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.     

Antagonists of chemoattractants reveal separate receptors for cAMP, folk acid and pterin in Dictyostelium

Adenosine 3′,5′-monophosphate (cAMP), folic acid and pterin are chemoattractants in the cellular slime molds. The cAMP analog, 3′-amino-cAMP, inhibits a chemotactic reaction to cAMP at a concentration at which the analog is chemotactically inactive. The antagonistic effect of 3′-amino-cAMP on the chemotactic activity of cAMP is competitive, which suggests that 3′-amino-cAMP antagonizes cAMP via the chemotactic receptor for cAMP. 3′-Amino-cAMP does not antagonize folic acid or pterin. The binding of folic acid to post-vegetative Dictyostelium discoideum cells is inhibited by low concentrations of 2-deamino-2-hydro folic acid (DAFA [7]). DAFA is neither chemotactically active, nor does it inhibit a chemotactic reaction to folic acid. This questions the involvement of the main folic acid cell surface-binding sites in the chemotactic response to folic acid. The pterin analog, 6-aminopterin, is an antagonist of pterin, but not of cAMP or folic acid. Our results show that cAMP, folic acid and pterin are detected by different receptors. Furthermore, they suggest that the antagonistic action of 3′-amino-cAMP and 6-aminopterin is localized in the signal transduction pathway at a step before the signals from the separate receptors have arrived at a single pathway.     

Gene regulation during dedifferentiation in Dictyostelium discoideum

During development of Dictyostelium discoideum, cells acquire the capacity to rapidly recapitulate morphogenesis. Therefore, when cells at the loose aggregate stage are disaggregated and challenged to reaggregate, they do so in a tenth of the original time. If loose aggregate cells are disaggregated and resuspended in buffered dextrose solution (erasure medium), they retain the capacity of rapid recapitulation for 80 min, then completely lose this capacity in a single, synchronous step referred to as the "erasure event." The erasure event sets in motion a program of dedifferentiation during which cells lose developmentally acquired characteristics at different times. The erasure event is inhibited by the addition of 10(-4) M cAMP to erasure medium. The synthesis of 33 growth-associated polypeptides, the synthesis of 53 development-associated polypeptides, and the level of 2 development-associated RNAs have been monitored during the erasure program and in cultures inhibited from erasing by the addition of 10(-4) M cAMP. Growth-associated polypeptides begin to be resynthesized and development-associated polypeptides exhibit dramatic decreases in rate of synthesis at different times throughout the first 240 min in erasure medium. Inhibiting the erasure event with cAMP has no major effect in the resynthesis of the majority of growth-associated polypeptides. Only one growth-associated polypeptide, V28, is completely inhibited by cAMP, suggesting that it may play a role in the erasure process. In contrast, inhibiting the erasure event with cAMP has a marked effect on the synthesis of development-associated polypeptides, causing a dramatic reduction in the rate at which synthesis decreases for 6 polypeptides, and completely inhibits the decrease in the synthetic rate of 8 polypeptides. The two development-associated RNAs, 16G1 and 10C3, exhibit two distinctly different patterns of loss during erasure, but in both cases cAMP added at time zero of the erasure process dramatically retards or inhibits loss. In addition, when cAMP is added just prior to the erasure event, it inhibits the erasure event and stimulates a rapid increase in the level of 16G1 RNA back to the developmental level. The level of 16G1 RNA then remains at this level for at least 400 min. When cAMP is added after the erasure event, it causes a low, transient increase in the level of 16G1 RNA. These results are considered both in relation to the program of erasure, and in relation to the role of cAMP in the expression of developmental genes during the forward program of development.     

Differentiation and dedifferentiation can function simultaneously and independently in the same cells in Dictyostelium discoideum

