, this implies that prespore cells moved faster than aggregation stage cells. At 12 hr of development cell movement velocity dropped again and became highly periodic. These measurements show that the relay system is characterized by a specific temporal evolution, which is closely correlated with cellular differentiation. The remarkable changes in cell movement speed and period indicate a qualitative change in signal and movement parameters which might well be caused by the observed switch from high- to low-affinity cAMP receptors during mound formation. This switch might be required to copy with the increase in cell density and most likely plays a crucial role in the process of cell sorting.

Traveling-Wave Chemotaxis.

A simple model is studied for the chemotactic movement of biological cells in the presence of a periodic chemical wave. It incorporates the feature of adaptation that may play an important role in allowing for ``rectified'' chemotaxis: motion opposite the direction of wave propagation. The conditions under which such rectification occurs are elucidated in terms of the form and speed of the chemical wave, the velocity of chemotaxis, and the time scale for adaptation. An experimental test of the adaptation dynamics is proposed.

Signal transduction and motility of Dictyostelium.

This review is concerned with the roles of cyclic GMP and Ca(2+) ions in signal transduction for chemotaxis of Dictyostelium. These molecules are involved in signalling between the cell surface cyclic AMP receptors and cytoskeletal myosin II involved in chemotactic cell movement. Evidence is presented for uptake and/or efflux of Ca(2+) being regulated by cyclic GMP. The link between Ca(2+), cyclic GMP and chemotactic cell movement has been explored using "streamer F" mutants whose primary defect is in the structural gene for the cyclic GMP-specific phosphodiesterase. This mutation causes the mutants to produce an abnormally prolonged peak of cyclic GMP accumulation in response to stimulation with the chemoattractant cyclic AMP. The production and relay of cyclic AMP signals is normal in these mutants, but certain events associated with movement are (like the cyclic GMP response) abnormally prolonged in the mutants. These events include Ca(2+) uptake, myosin II association with the cytoskeleton and regulation of both myosin heavy and light chain phosphorylation. These changes can be correlated with changes in the shape of the amoebae after chemotactic stimulation. Other mutants in which the accumulation of cyclic GMP in response to cyclic AMP stimulation was absent produced no myosin II responses. A model is described in which cyclic GMP (directly or indirectly via Ca(2+) regulates accumulation of myosin II on the cytoskeleton by regulating phosphorylation of the myosin heavy and light chain kinases.

Calcium, cyclic GMP and the control of myosin II during chemotactic signal transduction ofDictyostelium

Evidence is presented for Ca2+ and cyclic GMP being involved in signal transduction between the cell surface cyclic AMP receptors and cytoskeletal myosin II involved in chemotactic cell movement. Ca2+ is shown to be required for chemotactic aggregation of amoebae. The evidence for uptake and/or eflux of this ion being regulated by the nucleotide cyclic GMP is discussed. The connection between Ca2+, cyclic GMP and chemotactic cell movement has been explored using “streamer F” mutants. The primary defect in these mutants is in the structural gene for the cyclic GMP-specific phosphodiesterase which results in the mutants producing an abnormally prolonged peak of accumulation of cyclic GMP in response to stimulation with the chernoattractant cyclic AMP. While events associated with production and relay of cyclic AMP signals are normal, certain events associated with movement are (like the cyclic GMP response) abnormally prolonged in the mutants. These events include Ca2+ uptake, myosin II association with the cytoskeleton and inhibition of myosin heavy and light chain phosphorylation. These changes can be correlated with the amoebae becoming elongated and transiently decreasing their locomotive speed after chemotactic stimulation. Other mutants studied in which the accumulation of cyclic GMP in response to cyclic AMP stimulation was absent produced no myosin II responses. Models are described in which cyclic GMP (directly or indirectly via Ca2+) regulates accumulation of myosin II on the cytoskeleton by inhibiting phosphorylation of the myosin heavy and light chain kinases.

On Topological Simulations in Developmental Biology

Further study is made of the topological model framework for cell simulations that was introduced by Matela & Fletterick (1979, 1980). The role that cell-cell interactions play in development is studied via processes such as cell growth, cell division, cell movement, cell adhesion, cell differentiation, cell death, and the process of chemical diffusion. Two phenomena are employed in simple applications of the framework: cell cleavage and chemotactic cell movement. A model for the first steps of neural differentiation in the developing visual system of the Drosophila is introduced in a more comprehensive case study. The strengths and weaknesses of the topological approach are discussed.

Streamer F mutants and chemotaxis of Dictyostelium.

