Actin polymerization and pseudopod extension during amoeboid chemotaxis

Amoebae of the cellular slime mold Dictyostelium discoideum are an excellent model system for the study of amoeboid chemotaxis. These cells can be studied as a homogeneous population whose response to chemotactic stimulation is sufficiently synchronous to permit the correlation of the changes in cell shape and biochemical events during chemotaxis. Having demonstrated this synchrony of response, we show that actin polymerization occurs in two stages during stimulation with chemoattractants. The assembly of F-actin that peaks between 40 and 60 sec after the onset of stimulation is temporally correlated with the growth of new pseudopods. F-actin, which is assembled by 60 sec after stimulation begins, is localized in the new pseudopods that are extended at this time. Both stages of actin polymerization during chemotactic stimulation involve polymerization at the barbed ends of actin filaments based on the cytochalasin sensitivity of this response. We present a hypothesis in which actin polymerization is one of the major driving forces for pseudopod extension during chemotaxis. The predictions of this model, that localized regulation of actin nucleation activity and actin filament cross-linking must occur, are discussed in the context of current models for signal transduction and of recent information regarding the types of actin-binding proteins that are present in the cell cortex.     

Chemoattractant stimulation of TORC2 is regulated by receptor/G protein–targeted inhibitory mechanisms that function upstream and independently of an essential GEF/Ras activation pathway in Dictyostelium

Global stimulation of Dictyostelium with different chemoattractants elicits multiple transient signaling responses, including synthesis of cAMP and cGMP, actin polymerization, activation of kinases ERK2, TORC2, and phosphatidylinositide 3-kinase, and Ras-GTP accumulation. Mechanisms that down-regulate these responses are poorly understood. Here we examine transient activation of TORC2 in response to chemically distinct chemoattractants, cAMP and folate, and suggest that TORC2 is regulated by adaptive, desensitizing responses to stimulatory ligands that are independent of downstream, feedback, or feedforward circuits. Cells with acquired insensitivity to either folate or cAMP remain fully responsive to TORC2 activation if stimulated with the other ligand. Thus TORC2 responses to cAMP or folate are not cross-inhibitory. Using a series of signaling mutants, we show that folate and cAMP activate TORC2 through an identical GEF/Ras pathway but separate receptors and G protein couplings. Because the common GEF/Ras pathway also remains fully responsive to one chemoattractant after desensitization to the other, GEF/Ras must act downstream and independent of adaptation to persistent ligand stimulation. When initial chemoattractant concentrations are immediately diluted, cells rapidly regain full responsiveness. We suggest that ligand adaptation functions in upstream inhibitory pathways that involve chemoattractant-specific receptor/G protein complexes and regulate multiple response pathways.     

Effects of cytochalasin B on cell movements and chemoattractant-elicited actin changes of Dictyostelium

The actin-binding drug cytochalasin B (CB) was employed to study the stability and role of cytoskeletal actin following chemotactic stimulation of Dictyostelium discoideum. Intact amoebae were found to be impermeable to this drug, as shown by lack of inhibition of chemotactic movement in its presence and failure of [3H]CB to bind to intact amoebae. However, there were approx. 150 000 high affinity CB-binding sites per cell detectable after cell breakage and preparation of Triton-insoluble cytoskeletons. The effect of CB on cytoskeletons was to destabilize the second (25-45 sec) and third (60 sec) chemotactically-induced peaks of cytoskeletal actin accumulation and to reduce the actin levels to the low prestimulus amount. In contrast, the drug had no such action on the rapid (3-5 sec) actin peak. This peak appeared to be stable in the presence of CB added before or simultaneously with lysis of the cell. It was also observed that the instability of the second and third peaks to CB gradually decreased after cell lysis (as did the number of CB binding sites) such that if CB was added 5 min after lysis of the chemotactically stimulated amoebae it had no destabilizing effect. Evidence was obtained from experiments employing centrifugation of cytoskeletons at 100 000 g and from the use of the DNase I inhibition assay for G-actin, that the first (3-5 sec) actin peak of accumulation involved polymerization rather than just cross-linking of short filamentous actin fragments. The significance of these actin accumulation peaks is discussed and their timing correlated with events involved in chemotaxis.     

