Imaging Promoter Assay of Adenylyl Cyclase A Gene in Dictyostelium discoideum during Fruiting Body Formation by Dual-Color Bioluminescence Microscopy

Cyclic adenosine monophosphate (cAMP), which is derived from adenosine triphosphate through adenylyl cyclase A (acaA), acts as an intracellular secondary messenger and an extracellular chemotactic substance in important biological processes. In the social amoebae Dictyostelium discoideum, cAMP mediates cell aggregation, development, and differentiation to spore and stalk cells during fruiting body formation. The acaA gene is transcribed under the control of three different alternative promoters. This study aimed to develop a promoter assay for acaA in D. discoideum using bioluminescence microscopy. Here, we inserted green- and red-emitting luciferase genes into downstream of promoter regions 1 and 3, respectively. Promoter activities were visualized by bioluminescence microscopy. We confirmed the differential expression of acaA under the control of promoters 1 and 3 at the different stages of D. discoideum development. We also demonstrated the application of dual-color bioluminescence imaging in the development of an imaging promoter assay.

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Culmination in the slime mould Dictyostelium discoideum studied with a scanning electron microscope

Development ◽

10.1242/dev.35.2.323 ◽

1976 ◽

Vol 35 (2) ◽

pp. 323-333

Author(s):

D. J. Watts ◽

T. E. Treffry

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Dictyostelium Discoideum ◽

Fruiting Body ◽

Maximum Height ◽

Slow Process ◽

Fruiting Body Formation ◽

Basal Disc ◽

Body Formation ◽

Scanning Electron

Myxamoebae of Dictyostelium discoideum were allowed to develop on cellulose acetate filters, and specimens taken at various stages of fruiting body formation were prepared for study by scanning electron microscopy. In the immature fruiting body where the mass of pre-spore cells has just been lifted off the substratum by the developing stalk, the pre-spore cells are irregular in shape and are similar in appearance to cells in aggregates at earlier stages of development. As the stalk lengthens, the pre-spore cells gradually separate from one another and become rounded and elongate, but mature spores are not visible until the fruiting body reaches its maximum height. It is concluded that, contrary to previous reports, spore maturation is a slow process and is not completed until the sorus becomes pigmented. The mature stalk is surrounded by a smooth cellulose sheath but this does not envelop the cells of the basal disc, which remain discrete. The fruiting body is enclosed in a slime sheath and this may be important in holding together the mass of spores.

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In vitro stalk cell differentiation in wild-type and ‘slugger’ mutants of Dictyostelium discoideum

Development ◽

10.1242/dev.118.2.523 ◽

1993 ◽

Vol 118 (2) ◽

pp. 523-526 ◽

Cited By ~ 2

Author(s):

K. Inouye ◽

J. Gross

Keyword(s):

Dictyostelium Discoideum ◽

Fruiting Body ◽

Cell Maturation ◽

Stalk Cell ◽

Wild Type ◽

Fruiting Body Formation ◽

Body Formation ◽

Close Relationship ◽

Long Time

In ‘slugger’ mutants of Dictyostelium discoideum, aggregates of cells remain for an abnormally long time in the migratory phase under conditions where wild-type aggregates form fruiting bodies. In the present work, we have examined the relationship between the defect in fruiting body formation in these mutants and their ability to form mature stalk cells. We dissociated anterior cells from slugs of the mutants and their parents and tested their ability to form stalk cells when incubated at low density in the presence of (1) the stalk cell morphogen Differentiation Inducing Factor-1 (DIF-1) together with cyclic AMP, or (2) 8-Br-cAMP, which is believed to penetrate cell membrane and activate cAMP- dependent protein kinase (PKA). Most of the mutants were markedly defective in forming stalk cells in response to DIF-1 plus cAMP, confirming a close relationship between fruiting body formation and stalk cell maturation. On the other hand, many of these same mutants formed stalk cells efficiently in response to 8-Br-cAMP. This supports evidence for an essential role of PKA in stalk cell maturation and fruiting body formation. It also indicates that many of the mutants owe their slugger phenotype to defects in functions required for optimal adenylyl cyclase activity.

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