When developing cultures of Dictyostelium discoideum are disaggregated and morphogenesis is reinitiated, cells recapitulate the stages they had progressed through prior to disaggregation in a fraction of the original time. If developing cultures are disaggregated and the cells resuspended in nutrient medium, they retain this capacity for 1.5 hr and then synchronously and rapidly revert to the slow timing of log phase cells. Loss of the capacity to recapitulate morphogenesis rapidly is referred to as the “erasure event.” Following the erasure event, cells systematically lose developmentally acquired functions in a defined temporal sequence of dedifferentiation. Cells which have just passed through the erasure event can be stimulated to reenter the developmental program, even though they still possess several aggregation-associated functions acquired during the initial developmental program. In this report, we have tested whether cells stimulated to reenter the developmental program immediately after the erasure event progress along the same rate-limiting pathway leading to aggregation as they did during initial development and whether this rate-limiting pathway can run simultaneously with and independently of the sequence of dedifferentiation. Results are presented which demonstrate (1) that the erasure event resets the rate-limiting pathway for development back to zero and that erased cells reentering development progress along the same rate-limiting pathway as naive log phase cells, (2) that the loss of an aggregation-associated function late in the sequence of dedifferentiation is completely blocked by the addition of cycloheximide, but not cAMP, just prior to the expected time of loss, and (3) that differentiation and dedifferentiation can function simultaneously and independently in the same cells, even though the former leads to the acquisition and the latter to the loss of the same aggregation-associated functions (in this case EDTA-resistant adhesion and cAMP-stimulated motility).     

Relationship between adaptation of the folic acid and the cAMP mediated cGMP response in Dictyostelium

Chemotactic stimulation of post-vegetative Dictyostelium cells with folic acid or aggregative cells with cAMP results in a fast transient cGMP response which peaks at 10 s; basal levels are recovered in about 30–40 s. Stimulation with folic acid or cAMP rapidly desensitizes the cells for equal or lower concentrated stimuli. However, cells remain responsive for stimuli with higher concentration, which indicates that desensitization is caused by an adaptation process. Removal of the stimulus induces deadaptation, which for both cAMP and folic acid has first order kinetics with a half-life of 1.5 min.Cells were prepared which are simultaneously sensitive to folic acid and to cAMP. The cGMP responses to saturated folic acid and cAMP stimuli are not additive, which suggests that the transduction pathways of these signals meet each other at or before the guanylate cyclase. Cells which are adapted to folic acid are not adapted to cAMP and vice versa. This demonstrates that adaptation of Dictyostelium cells to chemotactic stimuli is localized at a step in the transduction chain before the transduced folic acid and cAMP signals combine in one pathway.     

Cyclic AMP inhibits dedifferentiation in the cellular slime mold Dictyostelium discoideum

When aggregating amoebas of the cellular slime mold Dictyostelium discoideum are disaggregated and morphogenesis is reinitiated, the amoebas will reaggregate in less than $\text{1}\text{10}\text{th}$ the original time. When aggregating amoebas are disaggregated and resuspended either in full nutrient medium or in buffered salts solution containing dextrose, they retain this developmentally acquired capacity to rapidly reaggregate for approximately 1 hr and then lose it completely in a synchronous and discrete step which we have referred to as the “erasure event.” In this report, it is demonstrated that micromolar concentrations of cAMP completely block this transition from the developmental to vegetative state, and that other cyclic nucleotides also inhibit it, but they do so at 20-fold higher concentrations. Neither the hydrolysis products of cAMP nor the vegetative chemoattractant folic acid inhibit dedifferentiation at concentrations as high as 10^?3M, demonstrating a specificity for cyclic nucleotides and cAMP in particular. The addition of cAMP at any time during the lag period preceding the erasure event inhibits it and addition immediately after the erasure event reverses it. Since cAMP may inhibit the transition from the developmental to vegetative state intracellularly or extracellularly, we have also examined the intracellular concentration of cAMP and the levels of cAMP binding sites on the cell surface during the erasure process. Evidence is presented that the majority of cAMP binding sites on the cell surface are not necessary for the inhibition of erasure by cAMP. The results of these latter studies are discussed in terms of alternative models for the involvement of cAMP in the transition from the developing to vegetative state.     

The developmental regulation of single-cell motility in Dictyostelium discoideum

The velocity of single amebae in the absence of a chemotactic signal has been analyzed during growth, development, rapid recapitulation, and dedifferentiation in the cellular slime mold Dictyostelium discoideum. It is demonstrated that (1) the velocity of axenically grown cells in half that of bacterially grown cells, (2) the velocity of bacterially grown cells decreased to roughly the same low level as axenically grown cells approximately 5 hr after the removal of exogeneous bacteria, (3) the velocity remains low for a 7-hr period preceding the onset of aggregation in both axenically and bacterially grown cells, (4) the velocity increases transiently at the onset of aggregation for both axenically and bacterially grown cells, (5) the velocity decreases to a very low level after the formation of loose aggregates and remains at that level at least through the early culminate I stage, (6) the velocity is not stimulated in 13-hr developing cells (finger stage) by inducing rapid recapitulation, (7) the velocity decreases after the erasure event in cultures of 7-hr developing cells (ripple stage) stimulated to undergo dedifferentiation, but the inhibition of the erasure event by the addition of 10(-4) M cAMP does not block this decrease. These results demonstrate that the basal level of single-cell motility in growing cultures is significantly influenced by the nutrient composition of the supporting medium, and that the transient increase in single-cell motility at the onset of aggregation is under the rigid control of the initial developmental program. Both rapid recapitulation and the program of dedifferentiation appear to have no influence on the basal level of single-cell motility.     

