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Eukaryotic Cell, February 2004, p. 61-71, Vol. 3, No. 1
1535-9778/04/$08.00+0     DOI: 10.1128/EC.3.1.61-71.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

The Ca2+/Calcineurin-Regulated cup Gene Family in Dictyostelium discoideum and Its Possible Involvement in Development

Barrie Coukell,* Yi Li,{dagger} John Moniakis,{ddagger} and Anne Cameron

Department of Biology, York University, Toronto, Ontario M3J 1P3, Canada

Received 26 September 2003/ Accepted 13 November 2003

Changes in free intracellular Ca2+ are thought to regulate several major processes during Dictyostelium development, including cell aggregation and cell type-specific gene expression, but the mechanisms involved are unclear. To learn more about Ca2+ signaling and Ca2+ homeostasis in this organism, we used suppression subtractive hybridization to identify genes up-regulated by high extracellular Ca2+. Unexpectedly, many of the genes identified belong to a novel gene family (termed cup) with seven members. In vegetative cells, the cup genes were up-regulated by high Ca2+ but not by other ions or by heat, oxidative, or osmotic stress. cup induction by Ca2+ was blocked completely by inhibitors of calcineurin and protein synthesis. In developing cells, cup expression was high during aggregation and late development but low during the slug stage. This pattern correlates closely with reported levels of free intracellular Ca2+ during development. The cup gene products are highly homologous, acidic proteins possessing putative ricin domains. BLAST searches failed to reveal homologs in other organisms, but Western analyses suggested that Cup-like proteins might exist in certain other cellular slime mold species. Localization experiments indicated that Cup proteins are primarily cytoplasmic but become cell membrane-associated during Ca2+ stress and cell aggregation. When cup expression was down-regulated by antisense RNA, the cells failed to aggregate. However, this developmental block was overcome by partially up-regulating cup expression. Together, these results suggest that the Cup proteins in Dictyostelium might play an important role in stabilizing and/or regulating the cell membrane during Ca2+ stress and/or certain stages of development.

* Corresponding author. Mailing address: Department of Biology, York University, 4700 Keele St., Toronto, ON, M3J 1P3, Canada. Phone: (416) 736-2100, ext. 33554. Fax: (416) 736-5698. E-mail: bcoukell{at}yorku.ca.

{dagger} Present address: Department of Medicine, Division of Rheumatology, Queen's University, Kingston, ON, K7L 3N6, Canada.

{ddagger} Present address: The Blueprint Initiative, Toronto, ON, M5G 1W7, Canada.

Eukaryotic Cell, February 2004, p. 61-71, Vol. 3, No. 1
1535-9778/04/$08.00+0     DOI: 10.1128/EC.3.1.61-71.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.





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