Quantitative analysis of cell motility and chemotaxis in Dictyostelium discoideum by using an image processing system and a novel chemotaxis chamber providing stationary chemical gradients

An image processing system was programmed to automatically track and digitize the movement of amebae under phase-contrast microscopy. The amebae moved in a novel chemotaxis chamber designed to provide stable linear attractant gradients in a thin agarose gel. The gradients were established by pumping attractant and buffer solutions through semipermeable hollow fibers embedded in the agarose gel. Gradients were established within 30 min and shown to be stable for at least a further 90 min. By using this system it is possible to collect detailed data on the movement of large numbers of individual amebae in defined attractant gradients. We used the system to study motility and chemotaxis by a score of Dictyostelium discoideum wild-type and mutant strains, including "streamer" mutants which are generally regarded as being altered in chemotaxis. None of the mutants were altered in chemotaxis in the optimal cAMP gradient of 25 nM/mm, with a midpoint of 25 nM. The dependence of chemotaxis on cAMP concentration, gradient steepness, and temporal changes in the gradient were investigated. We also analyzed the relationship between turning behavior and the direction of travel during chemotaxis in stable gradients. The results suggest that during chemotaxis D. discoideum amebae spatially integrate information about local increases in cAMP concentration at various points on the cell surface.     

The internal phosphodiesterase RegA is essential for the suppression of lateral pseudopods during Dictyostelium chemotaxis

Dictyostelium strains in which the gene encoding the cytoplasmic cAMP phosphodiesterase RegA is inactivated form small aggregates. This defect was corrected by introducing copies of the wild-type regA gene, indicating that the defect was solely the consequence of the loss of the phosphodiesterase. Using a computer-assisted motion analysis system, regA(-) mutant cells were found to show little sense of direction during aggregation. When labeled wild-type cells were followed in a field of aggregating regA(-) cells, they also failed to move in an orderly direction, indicating that signaling was impaired in mutant cell cultures. However, when labeled regA(-) cells were followed in a field of aggregating wild-type cells, they again failed to move in an orderly manner, primarily in the deduced fronts of waves, indicating that the chemotactic response was also impaired. Since wild-type cells must assess both the increasing spatial gradient and the increasing temporal gradient of cAMP in the front of a natural wave, the behavior of regA(-) cells was motion analyzed first in simulated temporal waves in the absence of spatial gradients and then was analyzed in spatial gradients in the absence of temporal waves. Our results demonstrate that RegA is involved neither in assessing the direction of a spatial gradient of cAMP nor in distinguishing between increasing and decreasing temporal gradients of cAMP. However, RegA is essential for specifically suppressing lateral pseudopod formation during the response to an increasing temporal gradient of cAMP, a necessary component of natural chemotaxis. We discuss the possibility that RegA functions in a network that regulates myosin phosphorylation by controlling internal cAMP levels, and, in support of that hypothesis, we demonstrate that myosin II does not localize in a normal manner to the cortex of regA(-) cells in an increasing temporal gradient of cAMP.     

Amebae of Dictyostelium discoideum respond to an increasing temporal gradient of the chemoattractant cAMP with a reduced frequency of turning: evidence for a temporal mechanism in ameboid chemotaxis

In an aggregation territory of Dictyostelium discoideum, outwardly moving, nondissipating waves of the chemoattractant cAMP sweep across each ameba. At the front of each wave, an ameba experiences an increasing temporal and a positive spatial gradient of cAMP. At the back of a wave, an ameba experiences a decreasing temporal and a negative spatial gradient of cAMP. Employing a perfusion chamber, we have mimicked the temporal dynamics of these waves in the absence of a spatial gradient and demonstrated that the frequency of lateral pseudopod formation and the frequency of turning are dramatically affected by the direction and dynamics of the temporal gradient. In addition, since an ameba will move in a directed fashion up a shallow, nonpulsatile gradient of cAMP, we also mimicked the increasing temporal gradient generated by an ameba moving up a shallow spatial gradient. The frequency of lateral pseudopod formation and the frequency of turning were depressed. Together, these results demonstrate that amebae can assess the direction of a temporal gradient of chemoattractant in the absence of a spatial gradient and alter both the frequency of pseudopod extension and turning, accordingly. Although these results do not rule out the involvement of a spatial mechanism in assessing a spatial gradient, they strongly suggest that the temporal dynamics of a cAMP wave or the temporal gradient generated by an ameba moving through a spatial gradient may play a major role in chemotaxis.     

