Abstract. Amebae of Dictyostelium exhibit a transient uptake of extracellular Ca 2+ rv5 s after activation of surface folate or cAMP receptors (Bumann, J., B. Wurster, and D. Malchow. 1984. J. Cell Biol. 98:173-178). To further characterize these Ca 2+ entry systems, we analyzed 45Ca2+ uptake by resting and activated amebae. Like the surface chemoreceptors, folate-and cAMP-induced Ca 2+ uptake responses were developmentally regulated; the former response was evident in vegetative but not aggregation-competent cells, whereas the latter response displayed the opposite pattern of expression. In contrast, other characteristics of these Ca2+-uptake pathways were remarkably similar. Both systems (a) exhibited comparable kinetic properties, (b) displayed a high specificity for Ca 2+, and (c) were inhibited effectively by Ruthenium Red, sodium azide, and carbonylcyanide m-chlorophenylhydrazone. These results, together with the finding that vegetative cells transformed with a plasmid expressing the surface cAMP receptor exhibit a cAMP-induced Ca 2+ uptake, suggest that different chemoreceptors activate a single Ca 2+ entry pathway. Additional pharmacological and ion competition studies indicated that receptor-mediated Ca 2+ entry probably does not involve a Na+/Ca 2+ exchanger or voltageactivated channels. Chemoattractant binding appears to generate intracellular signals that induce activation and adaption of the Ca2+-uptake response. Analysis of putative signaling mutants suggests that Ca 2÷ entry is not regulated by the guanine nucleotide-binding (G) protein subunits God or Got2, or by G protein-mediated changes in intracellular cAMP or guanosine 3,'5'-cyclic monophosphate (cGMP).I N many higher eukaryotic cells, hormones and neurotransmitters induce changes in cytosolic free Ca 2+ by promoting mobilization of sequestered intracellular Ca 2+ and/or by stimulating entry of extracellular Ca 2+ across the plasmalemma. Ca 2+ mobilization responses can be mediated by increases in cytosolic Ca 2+ (18) or by agonistinduced production of inositol 1,4,5-trisphosphate, which releases Ca 2+ from the ER (4) and other cellular compartments (27, 48). Enhanced uptake of extracellular Ca 2+ occurs following activation of voltage-regulated (40) or ligandgated (3) Ca 2+ channels, and likely serves a crucial role both in Ca 2+ signaling events and in replenishing agonist-depleted stores (for review, see reference 22). Importantly, many of these Ca2+-uptake systems, both voltage-dependent and voltage-independent, appear to be regulated by extracellular factors (for review, see reference 43). In addition, certain of these Ca 2+ channels appear to be modulated directly by guanine nucleotide-binding protein (G protein) t subunits while others are regulated indirectly by second messengers such as 1. Abbreviations used in this paper: CCCP, carbonylcyanide m-chlorophenylhydrazone; G protein, guanine nucleotide-binding protein.cAMP, guanosine 3',5'-cyclic monophosphate (cGMP), and inositol polyphosphates (43).In the lower eukaryote, Dictyostel...

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To find mutants of Dictyostelium discoideum that are unable to respond to exogenous cAMP signals (frigid mutants), amoebae of 2 18 independent aggregation-deficient mutants were treated in suspension with artificial pulses of cAMP and screened for the capacity to form EDTA-resistant cohesion sites. Eleven frigid mutants were identified and further characterized. Using parasexual genetic techniques, these strains were assigned to five complementation groups (fgdA-E) and the fgd loci were mapped in three linkage groups: fgdA and D in group 11, fgdC in group 111, andfgdB and E in group VII. Biochemical and physiological experiments with these strains indicated that fgd mutants are of two general types. When starved, strains in groups fgdB, D, and E failed to produce detectable levels of membrane-associated cAMP phosphodiesterase, surface cAMP receptors, or extracellular phosphodiesterase inhibitor, and the cells continued to respond chemotactically to folate. Thus, these strains are probably arrested in the vegetative phase or very early in development. In contrast, strains in groups fgdA and C produced low levels of cAMP receptors and secreted phosphodiesterase inhibitor. Moreover, after starvation, some of these mutants elicited a weak chemotactic response to CAMP. Therefore, unlike the former group of mutants, these strains appear to initiate development when starved, but the process is blocked at an early stage.

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Simple parasexual genetic techniques have been employed to extend the linkage analysis initiated in an earlier study (Coukell, 1975) of developmental mutations (agg mutations) in 40 independently isolated aggregation-deficient mutants of Dictyostelium discoideum. Using these techniques, agg mutations in 28 of the 40 mutants have been assigned to 4 linkage groups: 16 in group II, 1 in group III, 10 in group IV, and 1 in group VI. None of the agg mutations analyzed appear to map in linkage group I. In addition, a new temperature-sensitive growth locus, designated tsgJ, was mapped in group III. It was also found that diploid strains of D. discoideum are readily induced to undergo haploidization when grown on 0.1% p-fluorophenylalanine (PFP) at 25.5 degrees C. Growth of diploid strains on PFP had no effect on the type of segregant classes obtained (i.e. PFP does not induce mitotic crossing-over), the subsequent growth and/or development of the segregants, or the ability of the segregants to reform stable diploids.