Transduction of the chemotactic signal to the actin cytoskeleton of Dictyostelium discoideum

Dictyostelium discoideum amebae chemotax toward folate during vegetative growth and toward extracellular cAMP during the aggregation phase that follows starvation. Stimulation of starving amebae with extracellular cAMP leads to both actin polymerization and pseudopod extension (Hall et al., 1988, J. Cell. Biochem. 37, 285-299). We have identified an actin nucleation activity (NA) from starving amebae that is regulated by cAMP receptors and controls actin polymerization (Hall et al., 1989, J. Cell Biol., in press). We show here that NA from vegetative cells is also regulated by chemotactic receptors for folate. Our studies indicate that NA is an essential effector in control of the actin cytoskeleton by chemotactic receptors. Guided by a recently proposed model for signal transduction from the cAMP receptor (Snaar-Jagalska et al., 1988, Dev. Genet. 9, 215-225), we investigated which of three signaling pathways activates the NA effector. Treatment of whole cells with a commercial pertussis toxin preparation (PT) inhibited cAMP-stimulated NA. However, endotoxin contamination of the PT appears to account for this effect. The synag7 mutation and caffeine treatment do not inhibit activation of NA by cAMP. Thus, neither activation of adenylate cyclase nor a G protein sensitive to PT treatment of whole cells is necessary for the NA response. Actin nucleation activity stimulated with folate is normal in vegetative fgdA cells. However, cAMP suppresses rather than activates NA in starving fgdA cells. This indicates that the components of the actin nucleation effector are present and that a pathway regulating the inhibitor(s) of nucleation remains functional in starving fgdA cells. The locus of the fgdA defect, a G protein implicated in phospholipase C activation, is directly or indirectly responsible for transduction of the stimulatory chemotactic signal from cAMP receptors to the nucleation effector in Dictyostelium.     

A dedifferentiation-defective mutant of Dictyostelium that retains the capacity to aggregate in the absence of chemotaxis

During slime mold development, cells acquire the capacity to rapidly recapitulate morphogenesis in roughly a tenth the original time. When developing cells are disaggregated and refed, they completely loss this capacity in a rapid and synchronous step referred to as the “erasure event.” The erasure event sets in motion a program of dedifferentiation during which developmentally acquired functions are lost at different times. In this report, we describe the phenotype of HI4, which is a mutant partially defective in the dedifferentiation program but normal in all aspects of growth, morphogenesis, and rapid recapitulation. HI4 cells progress through the erasure event, losing in a relatively normal fashion (I) the capacity to rapidly recapitulate later stages of morphogenesis, (2) the capacity to release a cAMP signal, and (3) the capacity to respond chemotactically to a cAMP signal. However, erased HI4 cells abnormally retain the capacity to rapidly reaggregate, even though they have lost chemotactic functions. Erased HI4 cells also abnormally retain EDTA-resistant cohesion (contact sites A) and the surface glycoprotein gp80. It appears that erased HI4 cells rapidly reaggregate owing to random collisions followed by tight cell cohesion.     

Non-chemotactic Dictyostelium discoideum mutants with altered cGMP signal transduction

Folic acid and cAMP are chemoattractants in Dictyostelium discoideum, which bind to different surface receptors. The signal is transduced from the receptors via different G proteins into a common pathway which includes guanylyl cyclase and acto-myosin. To investigate this common pathway, ten mutants which do not react chemotactically to both cAMP and folic acid were isolated with a simple new chemotactic assay. Genetic analysis shows that one of these mutants (KI-10) was dominant; the other nine mutants were recessive, and comprise nine complementation groups. In wild-type cells, the chemoattractants activate adenylyl cyclase, phospholipase C, and guanylyl cyclase in a transient manner. In mutant cells the formation of cAMP and IP3 were generally normal, whereas the cGMP response was altered in most of the ten mutants. Particularly, mutant KI-8 has strongly reduced basal guanylyl cyclase activity; the enzyme is present in mutant KI-10, but can not be activated by cAMP or folic acid. The cGMP response of five other mutants is altered in either magnitude, dose dependency, or kinetics. These observations suggest that the second messenger cGMP plays a key role in chemotaxis in Dictyostelium.     