RasC plays a role in transduction of temporal gradient information in the cyclic-AMP wave of Dictyostelium discoideum

To define the role that RasC plays in motility and chemotaxis, the behavior of a rasC null mutant, rasC-, in buffer and in response to the individual spatial, temporal, and concentration components of a natural cyclic AMP (cAMP) wave was analyzed by using computer-assisted two-dimensional and three-dimensional motion analysis systems. These quantitative studies revealed that rasC- cells translocate at the same velocity and exhibit chemotaxis up spatial gradients of cAMP with the same efficiency as control cells. However, rasC- cells exhibit defects in maintaining anterior-posterior polarity along the substratum and a single anterior pseudopod when translocating in buffer in the absence of an attractant. rasC- cells also exhibit defects in their responses to both the increasing and decreasing temporal gradients of cAMP in the front and the back of a wave. These defects result in the inability of rasC- cells to exhibit chemotaxis in a natural wave of cAMP. The inability to respond normally to temporal gradients of cAMP results in defects in the organization of the cytoskeleton, most notably in the failure of both F actin and myosin II to exit the cortex in response to the decreasing temporal gradient of cAMP in the back of the wave. While the behavioral defect in the front of the wave is similar to that of the myoA-/myoF- myosin I double mutant, the behavioral and cytoskeletal defects in the back of the wave are similar to those of the S13A myosin II regulatory light-chain phosphorylation mutant. Expression array data support the premise that the behavioral defects exhibited by the rasC- mutant are the immediate result of the absence of RasC function.     

Frequency and orientation of pseudopod formation of Dictyostelium discoideum amebae chemotaxing in a spatial gradient: further evidence for a temporal mechanism

Amebae of Dictyostelium discoideum normally chemotax to aggregation centers by assessing the direction of outwardly moving, nondissipating waves of the chemoattractant cAMP. However, D. discoideum amebae can also assess the direction of a relatively stable spatial gradient. We demonstrate that amebae migrating towards the "source" of a stable, spatial gradient move faster, extend fewer pseudopodia, and turn less frequently than amebae migrating away from the "source" in the same spatial gradient. In addition, amebae extend lateral pseudopods in a polarized fashion from the anterior half of the cell, and do so as frequently towards the source as away from the source. However, those formed towards the source more often produce a turn than those formed away from the source. These results suggest that there may be two decision-making systems, one localized in the pseudopods, and one along the entire cell body; they support the suggestion that Dictyostelium amebae may employ a temporal mechanism to assess the direction of a spatial gradient of chemoattractant.     

Intracellular role of adenylyl cyclase in regulation of lateral pseudopod formation during Dictyostelium chemotaxis

Cyclic AMP (cAMP) functions as the extracellular chemoattractant in the aggregation phase of Dictyostelium development. There is some question, however, concerning what role, if any, it plays intracellularly in motility and chemotaxis. To test for such a role, the behavior of null mutants of acaA, the adenylyl cyclase gene that encodes the enzyme responsible for cAMP synthesis during aggregation, was analyzed in buffer and in response to experimentally generated spatial and temporal gradients of extracellular cAMP. acaA- cells were defective in suppressing lateral pseudopods in response to a spatial gradient of cAMP and to an increasing temporal gradient of cAMP. acaA- cells were incapable of chemotaxis in natural waves of cAMP generated by majority control cells in mixed cultures. These results indicate that intracellular cAMP and, hence, adenylyl cyclase play an intracellular role in the chemotactic response. The behavioral defects of acaA- cells were surprisingly similar to those of cells of null mutants of regA, which encodes the intracellular phosphodiesterase that hydrolyzes cAMP and, hence, functions opposite adenylyl cyclase A (ACA). This result is consistent with the hypothesis that ACA and RegA are components of a receptor-regulated intracellular circuit that controls protein kinase A activity. In this model, the suppression of lateral pseudopods in the front of a natural wave depends on a complete circuit. Hence, deletion of any component of the circuit (i.e., RegA or ACA) would result in the same chemotactic defect.     