Streamer F mutants have been found to be useful tools for studying the pathway of signal transduction leading to chemotactic cell movement. The primary defect in these mutants is in the structural gene for the cyclic GMP specific phosphodiesterase. This defect allows a larger and prolonged peak of cyclic GMP to be formed in response to the chemotactic stimulus, cyclic AMP. This characteristic aberrant pattern of cyclic GMP accumulation in the streamer F mutants has been correlated with similar patterns of changes in the influx of calcium from the medium, myosin II association with the cytoskeleton, myosin phosphorylation and a decrease in speed of movement of the amoebae. From these studies a sequence of events can be deduced that leads from cell surface cyclic AMP stimulation to cell polarization prior to movement of the amoebae in response to the chemotactic stimulus.

Migration of individual microvessel endothelial cells: stochastic model and parameter measurement

Analysis of cell motility effects in physiological processes can be facilitated by a mathematical model capable of simulating individual cell movement paths. A quantitative description of motility of individual cells would be useful, for example, in the study of the formation of new blood vessel networks in angiogenesis by microvessel endothelial cell (MEC) migration. In this paper we propose a stochastic mathematical model for the random motility and chemotaxis of single cells, and evaluate migration paths of MEC in terms of this model. In our model, cell velocity under random motility conditions is described as a persistent random walk using the Ornstein-Uhlenbeck (O-U) process. Two parameters quantify this process: the magnitude of random movement accelerations, alpha, and a decay rate constant for movement velocity, beta. Two other quantities often used in measurements of individual cell random motility properties--cell speed, S, and persistence time in velocity, Pv--can be defined in terms of the fundamental stochastic parameters alpha and beta by: S =square root (alpha/beta) and Pv = 1/beta. We account for chemotactic cell movement in chemoattractant gradients by adding a directional bias term to the O-U process. The magnitude of the directional bias is characterized by the chemotactic responsiveness, kappa. A critical advantage of the proposed model is that it can generate, using experimentally measured values of alpha, beta and kappa, computer simulations of theoretical individual cell paths for use in evaluating the role of cell migration in specific physiological processes. We have used the model to assess MEC migration in the presence of absence of the angiogenic stimulus acidic fibroblast growth factor (aFGF). Time-lapse video was used to observe and track the paths of cells moving in various media, and the mean square displacement was measured from these paths. To test the validity of the model, we compared the mean square displacement measurements of each cell with model predictions of that displacement. The comparison indicates that the O-U process provides a satisfactory description of the random migration at this level of comparison. Using nonlinear regression in these comparisons, we measured the magnitude of random accelerations, alpha, and the velocity decay rate constant, beta, for each cell path. We consequently obtained values for the derived quantities, speed and persistence time. In control medium, we find that alpha = 250 +/- 100 microns 2h-3 and beta = 0.22 +/- 0.03h-1, while in stimulus medium (control plus unpurified aFGF) alpha = 1900 +/- 720 microns 2h-3 and beta = 0.99 +/- 0.37h-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Photodynamically induced chemoresponses of the colorless flagellate, Astasia longa, in the presence of riboflavin

In the presence of the photosensitizer riboflavin, Astasia accumulates in illuminated fields at high fluence rates. The quenchers of riboflavin excited states, NaN3 and KI, abolish the photodynamic effect of riboflavin. Crocetin, a 1O2 quencher, does not influence the photodynamic action of riboflavin while 1,4-benzoquinone very strongly depresses its effect. This indicates a type I pathway forming H2O2 as a photoproduct. The photodynamic effect is abolished by the addition of 10-5 M catalase which breaks down H2O2. Astasia shows chemoaccumulations around the opening of a capillary filled either with riboflavin (under high intensity irradiation) or H2O2 which proves the hypothesis that the photobehavioral response in the presence of riboflavin is based on a chemoresponse toward H2O2, produced when the dye is irradiated. The accumulation around the capillary opening is not due to a direct chemotactic movement of cells but rather to a chemophobic response which prevents the cells from swimming away from the chemoattractant.

Relationships between the ligand specificity of cell surface folate binding sites, folate degrading enzymes and cellular responses in Dictyostelium discoideum

The affinity of four distinct types of folate binding sites and of two membrane-bound folate degrading enzymes for 15 folic acid derivatives was monitored. Apart from two types (A H and A L of binding sites, all binding classes or enzymes show different affinity patterns. This strongly suggests the observed binding sites to be nonidentical to the membrane-bound folate deaminase or folate C 9-N 10 cleaving enzyme. The analog specificity of the chemotactic response towards folates shows a strong resemblance to the specificity of binding to one of the binding classes: ‘B-sites’ ( p < 0.01%). Less or no correlation was observed between the other classes or the enzymes and the chemotactic response. This may indicate that the B-sites are involved in the transduction of an extracellular signal to chemotactic cell movement. Folates elicit secretion of cAMP. Recently, the activity of several folate derivatives to evoke a cAMP response was studied (Devreotes, P.N. (1983) Dev. Biol. 95, 154–162). Comparison of this activity and the specificity of the binding sites in this study, suggests that the ‘A-sites’ are involved in the cAMP response.