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Dictyostelium Development in the Absence of cAMP

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Science  11 Jul 1997:
Vol. 277, Issue 5323, pp. 251-254
DOI: 10.1126/science.277.5323.251

Abstract

Adenosine 3′,5′-monophosphate (cAMP) and cAMP-dependent protein kinase (PKA) are regulators of development in many organisms.Dictyostelium uses cAMP as an extracellular chemoattractant and as an intracellular signal for differentiation. Cells that are mutant in adenylyl cyclase do not develop. Moderate expression of the catalytic subunit of PKA in adenylyl cyclase–null cells led to near-normal development without detectable accumulation of cAMP. These results suggest that all intracellular cAMP signaling is effected through PKA and that signals other than extracellular cAMP coordinate morphogenesis in Dictyostelium.

In both vertebrates and invertebrates, the control of cAMP synthesis and its detection by cellular targets such as PKA are essential for many developmental processes (1). During Dictyostelium development, cAMP is used as a signal both inside and outside of cells (2). Pulses of extracellular cAMP are generated by the cells and are used for chemotaxis during aggregation, a process that brings 105 cells into a mound. Extracellular cAMP is detected by G protein–coupled cell surface receptors (3). This results in the chemotaxis of cells toward increased cAMP as well as propagation of the signal through the activation of ACA, the adenylyl cyclase that produces cAMP during development (4). PKA, the major downstream effector of the cAMP-ACA signaling pathway inside the cell, is activated when its regulatory subunit (PKA-R) binds cAMP and dissociates from the catalytic subunit (PKA-C) (5). Cells in which PKA-R is inactivated (6) or in which PKA-C is constitutively active (7, 8) develop rapidly, which suggests that PKA regulates the timing of development.

After aggregation, extracellular cAMP has been proposed to direct a number of processes, including the sorting of cell types into distinct tissues, slug migration, terminal cell differentiation, and the morphogenesis of the final fruiting body (9-11). To test the role of extracellular cAMP in development, we obviated the requirement for cAMP as a second messenger by rendering PKA constitutively active. By doing this in an ACA-null mutant, we produced cells that do not make cAMP but have the key intracellular cAMP pathway activated. Because PKA activation promotes many developmental events (7, 8, 11, 12), these cells allowed an assessment of the processes that are independent of cAMP.

We introduced an expression plasmid with an epitope-tagged PKA-C coding region under the transcriptional control of anactin15 promoter into acaA mutant cells by transformation (13). The actin15 promoter directs relatively constant amounts of transcription throughout development in all cells (14). After the primary transformants were cloned on growth plates, cells that had exhausted the bacterial food supply within some of the colonies were clearly undergoing development. The epitope-tagged PKA-C protein was produced by these developing transformants [acaA(PKA-C) cells] but was not detected in transformants that did not develop (13). Analysis of the DNA and RNA of several isolates ofacaA(PKA-C) cells confirmed that the acaA locus remained disrupted and that acaA mRNA did not accumulate at any time during development (13). We also measured PKA-C activity in cells that had been developing for 8 hours, as measured by phosphorylation of the peptide substrate Kemptide (15). The acaA(PKA-C) cells exhibited 4.3 times the activity (1.4 nmol min−1 mg−1) of the parental acaA cells and 1.6 times the activity of wild-type cells.

When acaA(PKA-C) cells were axenically grown, washed, and placed on Millipore filters, they underwent relatively normal development. They began to aggregate by 10 hours, forming broad “streams” of cells that persisted for several hours, such that mound formation was delayed by about 2 hours (Fig.1A). The mounds produced by theacaA(PKA-C) cells progressed through a normal sequence of developmental structures and formed fruiting bodies by 30 hours (Fig.1, B and C).

Figure 1

Development of wild-type and PKA-C–rescuedacaA cells. (A) Typical fields ofacaA mutant, acaA(PKA-C), and wild-type cells, 12 hours after they were spread on a Millipore filter to initiate development (27). (B andC) Multicellular structures ofacaA(PKA-C) cells (B) and wild-type cells (C) are shown with the time of development above each panel. The development of the acaA(PKA-C) cells is delayed relative to the wild-type cells, so similar morphological stages are shown for comparison. Scale bars, 250 μm.

To determine whether constitutive PKA activity restored cAMP signaling in the acaA(PKA-C) cells, we measured adenylyl cyclase activity and cAMP production in several ways. After cells were pulsed with cAMP for 5 hours, acaA(PKA-C) cells had <0.4% of the adenylyl cyclase activity of wild-type cells (16). During development on filters, adenylyl cyclase activity in wild-type cells increased to a maximum at 8 hours and then declined, as previously reported (17). The activity in acaA(PKA-C) cells and acaA mutant cells never rose above the background of the assay (16). We also directly measured the accumulation of cAMP in intact aggregation-competent cells (18). When cells were stimulated with 2′-deoxy-cAMP, cAMP rapidly accumulated in wild-type cells but remained at background in theacaA and acaA(PKA-C) cells (Fig.2A). During the development of wild-type cells, steady-state amounts of cAMP were highest during aggregation, declined until the time of culmination, and then increased during fruiting body formation (Fig. 2B). In the acaA(PKA-C) cells, cAMP was below detection limits at all times tested (18). These measurements of adenylyl cyclase activity and cAMP production are consistent with previous findings that ACA accounts for all of the detectable adenylyl cyclase activity during development (4). Thus, the development of acaA(PKA-C) cells is not attributable to the ACA-independent synthesis of cAMP.