Effects of cAMP on single cell motility in Dictyostelium

The motility of individual, aggregation-competent amebae of Dictyostelium has been analyzed at different concentrations of cAMP under both nongradient and gradient conditions. The following is demonstrated: (a) concentrations of cAMP greater than 10(-8) M inhibit motility in a concentration-dependent fashion, decrease the frequency but not the degree of turning, and cause rounding in cell shape; (b) no concentration of cAMP stimulates motility, or positive chemokinesis; (c) concentrations of cAMP that stimulate a maximal chemotactic response do not affect motility and concentrations of cAMP that maximally inhibit motility do not stimulate chemotaxis under gradient conditions; and (d) the concentrations of cAMP that inhibit motility are identical under gradient and nongradient conditions.     

Dictyostelium discoideum surface changes elicited by high concentrations of cAMP

The effects of high concentrations of cAMP on both morphological and biochemical development of Dictyostelium discoideum amebae are reported. Observations using light and scanning electron microscopy (SEM) indicate that the cells' response to such treatment varies with the length of time they had been starved prior to cAMP addition. Vegetative and early developmental amebae become rounded within a short period after treatment. Such cells are capable of undertaking a normal aggregation after a delay of a few hours. A substantial induction of phosphodiesterase activity is elicited from these cells by cAMP treatment but their levels of cAMP surface binding sites remain low. cAMP addition to aggregation competent cells causes amebae first to flatten and then to retract into spherical forms and group into small aggregates. No induction of phosphodiesterase activity is observed in such cells and the levels of cAMP binding sites present on the amebae decrease rapidly. The data are discussed in terms of the different states of cAMP-sensitivity between vegative and aggregation-competent amebae.     

Sensory adaptation of Dictyostelium discoideum cells to chemotactic signals

Postvegetative Dictyostelium discoideum cells react chemotactically to gradients of cAMP, folic acid, and pterin. In the presence of a constant concentration of 10(-5) M cAMP cells move at random. They still are able to respond to superimposed gradients of cAMP, although the response is less efficient than without the high background level of cAMP. Cells which are accommodated to 10(-5) M cAMP do not react to a gradient of cAMP if the mean cAMP concentration is decreasing with time. This indicates the involvement of adaptation in the detection of chemotactic gradients: cells adapt to the mean concentration of chemoattractant and respond to positive deviations from the mean concentration. Cells adapted to high cAMP concentrations react normally to gradients of folic acid or pterin. Adaptation to one of these compounds does not affect the response to the other attractants. This suggests that cAMP, folic acid, and pterin are detected by different receptors, and that adaptation is localized at a step in the transduction process before the signals from these receptors coincide into one pathway. I discuss the implications of adaptation for chemotaxis and cell aggregation.     

Dictyostelium discoideum chemotaxis: threshold for directed motion

The chemotactic response of Dictyostelium discoideum cells to stationary, linear gradients of cyclic adenosine 3',5'-monophosphate (cAMP) was studied using microfluidic devices. In shallow gradients of less than 10(-3) nM/microm, the cells showed no directional response and exhibited a constant basal motility. In steeper gradients, cells moved up the gradient on average. The chemotactic speed and the motility increased with increasing steepness up to a plateau at around 10(-1) nM/microm. In very steep gradients, above 10 nM/microm, the cells lost directionality and the motility returned to the sub-threshold level. In the regime of optimal response the difference in receptor occupancy at the front and back of the cell is estimated to be only about 100 molecules.     