Relationship of pseudopod extension to chemotactic hormone-induced actin polymerization in amoeboid cells

Aggregation-competent amoeboid cells of Dictyostelium discoideum are chemotactic toward cAMP. Video microscopy and scanning electron microscopy were used to quantitate changes in cell morphology and locomotion during uniform upshifts in the concentration of cAMP. These studies demonstrate that morphological and motile responses to cAMP are sufficiently synchronous within a cell population to allow relevant biochemical analyses to be performed on large numbers of cells. Changes in cell behavior were correlated with F-actin content by using an NBD-phallacidin binding assay. These studies demonstrate that actin polymerization occurs in two stages in response to stimulation of cells with extracellular cAMP and involves the addition of monomers to the cytochalasin D-sensitive (barbed) ends of actin filaments. The second stage of actin assembly, which peaks at 60 sec following an upshift in cAMP concentration, is temporally correlated with the growth of new pseudopods. The F-actin assembled by 60 sec is localized in these new pseudopods. These results indicate that actin polymerization may constitute one of the driving forces for pseudopod extension in amoeboid cells and that nucleation sites regulating polymerization are under the control of chemotaxis receptors.     

Actin dynamics rapidly reset chemoattractant receptor sensitivity following adaptation in neutrophils

Neutrophils are cells of the innate immune system that hunt and kill pathogens using directed migration. This process, known as chemotaxis, requires the regulation of actin polymerization downstream of chemoattractant receptors. Reciprocal interactions between actin and intracellular signals are thought to underlie many of the sophisticated signal processing capabilities of the chemotactic cascade including adaptation, amplification and long-range inhibition. However, with existing tools, it has been difficult to discern actin''s role in these processes. Most studies investigating the role of the actin cytoskeleton have primarily relied on actin-depolymerizing agents, which not only block new actin polymerization but also destroy the existing cytoskeleton. We recently developed a combination of pharmacological inhibitors that stabilizes the existing actin cytoskeleton by inhibiting actin polymerization, depolymerization and myosin-based rearrangements; we refer to these processes collectively as actin dynamics. Here, we investigated how actin dynamics influence multiple signalling responses (PI3K lipid products, calcium and Pak phosphorylation) following acute agonist addition or during desensitization. We find that stabilized actin polymer extends the period of receptor desensitization following agonist binding and that actin dynamics rapidly reset receptors from this desensitized state. Spatial differences in actin dynamics may underlie front/back differences in agonist sensitivity in neutrophils.     

Time-resolved responses to chemoattractant, characteristic of the front and tail of Dictyostelium cells

In a gradient of chemoattractant, Dictyostelium cells are orientated with their front directed toward the source and their tail pointing into the opposite direction. The front region is specified by the polymerization of actin and the tail by the recruitment of filamentous myosin-II. We have dissected these front and tail responses by exposing cells to an upshift of cyclic AMP. A sharp rise and fall of polymerized actin within 10s is accompanied by the recruitment of proteins involved in turning actin polymerization on or off. The cortical accumulation of myosin-II starts when the front response has declined, supporting the concept of divergent signal transmission and adaptation pathways.     

Adenosine 3',5'-monophosphate and testosterone responses of normal and desensitized Leydig cells to forskolin

Forskolin has a potent stimulatory effect on both cyclic AMP and testosterone formation by purified Leydig cells. Forskolin also markedly enhanced hCG-induced cyclic AMP formation, but maximal testosterone production remained unaltered. Cyclic AMP and testosterone responses of desensitized Leydig cells to in vitro hCG stimulation were completely lost. Cholera toxin-induced cyclic AMP formation was also reduced. However, forskolin was able to stimulate a 3.4-fold increment in cyclic AMP formation and potentiate hCG-induced cyclic AMP response by desensitized Leydig cells. The absolute cyclic AMP levels were significantly lower than in normal control cells. These results suggest that the catalytic unit remains intact in desensitized Leydig cells and the coupling between N-protein and catalytic unit is impaired. The N-protein is required for full expression of maximal response of Leydig cells to forskolin.     

