1 School of Life Sciences, Division of Cell and Developmental Biology, University of Dundee, Wellcome Trust Biocentre, Dow Street, Dundee, DD1 5EH, UK
2 Department of Biological Chemistry, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA
*Author for correspondence (e-mail: c.j.weijer{at}dundee.ac.uk)
Accepted March 26, 2001
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SUMMARY |
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Key words: cAMP receptor, Receptor affinity, cAMP relay, Wave propagation, Chemotaxis, Cell movement
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INTRODUCTION |
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The social amoebae Dictyostelium discoideum provide a powerful system for studying the role of receptor subtypes (of different affinities) in controlling the dynamics of cell-cell signalling during multicellular development. The starvation-induced chemotactic aggregation of individual cells is controlled by propagating waves of the chemo-attractant cyclic AMP (Parent and Devreotes, 1996). The cAMP signals are periodically initiated by cells in aggregation centres and relayed by surrounding cells, to result in outward propagating waves of cAMP, which direct inward movement of the cells towards the aggregation centre. The cells detect the signal via specific cAMP receptors, activate the aggregation-specific adenylylcyclase (ACA) and produce and secrete additional cAMP. cAMP binding to the receptors also triggers an adaptation process, which results in a cessation of ACA activation and a shutdown of cAMP production. A highly specific secreted cAMP phosphodiesterase causes the extracellular cAMP levels to fall. When cAMP levels fall, the cells de-adapt and become responsive to new signals coming from the centre (Parent and Devreotes, 1999). Cells move up cAMP gradients as long as the signal is increasing over time (Wessels et al., 1996). cAMP not only controls cell movement during aggregation but is also a major regulator controlling the expression of developmentally regulated and cell-type specific genes (Firtel, 1996; Gerisch, 1987). cAMP waves also control the movement of the cells in all the later stages of development (Dormann et al., 2000). The dynamics of the cAMP signals are essential for the proper control of cell movement and gene expression and therefore for development.
During aggregation the geometry and dynamics of the cAMP waves are accurately reflected by their associated darkfield waves, which are caused by cell shape changes in response to propagating cAMP waves (Alcantara and Monk, 1974; Tomchik and Devreotes, 1981). Darkfield waves can be observed in mounds as well, and we have shown recently that they reflect cAMP waves (Patel et al., 2000; Rietdorf et al., 1996; Siegert and Weijer, 1995).
During development the cAMP signal is detected and transduced by a family of at least four cAMP receptors (cAR1-cAR4), which differ in their expression levels and patterns. The cAMP receptor types expressed sequentially during development have decreasing affinities for cAMP (Table 1), possibly to enable the organism to cope with an increase in extracellular cAMP concentrations during the formation of the multicellular structures (Abe and Yanagisawa, 1983; Kim et al., 1998). The high affinity receptor cAR1 is the first to be expressed during early aggregation, it is the primary receptor responsible for aggregation since cells lacking cAR1 fail to aggregate (Sun and Devreotes, 1991; Sun et al., 1990). cAR1 has two distinct affinity states of 30 and
300 nM under physiological conditions (Johnson et al., 1992). These different affinity states most likely reflect covalent modifications (e.g. phosphorylation) and/or the interaction with intracellular effectors (e.g. G proteins). cAR1 continues to be expressed in later development in all cells. During later aggregation a small number of cAR3 receptors are expressed. Deletion of cAR3 has no obvious phenotype (i.e. cAR3 null cells are still able to complete development and form fruiting bodies (Johnson et al., 1993)). In the slug, the expression of cAR3 becomes confined to the prespore cells (Yu and Saxe, 1996). cAR2 is first expressed at the mound stage where it is restricted to cells in the forming prestalk zone. The affinity of cAR2 is low and can hardly be measured by a standard cAMP binding assay. However, the measurement of the cAMP-dependent phosphorylation of the receptors cytoplasmic tail shows an EC50 of
50 µM, at least 1000-fold higher than the EC50 of cAR1 (Kim et al., 1998). Deletion of cAR2 arrests development at the mound stage (Saxe et al., 1993). At the slug stage the low affinity receptor cAR4 is expressed in a prestalk-specific manner. Deletion of cAR4 leads to defects during culmination (Louis et al., 1994).