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.     

Guanylate Cyclase Activity in Dictyostelium discoideum and Its Increase during Cell Development

Following consumption of the food supply, cells of the cellular slime mould Dictyostelium discoideum aggregate and form a multicellular organism. The mechanism for cell aggregation is chemotaxis. The chemotactic signal in D. discoideum is released periodically from aggregation centers and propagated from cell to cell. cAMP mediates cell aggregation by acting as chemotactic attractant and as propagator of the signal. cAMP signals are measured by cell-surface receptors. Recent evidence indicates a role for cGMP during cAMP-mediated cell aggregation in D. discoideum .
During cell differentiation to aggregation competence, cAMP binding sites appear at the cell surface, and the activity of the enzymes adenylate cyclase and phosphodiesterase increases several-fold. In the present work we investigate the synthesis of cGMP in D. discoideum . Conditions for the assay of guanylate cyclase in cell homogenates are described. Guanylate cyclase activity was followed during cell differentiation to aggregation competence and found to increase fourfold. These results indicate that cGMP is involved in cell differentiation of D. discoideum . In contrast to adenylate cyclase, which is activated by cAMP, guanylate cyclase was under our conditions activated neither by cAMP, nor by folic acid.     

Cell motility and chemotaxis in Dictyostelium amebae lacking myosin heavy chain

Dictyostelium amebae have been engineered by homologous recombination of a truncated copy of the myosin heavy chain gene (heavy meromyosin (HMM) cells) and by transformation with a vector encoding an antisense RNA to myosin heavy chain mRNA (mhcA cells) so that they lack native myosin heavy chain protein. In the former case, cells synthesize only the heavy meromyosin portion of the protein and in the latter case they synthesize negligible amounts of the protein. Surprisingly, it was demonstrated that both cell lines are viable and motile. In order to compare the motility of these cells with normal cells, the newly developed computer-assisted Dynamic Morphology System (DMS) was employed. The results demonstrate that the average HMM or mhcA ameba moves at a rate of translocation less than half that of normal cells. It is rounder and less polar than a normal cell, and exhibits a rate of cytoplasmic expansion and contraction roughly half that of normal cells. In a spatial gradient of cAMP, the average ameba of HMM or mhcA exhibits a chemotactic index of +0.10 or less, compared to the chemotactic index of +0.50 exhibited by normal cells. Finally, the initial area, rate of expansion, and final area of pseudopods are roughly half that of normal cells. The five fastest HMM amebae (out of 35 analyzed in detail) moved at an average rate of translocation equal to that of normal amebae, and exhibited an average chemotactic index of +0.34. In addition, the average rate of cytoplasmic flow in fast HMM cells was equal to that of the average normal ameba. However, fast HMM amebae still exhibited the same defects in pseudopod formation that were exhibited by the entire HMM cell population. These results suggest that myosin heavy chain is involved in the "fine tuning" and efficiency of pseudopod formation, but is not essential for the basic behavior of pseudopod expansion.     

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 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.     

Chemotactic response of wild-type and aggregation-defective mutants of Polysphondylium violaceum

Abstract. The aggregation-specific chemoattractant for Polysphondylium violaceum is N-propionyl-γ-L-glutamyl-L-ornithine-δ-lactam ethyl ester, or glorin. Wild-type amoebae allowed to develop in liquid culture acquire increased ability to respond to glorin shortly after starvation, i.e., just prior to the time they become aggregation competent. Similarly, as development proceeds, the amoebae show decreased sensitivity to folic acid, but they show almost no response to cyclic AMP at any time during their development in liquid culture. The optimum concentrations for the chemotactic response are 10^-8 M for glorin and 10^-5–10^-6 M for folic acid. A class of aggregation-defective mutants, aggA , will not aggregate in the absence of an excreted pheromone, D factor. During development in liquid culture in the presence or absence of D factor, these aggA mutants show a chemotactic response similar to that of wild-type amoebae to folic acid and glorin. However, D factor does enhance the chemotactic response of aggA mutants to glorin. In the absence of D factor, mutant amoebae will form fruiting bodies if exposed to a chemotactic gradient of either folic acid or glorin. Under these conditions, the mutant amoebae circumvent the requirement for D factor in order to develop.     

