Dictyostelium cells express a G-protein-coupled adenylyl cyclase, ACA, during aggregation and an atypical adenylyl cyclase, ACG, in mature spores. The ACG gene was disrupted by homologous recombination. acg- cells developed into normal fruiting bodies with viable spores, but spore germination was no longer inhibited by high osmolarity, a fairly universal constraint for spore and seed germination. ACG activity, measured in aca-/ACG cells, was strongly stimulated by high osmolarity with optimal stimulation occurring at 200 milliosmolar. RdeC mutants, which display unrestrained protein kinase A (PKA) activity and a cell line, which overexpresses PKA under a prespore specific promoter, germinate very poorly, both at high and low osmolarity. These data indicate that ACG is an osmosensor controlling spore germination through activation of protein kinase A.

Cellular communication dictates all stages of growth and development in the cellular slime molds. Dictyostelium discoideum utilizes a number of signal molecules for cell-to-cell communication, including growth and density factors, cAMP, ammonia, differentiation-inducing factor, discadenine, and spore autoactivator. A source and sink model is presented in which the assimilation of ammonia plays a major role in determining cell fate and pattern formation. This model emphasizes a recycling of ammonia by prespore cells, the accumulation of free hydrophilic and neutral amino acids, and their incorporation into proteins associated with sporulation and (or) germination. If spore cAMP signalling is regulated by the relative concentrations of discadenine and autoactivator, and its disruption triggers the initiation of the spore germination cascade, then the accumulation of intracellular cAMP may be necessary for both sporulation and dormancy maintenance.

Signalling mechanisms involving CAMP have a well-documented role in the coordination of multicellular development and differentiation leading to spore formation in the social amoeba, Dictyostelium discoideum. The involvement of CAMP in the poorly understood developmental stages of spore dormancy and germination have been investigated in this study. Dormant spores contained up to l l -f o l d more cAMP than nascent amoebae. The spore CAMP levels were not constant, but typically underwent a surge at 14-18 d when spores acquired the ability to germinate spontaneously. The high CAMP levels decreased only during successful spore germination, i.e. emergence of nascent amoebae. The temporal pattern of CAMP decrease was complex and unique to the method of spore activation, supporting our hypothesis that exogenously (e.g. heat) activated and autoactivated spores germinate by different mechanisms. During heat-induced activation, transcription of acg (a gene encoding adenylyl cyclase associated with germination) correlated well with spore CAMP content. Young wild-type spores, incapable of spontaneous germination, maintained a uniformly high CAMP level, and spore CAMP levels also remained high if germination was inhibited. When activated spores were deactivated by applying increased osmotic pressure, CAMP concentrations rose and ultimately levelled off a t the high levels typical of dormant spores. The correlation between high CAMP and failure to germinate was also evident when autoactivation was inhibited by the CAMP analogue, 8-bromo-CAMP. Also, spores from a strain (HTY217) with unrestrained protein kinase A activity were incapable of spontaneous germination. Overall, our experiments provide evidence for continued CAMP signalling in spores up to 18 d after sporulation and for linkages between elevated CAMP, spore deactivation and inhibition of spontaneous germination.

RasG protein levels in dormant and germinating spores of Dictyostelium discoideum strains JC1 and SG1 were estimated by Western blotting. RasG levels were very low in dormant spores and remained low during the lag period, regardless of whether spores were heat activated or treated with autoactivator during the early stages of spore germination. RasG levels increased late during spore swelling just prior to the emergence stage of germination. These data are consistent with a requirement for RasG during vegetative growth.