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To dissect the influence of different receptors we have investigated the dynamics of darkfield wave initiation and propagation in mutants that overexpress either cAR1, affinity mutants of cAR1, cAR2, cAR3 or the cAR1/cAR2 chimera N272 in a cAR1/cAR3 double null background (RI9), which in itself is completely unresponsive to stimulation with cAMP. The cAMP sensitivity of the investigated receptors can be put in the following order: cAR1 (highest affinity)>cAR3>N272>IIIb21>cAR2 (lowest affinity) (Table 1). We have carefully quantified the optical density wave frequencies and propagation speeds and found that lower affinity receptors result in lower frequencies of wave initiation. Surprisingly, once initiated, wave speed was not strongly dependent on receptor affinity. In the mound stage the dependence of wave frequency on receptor affinity persisted, although it was less pronounced than during aggregation.
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MATERIALS AND METHODS |
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Videomicroscopy
Optical density waves during the early stages of aggregation were monitored using darkfield optics (Gross et al., 1976). A single plano-convex lens with a diameter of 10 cm (f=15 cm) was used, similar to a previous set-up (Siegert and Weijer, 1989). A CCD camera (JAI 1020) with a zoom objective (Cosmicar TV Zoom 12.5-75 mm) and a 2x magnification lens was used to acquire the images.
The signal waves in aggregation streams and mounds were observed on an Axiovert 25 microscope (Zeiss, objectives: CP-Achromat 10x/NA 0.25 Ph1 Var1, Plan-Neofluar 2.5x/NA 0.0075). The annular stop slider at the condenser was arrested in the middle position between brightfield and phase-contrast (Ph1) to create the oblique illumination that is essential for the detection of optical density waves in these stages (Siegert and Weijer, 1995). A JAI 2040 camera was attached to the camera port via a 0.5x video adapter (Zeiss).
The video signals were digitised with an IC-PCI-board (AM-CLR acquisition module, Imaging Technology Inc.). The control of the image capturing board, the recording of time-lapse sequences and the subsequent analysis were performed with the Optimas software (Optimas Corporation, Version 5.2).
Analysis of image sequences
Time-space plots were generated to measure velocity and period of optical density waves during both early aggregation and mound stage (Siegert and Weijer, 1989; Siegert and Weijer, 1995). Basically, the grey values along a fixed line were stored for all the images of a sequence. The position of this line was such that waves travelled perpendicular to it. Afterwards these lines were displayed beneath each other in their temporal order, thus generating a time axis in Y-direction. The wave velocity could be derived from the slope of the resulting wave bands, and the period derived by measuring the distance between consecutive waves in Y-direction.
A new method was developed to determine the velocity of dark-field waves in the mutants where waves were initiated only once at random positions. We basically calculated the distance that the wave travelled over a period of time by using image multiplication to superimpose two images taken some time apart (Russ, 1995). If the time interval between these images was chosen appropriately, distinct wave fronts appeared and the distance covered during this time interval could be measured to give an estimate of the speed of wave propagation. Image subtraction of pairs of successive images was routinely used to enhance the visibility of optical density waves especially in the mound stage to determine the exact wave geometrys (Siegert and Weijer, 1995). This method detects the differences between images, the propagating waves, while unchanged structures and background are effectively suppressed.
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RESULTS |
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We next investigated N272/RI9, this line expresses a chimeric cAR1/cAR2 receptor with an EC50 of around 1 µM cAMP (Table 1; Kim et al., 1998). This strain produced concentric waves, which again originated at different random positions (Fig. 1E). The centres fired only once from a given position as can be seen in the time-space plot (Fig. 1J). Since the cells did not generate signals coming from a fixed centre, they randomly moved up wave fronts coming from different directions and, as a result, could not aggregate. This behaviour is similar to that shown by the car1/RI9 cells during early development. However, in N272/RI9, fixed centres never appeared. Instead, the darkfield waves disappeared completely after 10.6±1.0 hours (n=6). The time-space-plot shows that this was a sudden process, which affected all the cells on the plate simultaneously (Fig. 1J), indicative of a precisely timed process. It seems most likely that the actin15 promoter loses its activity during later development and that the number of N272 receptors decreases below a critical level, insufficient to sustain further wave propagation. Further development was nearly arrested at this stage. In some cases a few aggregation centres per plate emerged after a delay of several hours. Most dispersed again after a number of hours but some persisted and managed to form tiny fruiting bodies.