Chemoresponsiveness to cAMP and Folic Acid during Growth, Development, and Dedifferentiation in Dictyostelium discoideum

Chemoresponsiveness to cAMP and to folic acid are monitored in growing, developing, and dedifferentiating amebae of the cellular slime mold Dictyostelium discoideum . Two semiquantitative assays are employed, one measuring the directed movement of cells up a gradient of chemoattractant ('chemotaxis' assay) and the other measuring the outward spreading of cells in response to a chemical stimulant distributed equally throughout the substratum ('spreading' assay). Vegetative amebae possess relatively insignificant levels of chemotactic responsiveness to cAMP. Six h after the initiation of development, at approximately the same time as the onset of aggregation, cells rapidly acquire chemotactic responsiveness to cAMP. During 'erasure', a dedifferentiation induced by resuspending aggregating cells in fresh nutrient medium, chemotactic responsiveness to cAMP is lost just after the erasure event. By the same chemotactic assay, it is demonstrated that vegetative amebae possess a significant level of chemotactic responsiveness to folic acid. Two h after the initiation of development, cells completely lose chemotactic responsiveness to folic acid. During erasure, cells reacquire chemotactic responsiveness to folic acid at approximately the same time that they lose responsiveness to cAMP.
Dramatically different results are obtained by the spreading assay. When cells lose chemotactic responsiveness to folic acid early in development and when erasing cells lose chemotactic responsiveness to cAMP, they retain the spreading response to the two stimulants, respectively. The different results obtained for chemoreception employing the two assays are discussed in terms of molecular mechanisms, and a testable hypothesis is proposed for the possible roles of chemoresponsiveness and erasure in late morphogenesis.     

Chemoresponsiveness to cAMP and folic acid during growth, development, and dedifferentiation in Dictyostelium discoideum

Chemoresponsiveness to cAMP and to folic acid are monitored in growing, developing, and dedifferentiating amebae of the cellular slime mold Dictyostelium discoideum. Two semiquantitative assays are employed, one measuring the directed movement of cells up a gradient of chemoattractant ('chemotaxis' assay) and the other measuring the outward spreading of cells in response to a chemical stimulant distributed equally throughout the substratum ('spreading' assay). Vegetative amebae possess relatively insignificant levels of chemotactic responsiveness to cAMP. Six h after the initiation of development, at approximately the same time as the onset of aggregation, cells rapidly acquire chemotactic responsiveness to cAMP. During 'erasure', a dedifferentiation induced by resuspending aggregating cells in fresh nutrient medium, chemotactic responsiveness to cAMP is lost just after the erasure event. By the same chemotactic assay, it is demonstrated that vegetative amebae possess a significant level of chemotactic responsiveness to folic acid. Two h after the initiation of development, cells completely lose chemotactic responsiveness to folic acid. During erasure, cells reacquire chemotactic responsiveness to folic acid at approximately the same time that they lose responsiveness to cAMP. Dramatically different results are obtained by the spreading assay. When cells lose chemotactic responsiveness to folic acid early in development and when erasing cells lose chemotactic responsiveness to cAMP, they retain the spreading response to the two stimulants, respectively. The different results obtained for chemoreception employing the two assays are discussed in terms of molecular mechanisms, and a testable hypothesis is proposed for the possible roles of chemoresponsiveness and erasure in late morphogenesis.     

Pseudopodium activation and inhibition signals in chemotaxis by Dictyostelium discoideum amoebae

The behaviour of Dictyostelium discoideum amoebae has been studied in natural cAMP waves and in controlled spatial and temporal gradients. Chemoattractant gradients induce responses which indicate that amoebae spatially compare concentration increases at different points on the cell surface. This allows them to respond to the relative spatial and temporal gradients in a manner that is little affected by the absolute attractant concentration over several orders of magnitude. The changes in turning behaviour, motility and morphology that are induced by attractant gradients are consistent with transduction of stimuli into two intracellular signals - one activating and the other inhibiting pseudopodium formation. The former measures the present attractant concentration at particular points on the cell surface - the local, current signal. The latter measures the average attractant concentration over the whole cell surface during the recent past - the global, past signal. Both signals may be part of a normal pseudopodium autoactivation and inhibition system responsible for amoeboid morphology and motility. Attractants could modulate this system to generate the complex behavioural responses observed.     