Reactivation of Desensitized Formyl Peptide Receptors by Platelet Activating Factor: A Novel Receptor Cross Talk Mechanism Regulating Neutrophil Superoxide Anion Production

Neutrophils express different chemoattractant receptors of importance for guiding the cells from the blood stream to sites of inflammation. These receptors communicate with one another, a cross talk manifested as hierarchical, heterologous receptor desensitization. We describe a new receptor cross talk mechanism, by which desensitized formyl peptide receptors (FPR_des) can be reactivated. FPR desensitization is induced through binding of specific FPR agonists and is reached after a short period of active signaling. The mechanism that transfers the receptor to a non-signaling desensitized state is not known, and a signaling pathway has so far not been described, that transfers FPR_des back to an active signaling state. The reactivation signal was generated by PAF stimulation of its receptor (PAFR) and the cross talk was uni-directional. LatrunculinA, an inhibitor of actin polymerization, induced a similar reactivation of FPR_des as PAF while the phosphatase inhibitor CalyculinA inhibited reactivation, suggesting a role for the actin cytoskeleton in receptor desensitization and reactivation. The activated PAFR could, however, reactivate FPR_des also when the cytoskeleton was disrupted prior to activation. The receptor cross talk model presented prophesies that the contact on the inner leaflet of the plasma membrane that blocks signaling between the G-protein and the FPR is not a point of no return; the receptor cross-talk from the PAFRs to the FPR_des initiates an actin-independent signaling pathway that turns desensitized receptors back to a signaling state. This represents a novel mechanism for amplification of neutrophil production of reactive oxygen species.     

Regulatory light chains in myosins.

Scallop myosin molecules contain two moles of regulatory light chains and two moles of light chains with unknown function. Removal of one of the regulatory light chains by treatment with EDTA is accompanied by the complete loss of the calcium dependence of the actin-activated ATPase activity and by the loss of one of the two calcium binding sites on the intact molecule. Such desensitized preparations recombine with one mole of regulatory light chain and regain calcium regulation and calcium binding. The second regulatory light chain may be selectively obtained from EDTA-treated scallop muscles by treatment with the Ellman reagent (5,5′-dithiobis(2-nitrobenzoic acid)): treatment with this reagent, however, leads to an irreversible loss of ATPase activity. The light chains obtained by treatment with EDTA and then DTNB are identical in composition and function. A different light chain fraction obtained by subsequent treatment with guanidine-HCl does not bind to desensitized or intact myoflbrils and has no effect on ATPase activity.Regulatory light chains which bind to desensitized scallop myofibrils with high affinity and restore calcium control were found in a number of molluscan and vertebrate myosins, including Mercenaria, Spisula, squid, lobster tail, beef heart, chicken gizzard, frog and rabbit. Although these myosins all have a similar subunit structure and contain about two moles of regulatory light chain, only scallop myosin or myofibrils can be desensitized by treatment with EDTA.There appear to be two classes of regulatory light chains. The regulatory light chains of molluscs and of vertebrate smooth muscles restore full calcium binding and also resensitize purified scallop myosin. The regulatory light chains from vertebrate striated, cardiac, and the fast decapod muscles, on the other hand, have no effect on calcium binding and do not resensitize purified scallop myosin unless the myosin is complexed with actin. The latter class of light chains is found in muscles where in vitro functional tests failed to detect myosin-linked regulation.     