The cAR2/RI9 mutant, overexpressing the low affinity receptor cAR2 didnt show any signs of darkfield waves at all and was unable to aggregate (data not shown). These cells were not sensitive enough to detect the small changes in the extracellular cAMP that normally initiate aggregation. Attempts to stimulate these cells with cAMP to induce mound formation failed. We conducted chemotaxis assays with a cAMP-filled glass micropipette placed in a field of aggregation-competent cells. At high cAMP concentrations (10-2-10-1 M cAMP) the cells were attracted by the electrode, as can be seen by the pile of cells that has accumulated around the tip of the glass electrode (Fig. 3A). At concentrations that elicit a positive response in DH1 or cAR1/RI9 (10-4 M cAMP), the cAR2/RI9 cells show no reaction towards the electrode (Fig. 3B). Synergy experiments of 5% fluorescence-labelled cAR2/RI9 cells with 95% unlabelled DH1 cells showed that a small number of cAR2/RI9 cells managed to co-aggregate with DH1, whereas labelled RI9 cells, which are completely unresponsive to cAMP, did not co-aggregate (data not shown). This clearly shows that the cAR2/RI9 cells can respond to signals secreted by the parent strain DH1, most likely cAMP, albeit not very efficiently.
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We had previously suggested that the formation of multi-armed spirals could be due to the expression of the low affinity receptor cAR2 at the mound stage (Vasiev, 1997). To test this possibility we examined darkfield wave propagation in mounds of cAR2 null mutants. The development of these cells is arrested or severely delayed at the mound stage (Saxe et al., 1993). In mounds of cAR2 null strains there were clear multi-armed spirals (Fig. 5A,C). The other receptor, whose expression starts at the early mound stage is cAR3. There were multi-armed spirals in the cAR3null strain as well (Fig. 5B,D). These observations clearly show that the sudden appearance of multi-armed spiral waves is not linked to the expression of either cAR2 or cAR3, as originally proposed. However, multi-armed spirals were less frequent in the cAR1/RI9 and cAR3/RI9 strains, suggesting that high levels of expression of a high affinity receptor can suppress multi-armed spiral formation. Since the number of cAR1 receptors decreases in wild-type cells after aggregation, this decrease in high affinity receptors could be responsible for lowering the excitability and the appearance of multi-armed spiral waves.
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cAMP receptor affinity does not determine wave propagation speed
We measured wave propagation speed and period during the aggregation stage and in mounds. The velocity of the darkfield waves seemed to be quite constant over time in the three mutants cAR1, cAR3 and N272 and even their average velocities were similar (cAR1 204±33 µm/minute; cAR3 231±39 µm/minute; N272 215±34 µm/minute). Thus wave propagation velocity is not determined by receptor affinity. Fig. 8A shows the distribution of the wave velocities. The data were grouped into classes with a width of 25 µm/minute and normalised to account for the different numbers of measurements per mutant. The values for the mutants peaked in the two classes ranging from 200 to 250 µm/minute. The parental strain DH1 showed a much broader distribution instead and the peak was shifted towards the higher values. This reflects the fact that the velocity of DH1 darkfield waves changed during the course of development. It decreased from maximum values of more than 500 µm/minute to around 150 µm/minute, resembling the Ax2 and Ax3 strains, where this process has been well documented (Rietdorf et al., 1996; Siegert and Weijer, 1989).
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The receptor subtypes have different affinities for cAMP, but there are also structural differences between them, which may affect their function. To further investigate the relationship between wave initiation frequency and affinity, we studied mutants that express receptors in which the affinity for cAMP had been changed by single amino acid substitutions. We investigated mutants IIb21, IIb22 and IIIb22 (Kim et al., 1997) and found that only IIIb21 produced darkfield waves (Fig. 9A). This receptor has been shown to have an affinity of around 10 µM, slightly higher than N272 (Table 1). This mutant initiated random waves and never fired from the same centre twice. Although the wave propagation speed is only marginally slower than that from the mutant N272, the average local wave period was at least double (Fig. 9C,D). The waves eventually disappeared, as in the case of N272 (Fig. 9B). Sometimes small cell accumulations formed that looked like early mounds, although we have never observed any OD waves in these structures. This strain never develops into fruiting bodies under our conditions. These results clearly show that the differences in wave parameters are primarily linked to the affinity of the cAMP receptor and not to small structural differences between the receptors.
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DISCUSSION |
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Remarkably, none of the mutants investigated could initiate waves with the same frequency as the parent strains Ax3 and DH1. Since cAR1 is the major receptor expressed during aggregation, one would expect that expression of the cAR1 receptor in the RI9 mutant would result in a phenotype which resembled that of the parent strain DH1. This is not the case, DH1 emits periodically concentric darkfield waves from aggregation centres at fixed locations (Fig. 1B,G), whereas cAR1/RI9 initiates waves at lower frequency in completely random locations during early aggregation, typical of the behaviour observed in the strains expressing the low affinity receptors. Expression under the control of the actin15 promoter results in at least fivefold overexpression of cAR1 during early aggregation, as determined by cAMP-binding studies (Table 1; Johnson et al., 1991). The observed reduction in wave initiation frequency suggests that overexpression of cAR1 could have a partial dominant negative effect on cAMP relay in vivo. This effect is not observed in vitro under saturating assay conditions where the cells are able to give normal amplitude responses (Kim et al., 1998). It therefore seems likely that the great number of receptors sequester limiting amounts of cAMP, without being able to transduce the signal efficiently to activate adenylylcyclase. Possibly not all receptors are coupled to intracellular signal transduction components such as G proteins during the early stages of aggregation, where these components may be limiting.