Diverse chemotactic responses of Dictyostelium discoideum amoebae in the developing (temporal) and stationary (spatial) concentration gradients of folic acid,cAMP, Ca(2+) and Mg(2+)

The responses of Dictyostelium discoideum amoebae to developing (temporal) and stationary (spatial) gradients of folic acid, cAMP, Ca(2+), and Mg(2+) were studied using the methods of computer-aided image analysis. The results presented demonstrate that the new type of experimental chambers used for the observation of single cells moving within the investigated gradients of chemoattractants permit time lapse recording of single amoebae and determination of the trajectories of moving cells. It was found that, besides folic acid and cAMP (natural chemoattractants for Dictyostelium discoideum amoebae), also extracellular Ca(2+) and Mg(2+) are potent inducers of these cells' chemotaxis, and the amoebae of D. discoideum can respond to various chemoattractants differently. In the positively developing gradients of folic acid, cAMP, Ca(2+), and Mg(2+) oriented locomotion of amoebae directed towards the higher concentration of the tested chemoattractants was observed. However, in the negatively developing (temporal) and stationary linear (spatial) gradients, the univocal chemotaxis of amoebae was recorded only in the case of the Mg(2+) concentration gradient. This demonstrates that amoebae can respond to both developing and stationary gradients, depending upon the nature of the chemoattractant. We also investigated the effects of chosen inhibitors of signalling pathways upon chemotaxis of D. discoideum amoebae in the positively developing (temporal) gradients of tested chemoattractants. Verapamil was found to abolish the chemotaxis of amoebae only in the Ca(2+) gradients. Pertussis toxin suppressed the chemotactic response of cells in the gradients of folic acid and cAMP but did not prevent chemotaxis in those of Ca(2+) and Mg(2+), while quinacrine inhibited chemotaxis in the gradients of folic acid, cAMP, and Ca(2+) but only slightly affected chemotaxis in the Mg(2+) gradient. None of the tested inhibitors causes inhibition of cell random movement, when applied in isotropic solution. Also EDTA and EGTA up to 50 mM concentration did not inhibit locomotion of amoebae in control isotropic solutions.     

Dictyostelium Chemotactic Response to Spatial and Temporal Gradients. Theories of the Limits of Chemotactic Sensitivity and of Pseudochemotaxis

Four aspects of ameboid cell chemotaxis are discussed: 1) Ameboid cells (Dictyostelium discoideum, leukocytes) might orient to chemotaxin gradients by sensing a spatial gradient or a temporal change in the concentration. Using a moving micropipette source of cAMP, we show the D discoideum cells can orient toward a gradient in which the concentration is everywhere decreasing with time–implying a spatial mechanism. 2) The number of molecules N that must be released by a source to orient a cell is limited by the natural concentration “noise” due to diffusion. N is shown to be simply related to the cell size and the distance from the source. 3) We show that previous diffusion equations for cell population movement have not taken the speed variations (klinokinesis) into account properly, and we present a new result that does. 4) We briefly discuss reaction-diffusion models of cell orientation.     

Myosin II heavy chain null mutant of Dictyostelium exhibits defective intracellular particle movement

Both cellular motility and intracellular particle movement are compared between normal Dictyostelium amebae of strain AX4 and amebae of a myosin II heavy chain null mutant, HS2215, using the computer assisted "Dynamic Morphology System." In AX4 cells rapidly translocating in buffer, cytoplasmic expansion is apical and the majority of intracellular particles move anteriorly, towards the site of expansion. When these cells are pulsed with 10(-6) M cAMP, the peak concentration of the natural cAMP wave, cells stop translocating and average particle velocity decreases threefold within 2-4 s after cAMP addition. After 8 s, there is a partial rebound both in cytoplasmic expansion and particle velocity, but in both cases, original apical polarity is lost. In HS2215 cells in buffer, both cellular translocation and average particle velocity are already at the depressed levels observed in normal cells immediately after cAMP addition, and no anterior bias is observed in either the direction of cytoplasmic expansion or the direction of particle movement. The addition of cAMP to myosin-minus cells results in no additional effect. The results demonstrate that myosin II is necessary for (a) the rapid rate of intracellular particle movement, (b) the biased anterior directionality of particle movement, and (c) the rapid inhibition of particle movement by cAMP.     

