Regulatory role of the G alpha 1 subunit in controlling cellular morphogenesis in Dictyostelium.
To determine the function of the Dictyostelium G alpha 1 subunit during aggregation and multicellular development, we analyzed the phenotypes of g alpha 1 null cells and strains overexpressing either wild-type G alpha 1 or two putative constitutively active mutations of G alpha 1. Strains overexpressing the wild-type or mutant G alpha 1 proteins showed very abnormal culmination with an aberrant stalk differentiation. The similarity of the phenotypes between G alpha 1 overexpression and expression of a putative constitutively active G alpha 1 subunit suggests that these phenotypes are due to increased G alpha 1 activity rather than resulting from a non-specific interference of other pathways. In contrast, g alpha 1 null strains showed normal morphogenesis except that the stalks were thinner and longer than those of wild-type culminants. Analysis of cell-type-specific gene expression using lacZ reporter constructs indicated that strains overexpressing G alpha 1 show a loss of ecmB expression in the central core of anterior prestalk AB cells. However, expression of ecmB in anterior-like cells and the expression of prestalk A-specific gene ecmA and the prespore-specific gene SP60/cotC appeared normal. Using a G alpha 1/lacZ reporter construct, we show that G alpha 1 expression is cell-type-specific during the multicellular stages, with a pattern of expression similar to ecmB, being preferentially expressed in the anterior prestalk AB cells and anterior-like cells. The developmental and molecular phenotypes of G alpha 1 overexpression and the cell-type-specific expression of G alpha 1 suggest that G alpha 1-mediated signaling pathways play an essential role in regulating multicellular development by controlling prestalk morphogenesis, possibly by acting as a negative regulator of prestalk AB cell differentiation. During the aggregation phase of development, g alpha 1 null cells display a delayed peak in cAMP-stimulated accumulation of cGMP compared to wild-type cells, while G alpha 1 overexpressors and dominant activating mutants show parallel kinetics of activation but decreased levels of cGMP accumulation compared to that seen in wild-type cells. These data suggest that G alpha 1 plays a role in the regulation of the activation and/or adaptation of the guanylyl cyclase pathway. In contrast, the activation of adenylyl cyclase, another pathway activated by cAMP stimulation, was unaffected in g alpha 1 null cells and cell lines overexpressing wild-type G alpha 1 or the G alpha 1 (Q206L) putative dominant activating mutation.(ABSTRACT TRUNCATED AT 400 WORDS)
Dharmawardhane
S
S
Department of Biology, University of California, San Diego, La Jolla 92093-0634.
Cubitt
A B
AB
Clark
A M
AM
Firtel
R A
RA
D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D004023:Dictyostelium / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics / Q000502:physiology*; D015870:Gene Expression; D017125:Genes, Protozoan; D009024:Morphogenesis; D010641:Phenotype; D011233:Precipitin Tests; D015398:Signal Transduction
D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D006152:Cyclic GMP