The cAR3/RI9 strain could set up stable aggregation centres emitting large spiral waves, which organised in large aggregation territories in agreement with earlier observations (Kim et al., 1998). From theoretical considerations it is known that spirals can persist in excitable media, whereas concentric waves need at least pacemaker cells in the centre (Durston, 1973). Since we never observed concentric waves during aggregation in cAR3/RI9, we consider it most likely that these cells are only excitable, in contrast to wild-type cells, which are in an oscillatory mode during aggregation. Although biochemical experiments have shown that the affinities of the cAR1 and cAR3 receptors are only slightly different, these in vivo experiments show that differences in receptor structure may lead to physiological significant changes in signal transduction efficiency not previously detected in biochemical experiments.
Influence of cAR affinity on centre formation
Our results show that there exists a close correlation between the frequency of wave initiation and the formation of stable aggregation centres. All the mutants that show a low frequency of wave initiation also have difficulty in forming stable signalling centres (Fig. 1E; Fig. 9B). During development, cells go from being non-excitable by cAMP at the vegetative stage, to being excitable during early aggregation, to being able to produce cAMP in an oscillatory manner (Gerisch, 1982; Lax, 1979). This evolution of the signalling system reflects the pulse-controlled increase in expression of essential components of the signalling system such as cAMP receptors, G2, cAMP phosphodiesterase and the aggregation-stage-specific adenylylcyclase (ACA) (Aubry and Firtel, 1999; Parent and Devreotes, 1996). Not all cells go through this developmental pathway at the same pace, resulting in considerable heterogeneity in the individual cells make-up of signal transduction components (Weeks and Weijer, 1994; Weijer et al., 1984a; Weijer et al., 1984b). Aggregation centres therefore most likely arise in random positions, where, by chance, a number of cells have come into close vicinity that secrete enough cAMP and are sensitive enough to cAMP to trigger autocatalytic cAMP production and initiate a cAMP wave. As the wave propagates outward, surrounding cells move towards the source of the signal, thus resulting in accumulation of cells at the site of wave initiation. Since more cells can produce more cAMP faster, this increases the probability to initiate a second cAMP wave from the same centre. The mutant strains that express the low affinity receptors take longer before they can raise the extracellular cAMP to levels where it comes into the regime of autocatalysis again. During this time the cells start to disperse again owing to their continuous random movement. This dispersal then results in a destabilisation of the centre and a new centre will be formed by chance somewhere else. Thus, centre stabilisation may require a fine balance between the period of the cAMP signals and its resulting chemotactic accumulation of cells in the centre and dispersal of the cells in the centre due to random movement.
The regulation of wave propagation speed during development
During normal development, wave velocity decreases from 800 µm/minute to 250 µm/minute (Rietdorf et al., 1996; Siegert and Weijer, 1989). This change in wave propagation speed was not observed in any of the cAR mutant strains, which always propagate waves at
250 µm/minute (Fig. 8A). During the early development of wild-type strains the first receptor to be expressed is cAR1. Initially, the number of cAR1 receptors is low but then their numbers rapidly increase since their expression is under the feedback control of the cAMP pulses. During this time the wave propagation speed decreases rapidly. The cAR mutants investigated in this study express cAR receptors at already higher numbers from the beginning of development (Table 1) and dont show this decrease in speed. This would suggest that high receptor numbers somehow result in lower wave propagation speed by an as yet unknown mechanism. In mounds, the wave propagation speed is low (60 µm/minute, Fig. 8B). The cells are now in tight contact and the extracellular space between the cells is small. We think it likely that the densities of cAR receptors and extracellular phosphodiesterase become so high that the cAMP produced by a given cell is only able to stimulate its immediate neighbours. Under these conditions wave propagation speed is set by the delay between a cell detecting cAMP at its front end and being able produce and secrete enough cAMP to stimulate the cell immediately behind it. This delay therefore should be less then 10 seconds to result in the wave propagation speeds observed.
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ACKNOWLEDGMENTS |
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