Localization of adenylate cyclase during development of Dictyostelium discoideum

Abstract. Cyclic AMP is known to function as the chemo-tactic signal during aggregation of single-celled amoebae of the cellular slime mold Dictyosteliwn discoideum. Evidence from several laboratories has accumulated suggesting that cAMP also acts as a regulatory molecule during Dictyostelium multicellular differentiation. We have used ultra-microtechniques and a sensitive radioimmunoassay in the localization of adenylate cyclase, the cAMP synthetic enzyme, during the development of Dictyostelium. We demonstrate that adenylate cyclase activity is localized in the pre-spore cells of the culminating individual with no activity detectable in the prestalk region. We show that this lack of activity in the stalk may be due to a masking by an endogenous inhibitor of the enzyme. Within the spore mass we found an increasing gradient of enzyme activity toward the base. These data, along with that from the localization of cyclic nucleotide phosphodiesterase, indicate that an enzymatic potential exists for the creation of cAMP gradients during development in the organism. Such a gradient may provide positional information necessary to direct the terminal differentiation of spore and stalk cells.     

The effect of amino acids on the motile behavior of Bacillus subtilis

Constant levels of amino acids enhanced the velocity of Bacillus subtilis 60015 cells about 2-fold and stimulated the response in motility assays. The stimulation of velocity did not occur via the receptors for chemotaxis. Cysteine and methionine, general inhibitors of chemotaxis, both completely inhibited the smooth response in a temporal gradient of attractant. After methionine starvation B. subtilis 60015 showed no measurable response in a temporal gradient of attractant, this in contrast to the effect observed with some other bacteria. Addition of methionine to starved cells restored the response toward attractant. Revertants of B. subtilis 60015 for methionine requirement could not be starved and showed a normal behavior toward temporal gradients of attractant.Abbreviation O.D.₆₀₀ optical density measured at 600 nm     

The effects of inhibitors of adenylate cyclase and phosphodiesterase on D. discoideum aggregation.

The action of two adenine analogues on the aggregation of D. discoideum amebae was examined. SQ22536 and SQ20009 are inhibitors of adenylate cyclase and phosphodiesterase, respectively, in higher eukaryotes. Both compounds are shown here to inhibit the differentiation of cells to aggregation-competence. SQ22536-treated cells exhibited normal accumulation of their adenylate cyclase activity as measured in cell lysates but the amebae did not synthesize cAMP. The ability of this drug to compete for cAMP surface-binding sites, and the observation that the effects of the drug could be reversed by imposed pulses of cAMP, suggest that SQ22536 functions as a cAMP antagonist. The effects of SQ20009 on cell differentiation did not appear to be mediated by an inhibition of phosphodiesterase activity or cAMP binding to the cell surface. Amebae were arrested at a very early stage in development and remained unresponsive to external cAMP.     

Vascular Smooth Muscle Cell Durotaxis Depends on Substrate Stiffness Gradient Strength

Mechanical compliance is emerging as an important environmental cue that can influence certain cell behaviors, such as morphology and motility. Recent in vitro studies have shown that cells preferentially migrate from less stiff to more stiff substrates; however, much of this phenomenon, termed durotaxis, remains ill-defined. To address this problem, we studied the morphology and motility of vascular smooth muscle cells on well-defined stiffness gradients. Baselines for cell spreading, polarization, and random motility on uniform gels with moduli ranging from 5 to 80 kPa were found to increase with increasing stiffness. Subsequent analysis of the behavior of vascular smooth muscle cells on gradient substrata (0-4 kPa/100 μm, with absolute moduli of 1-80 kPa) demonstrated that the morphology on gradient gels correlated with the absolute modulus. In contrast, durotaxis (evaluated quantitatively as the tactic index for a biased persistent random walk) and cell orientation with respect to the gradient both increased with increasing magnitude of gradient, but were independent of the absolute modulus. These observations provide a foundation for establishing quantitative relationships between gradients in substrate stiffness and cell response. Moreover, these results reveal common features of phenomenological cell response to chemotactic and durotactic gradients, motivating further mechanistic studies of how cells integrate and respond to multiple complex signals.