Activated Galpha subunits can inhibit multiple signal transduction pathways during Dictyostelium development.

Mutations impairing the GTPase activity of G protein Galpha subunits can result in activated Galpha subunits that affect signal transduction and cellular responses and, in some cases, promote tumor formation. An analogous mutation in the Dictyostelium Galpha4 subunit gene (Q200L substitution) was constructed and found to inhibit Galpha4-mediated responses to folic acid, including the accumulation of cyclic nucleotides and chemotactic cell movement. The Galpha4-Q200L subunit also severely inhibited responses to cAMP, including cyclic nucleotide accumulation, cAMP chemotaxis, and cellular aggregation. An analogous mutation in the Galpha2 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 Galpha5 subunit with the analogous alteration (Q199L substitution). All aggregation-defective Galpha mutants were capable of multicellular development after a temporary incubation at 4 degrees C and this development was found to be dependent on wild-type Galpha4 function. This study indicates that mutant Galpha subunits can inhibit signal transduction pathways mediated by other Galpha subunits.     

Ammonia differentially suppresses the cAMP chemotaxis of anterior-like cells and prestalk cells indictyostelium discoideum

A drop assay for chemotaxis to cAMP confirms that both anterior-like cells (ALC) and prestalk cells (pst cells) respond to cAMP gradients. We present evidence that the chemotactic response of both ALC and pst cells is suppressed by ammonia, but a higher concentration of ammonia is required to suppress the response in pst cells. ALC show a chemotactic response to cAMP when moving on a substratum of prespore cells in isolated slug posteriors incubated under oxygen. ALC chemotaxis on a prespore cell substratum is suppressed by the same concentration of ammonia that suppresses ALC chemotaxis on the agar substratum in drop assays. Chemotaxis suppression is mediated by the unprotonated (NH₃) species of ammonia. The observed suppression, by ammonia, of ALC chemotaxis to cAMP supports our earlier hypothesis that ammonia is the tip-produced suppressor of such chemotaxis. We discuss implications of ammonia sensitivity of pst cells and ALC with regard to the movement and localization of ALC and pst cells in the slug and to the roles played by ALC in fruiting body formation. In addition, we suggest that a progressive decrease in sensitivity to ammonia is an important part of the maturation of ALC into pst cells.     

Loss and resynthesis of a developmentally regulated membrane protein (gp80) during dedifferentiation and redifferentiation in Dictyostelium

When developing cultures of Dictyostelium discoideum are disaggregated and resuspended in nutrient medium, they lose the capacity to rapidly reaggregate after 90 min, in a rapid and synchronous step referred to as the "erasure event." They then proceed to lose remaining developmentally acquired functions in a program of dedifferentiation culuminating with the loss of EDTA-resistant cohesion roughly 5 hr later. Immediately following the erasure event, cells can be stimulated to reenter the developmental program even though they still possess a number of developmentally acquired functions. These cells therefore appear to undergo dedifferentiation and redifferentiation simultaneously (D. R. Soll and L. H. Mitchell, 1982, Dev. Biol. 91, 183-190). In this report, we have employed an antiserum made against a developmentally acquired membrane glycoprotein, gp80, to examine whether gp80 is lost during dedifferentiation and whether it is either reutilized or resynthesized during redifferentiation. Results are presented which demonstrate that (1) when 9-hr developing cells are disaggregated and resuspended in nutrient medium, gp80 continues to accumulate for several hours after the erasure event, then is lost at roughly the same time as EDTA-resistant cohesion; (2) when cells are stimulated to reenter the developmental program immediately after the erasure event, both gp80 and EDTA-resistant cohesion are still lost according to the program of dedifferentiation, but are then reacquired soon afterwards according to the program of redifferentiation; (3) during redifferentiation, cells do not reutilize gp80 which had been synthesized during initial development; rather they synthesize gp80 de novo; and (4) developing cells of a dedifferentiation-defective variant, HI4, when disaggregated and resuspended in nutrient medium, retain gp80, EDTA-resistant cohesion, and the capacity to rapidly reinitiate aggregation for at least 12 hr. This last result indicates that the loss of gp80 is regulated by the dedifferentiation process and is not an independent response to disaggregation or the reintroduction of nutrients. Together, these results reinforce the conclusion that dedifferentiation and redifferentiation can function independently and simultaneously in the same cells.