Insulin induces epidermal growth factor (EGF) receptor clustering and potentiates EGF-stimulated DNA synthesis in swiss 3T3 cells: a mechanism for costimulation in mitogenic synergy

In many cellular systems, activation with more than one ligand can produce a cellular response that is greater than the sum of the individual responses to the ligands. This synergy is sometimes referred to as coactivation. In Swiss 3T3 fibroblasts, activation of the epidermal growth factor (EGF) receptor produces a weak induction of DNA synthesis. Insulin has no stimulatory effect on this response. However, in combination, EGF and insulin synergize to cause a large induction of S phase. The underlying cellular biochemistry of this effect has been examined. The data indicate that phospholipase C activation is a major component of agonist-induced DNA synthesis. In contrast, activation of p70 S6 kinase by single agonists was inversely related to their ability to stimulate DNA synthesis. Therefore, it was examined whether stimulation of Swiss 3T3 cells with insulin causes changes in the subcellular distribution of EGF receptors and phospholipase Cgamma1 that could potentially explain the observed synergy or costimulation. It was found that insulin effectively induced the accumulation of EGF receptors on the actin arc of cells without activation of the EGF receptor. In contrast, EGF, when added for several hours, did not cause accumulation of the EGF receptor at this site. However, both EGF and insulin stimulated the accumulation of phospholipase Cgamma1 at the actin arc, which was coincident with the EGF receptor in the case of insulin- stimulated cells. Therefore, it is suggested that the insulin-induced coclustering of the EGF receptor with phospholipase Cgamma1 at the actin arc may allow for greater efficiency of signal transduction, resulting in the synergy observed for these two hormones in stimulation of DNA synthesis.     

The rapid desensitization of receptors for platelet derived growth factor, bradykinin and ATP: studies on individual cells using quantitative digital video fluorescence microscopy

The rise in free cytosolic Ca2+ of individual response to growth factors was studied in serum starved cultures of 3T3 fibroblasts. Quantitative digital video fluorescence microscopy revealed that with platelet derived growth factor (PDGF) there was a lag period between stimulation and Ca2+ response, with considerable cell-to-cell variation, whereas ATP, bradykinin and fetal calf serum induced an immediate, synchronous response. A coverslip with attached cells was mounted on a small flow chamber, allowing complete change of medium in 2 sec. Using this technique, homologous desensitization to a second addition of agonist 2 min after removal of the first addition was found for all agonists. Unusual heterologous desensitization was observed in that PDGF desensitized the cells to the other agonists, yet the reverse did not occur.     

Adaptational Modifications of the Vestibular Postural Response in Humans under Conditions of Horizontal Visual Reversal

In healthy volunteers, we recorded stabilograms and studied postural responses evoked by galvanic stimulation of the labyrinth (binaurally applied 1-mA current, 4 sec) with the subjects' eyes open and closed and under conditions of reversed visual perception. Horizontal reversal of the visual space was provided by using spectacles with the Dove's prisms. In series consisting of 10 sequential tests with eyes open, we observed a gradual drop in the response amplitude, while there were practically no changes in the maximum velocity of the displacement. Postural responses with eyes closed were considerably greater than those with eyes open, but their amplitude and velocity demonstrated no changes with sequential tests. Under conditions of reversal of the visual perception, both the amplitude and maximum velocity of the postural responses decreased with successive testing. Under the above conditions, at the beginning of a test series responses to vestibular stimulation were greater than those with eyes closed, but in repeated tests they decreased and attained the same magnitude as in the tests with eyes closed. Therefore, the effect of short-term adaptation to visual reversal on the system controlling vertical posture resulted in simple rejection of the information coming via the visual input. In another experimental mode, we studied the adaptation effects at longer (3 h long) visual reversal. Postural responses to galvanic stimulation of the labyrinth (monaurally applied, 2-mA current, 4 sec) were tested with 1-h-long intervals; tests with visual reversal and with eyes closed were made in a random order with each other. A 3-h-long interval with the prismatic spectacles on did not modify the amplitude and velocity of the vestibular postural responses when the tests were made with the eyes closed. When the tests were performed with the eyes open, but in the inverting spectacles, postural responses significantly decreased (by about 50-60%) to the 2nd and 3rd h of the experiment. Such selective suppression of the vestibular input under conditions of visual reversal can be interpreted as a result of adaptational transformation of the visual-vestibular relation directed toward minimization of the visual-vestibular conflict.     