Figure 2

cAMP produced by cAMP-stimulated cells and by cells developing on filters. (A) Production of cAMP from wild-type cells (solid circles), acaA mutant cells (open squares), or acaA(PKA-C) cells (solid squares) after stimulation with 2′-deoxy-cAMP (18). Means and SEs are shown for three experiments performed in triplicate. Absence of error bars indicates that the error was smaller than the symbol. (B) Total concentration of cAMP present at various times for wild-type cells (solid circles) oracaA(PKA-C) cells (solid squares) developing on filters (18). One representative experiment is shown.

Pulsatile cAMP signaling and chemotaxis is thought to be required for the recruitment of cells from a distance, which enables properly sized aggregates to form irrespective of the initial cell density (19). Because acaA(PKA-C) cells aggregate without producing detectable cAMP, we examined the cell density dependence of their aggregation. The acaA(PKA-C) cells aggregated at the cell density that is normally used for development on an agar surface (Table 1, second highest density). The aggregates were of normal size, and most went on to form fruiting bodies. However, at cell densities of 5.5 × 105cells/cm2 and lower, the acaA(PKA-C) cells failed to aggregate, whereas the wild-type cells formed aggregates of normal size and roughly in proportion to the total number of cells in the developing field (Table 1). These results directly confirm the requirement of cAMP signaling for the production of properly sized aggregates by cells at low density. Certain mutant cells have been shown to aggregate without pulsatile cAMP signaling in the presence of high, constant amounts of cAMP (4) or nonhydrolyzable cAMP derivatives (20). Our results show that cells at high density are able to aggregate without cAMP as long as PKA is active.

Table 1

Cell density dependence of aggregation. Means (±SD) of the total number of aggregates observed at 14 hours are shown at each cell density for three spots of cells developing on the same buffered-agar plate (29). Data from a typical experiment are shown. Most of the aggregates in every sample continued through development and formed fruiting bodies.

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Within aggregates, randomly distributed prestalk cells move to the apex, where they form a tip that can lead prespore cells on extended migrations as slugs (21). It has been assumed that these cell movements result from chemotactic responses to extracellular cAMP (9); hence, we tested whether acaA(PKA-C) cells formed prestalk regions and migrating slugs. Neutral red preferentially stains prestalk cells and is not transferred between cells (22). As observed by neutral red staining, prestalk cells sorted to the apex of acaA(PKA-C) cell aggregates in a manner indistinguishable from that of wild-type cells (23). These aggregates formed slugs with normal proportions of prestalk cells in their tips, and they migrated at the normal rate (23). These results rule out any essential role for extracellular cAMP in cell sorting or slug migration.

We also examined several molecular and cellular markers of development. The expression of pdsA was restored in theacaA(PKA-C) cells; however, a delay in the transition from the 1.9-kb transcript to the 2.4-kb transcript was evident (24) (Fig. 3A). Expression of the carA gene in acaA(PKA-C) cells appeared normal. The acaA gene was expressed during aggregation of the wild-type cells starting at 6 hours and, as expected, was not expressed in either of the acaA mutants. The expression patterns of the cell type–specific genes cotA,ecmA, and spiA in acaA(PKA-C) cells were remarkably similar to those of wild-type cells (Fig. 3B). The correct temporal regulation of cotA and ecmA in the acaA(PKA-C) cells indicates that specific temporal or spatial regulation of PKA is not required for the timing of cell type–specific gene expression. Later in development, the onset of expression of the sporulation-specific gene spiA was delayed by about 4 hours in the acaA(PKA-C) cells, consistent with the delay in the completion of development in these cells (Fig. 3B). The acaA(PKA-C) cells produced the same number of spores per fruiting body as were produced by wild-type cells (25), and the acaA(PKA-C) spores were viable, as judged by their ability to germinate and form colonies on bacterial growth plates (26). The formation by acaA(PKA-C) cells of a stalk capable of carrying a normal-size sorus above the substratum (Fig. 1B) signified proper stalk cell production. Thus, because PKA-C activation overcomes the requirement for cAMP signaling after aggregation, all responses to extracellular cAMP later in development must be mediated by PKA.

Figure 3

Gene expression in the PKA-C–rescuedacaA cells. Northern blots of mRNA purified from cells at the indicated times of development on filters are shown after hybridization with several gene probes and autoradiography (13). (A) Expression of aggregation-stage genes. carA encodes the major aggregation-stage cAMP receptor cAR1 (3), pdsA encodes an extracellular phosphodiesterase (30), andacaA encodes the adenylyl cyclase ACA (4), which is disrupted in the acaAmutant and acaA(PKA-C) cells. (B) Expression of cell type–specific genes.cotA is a prespore-specific gene (31),ecmA is a prestalk-specific gene (32), andspiA is a sporulation-specific gene (33). In this experiment, the wild-type cells andacaA(PKA-C) cells formed fruiting bodies by 26 and 30 hours, respectively, and the acaA mutant cells showed no visible signs of development.

How can our results be rationalized, given that work over the past 30 years has shown that extracellular cAMP is required for development? It is unlikely that adventitious regulatory mechanisms are induced by PKA-C overexpression, substitute for cAMP signaling, and recapitulate the wild-type developmental program. Instead, theacaA(PKA-C) cells reveal that other signaling systems can coordinate development in the absence of extracellular cAMP. These other signaling systems may provide essential regulation that is only observed to depend on cAMP-mediated temporal control of events in most experimental tests. Perhaps extracellular cAMP signals, transduced through PKA, are required to initiate events at critical points in development, gating the tempo of the cAMP-independent regulatory events. In this view, activation of PKA-C would have the effect of relieving a series of rate-limiting steps within a complex regulatory network, the downstream components of which remain largely unexplored.

  • * To whom correspondence should be addressed. E-mail: akuspa{at}bcm.tmc.edu

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