Imprecision of Adaptation in Escherichia coli Chemotaxis

Adaptability is an essential property of many sensory systems, enabling maintenance of a sensitive response over a range of background stimulus levels. In bacterial chemotaxis, adaptation to the preset level of pathway activity is achieved through an integral feedback mechanism based on activity-dependent methylation of chemoreceptors. It has been argued that this architecture ensures precise and robust adaptation regardless of the ambient ligand concentration, making perfect adaptation a celebrated property of the chemotaxis system. However, possible deviations from such ideal adaptive behavior and its consequences for chemotaxis have not been explored in detail. Here we show that the chemotaxis pathway in Escherichia coli shows increasingly imprecise adaptation to higher concentrations of attractants, with a clear correlation between the time of adaptation to a step-like stimulus and the extent of imprecision. Our analysis suggests that this imprecision results from a gradual saturation of receptor methylation sites at high levels of stimulation, which prevents full recovery of the pathway activity by violating the conditions required for precise adaptation. We further use computer simulations to show that limited imprecision of adaptation has little effect on the rate of chemotactic drift of a bacterial population in gradients, but hinders precise accumulation at the peak of the gradient. Finally, we show that for two major chemoeffectors, serine and cysteine, failure of adaptation at concentrations above 1 mM might prevent bacteria from accumulating at toxic concentrations of these amino acids.     

Characterization of TPM1 disrupted yeast cells indicates an involvement of tropomyosin in directed vesicular transport

Disruption of the yeast tropomyosin gene TPM1 results in the apparent loss of actin cables from the cytoskeleton (Liu, H., and A. Bretscher. 1989. Cell. 57:233-242). Here we show that TPM1 disrupted cells grow slowly, show heterogeneity in cell size, have delocalized deposition of chitin, and mate poorly because of defects in both shmooing and cell fusion. The transit time of alpha-factor induced a-agglutinin secretion to the cell surface is longer than in isogenic wild-type strains, and some of the protein is mislocalized. Many of the TPM1-deleted cells contain abundant vesicles, similar in morphology to late secretory vesicles, but without an abnormal accumulation of intermediates in the delivery of either carboxypeptidase Y to the vacuole or invertase to the cell surface. Combinations of the TPM1 disruption with sec13 or sec18 mutations, which affect early steps in the secretory pathway, block vesicle accumulation, while combinations with sec1, sec4 or sec6 mutations, which affect a late step in the secretory pathway, have no effect on the vesicle accumulation. The phenotype of the TPM1 disrupted cells is very similar to that of a conditional mutation in the MYO2 gene, which encodes a myosin-like protein (Johnston, G. C., J. A. Prendergast, and R. A. Singer. 1991. J. Cell Biol. 113:539-551). The myo2-66 conditional mutation shows synthetic lethality with the TPM1 disruption, indicating that the MYO2 and TPM1 gene products may be involved in the same, or parallel function. We conclude that tropomyosin, and by inference actin cables, may facilitate directed vesicular transport of components to the correct location on the cell surface.     

Unit responses of the cerebellar cortex to stimulation of the vagus nerve and nerves of the limbs

Responses of 74 cerebellar cortical neurons to electrical stimulation of the vagus nerve and its gastric branches and also of the limb nerves were recorded extracellularly in experiments on rats anesthetized with chloralose and pentobarbital. Phasic and tonic unit responses were similar to all types of stimulation, but fewer neurons responded to stimulation of the vagus nerve than to stimulation of the limb nerves. Monosensory neurons responding only to visceral stimulation, were not found. Among cells giving a phasic response, some had a short and others a long latent period. The latent period of responses of all types was longer to vagal stimulation than to stimulation of the limb nerves. The maximal frequency of rhythmic responses to vagal stimulation was 8/sec, compared with 16/sec for stimulation of the limb nerves. Tonic responses of the neurons were excitatory, inhibitory, and mixed. The type of unit response could vary depending on the frequency and repetitiveness of the stimuli but was otherwise independent of the type of stimulation.N. I. Pirogov Vinnitsa Medical Institute. I. M. Sechenov Institute of Evolutionary Biochemistry and Physiology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 4, No. 5, pp. 471–479, September–October, 1972.     

Afferent connections to the fast conduction pathway in the central nervous system of the leech Hirudo medicinalis

A burst of all-or-none action potentials, identical in size and shape, can be recorded from the ventral cord of H. medicinalis following both photic and mechanical stimulation of the skin. This response propagates both anteriorly and posteriorly from its point of origin at the same conduction velocity of about 1.3 m/sec. The action potential elicited by electrical stimulation of the cord collides by refractoriness with the action potentials elicited in response to photic and mechanical stimulation. The cord response to photic and mechanical stimulation is reversibly suppressed by perfusion with high Mg++ solutions, whereas the afferent discharges recorded from the segmental nerves remain unaffected. Lesion experiments show that the cord responses to mechanical and photic stimuli, travel along the median connective (Faivre's nerve). It is concluded that afferent impulses originating from mechanoreceptors and photoreceptors converge with chemical excitatory synapses onto a fast conducting pathway in the ventral cord. This fast conducting pathway is coextensive with the one which is excited by electrical stimulation of the ventral cord (1, 3).     

Cyclic 3', 5'-AMP relay dictyostelium discoideum. V. Adaptation of the cAMP signaling response during cAMP stimulation

In dictyostelium discoideum, extracellular cAMP activates adenylate cyclase, which leads to an increase in intracellular cAMP and the rate of cAMP secretion. The signaling response to a constant cAMP stimulus is terminated after several minutes by an adaptation mechanism. The time- course of adaptation stimuli of 10(-6) or 10(-7) M cAMP was assessed. We used a perfusion technique to deliver defined cAMP stimuli to [(3)H]adenosine-labeled amoebae and monitored their secretion of [(3)H]cAMP. Amoebae were pretreated with 10(-6) or 10(-7) M cAMP to periods of 0.33-12 minutes, and then immediately given test stimuli of 10(-8) M to 2.5 x 10(-7) M cAMP. The response to a given test stimulus was progressively attenuated and finally extinguished as the duration of the pretreatment stimulus increased. During concentration of the test stimulus. The responses to test stimuli of 10(-8), 5 x 10(-8), 10(-7), or 2.5 x 10(-7) M cAMP were extinguished after approximately 1, 2.25,2.5, and 10 min, respectively. 1.5 min of stimulation with 10(-7) M cAMP was necessary to extinguish the response of a test stimulus of 10(-8) M cAMP. Our data suggest that adaptation begins within 20 s of stimulation, rises rapidly for approximately 2.5 min, and reaches a plateau after approximately 10 min. The absolute rate of rise was faster during pretreatment with 10(-6) than with 10(-7) M cAMP. These results support a working hypothesis in which the occupancy of surface cAMP receptors leads to changes in two opposing cellular processes, excitation and adaptation, that control the activity of D. discoideum adenylate cyclase.     

The adaptation of the frog tongue to various taste solutions: the effect on gustatory neural responses to bitter stimuli

1. After the frog tongue was adapted for 10 sec to various salts and sugars, the initial phasic component of gustatory neural responses to almost all of quinine hydrochloride (Q-HCl), quinine sulfate (Q-H2SO4). Brucine, caffeine and picric acid was suppressed. 2. Following 10 sec adaptation to acetic acid, the phasic responses to Q-HCl and Q-H2SO4 were unchanged, those to brucine and caffeine were enhanced, and that to picric acid was depressed slightly. 3. The response to any one of Q-HCl, Q-H2SO4. brucine and caffeine was suppressed after adaptation to the other three, while those to picric acid and nicotine were unchanged or enhanced after adaptation to another bitter solution.