Project name: Dictyostelium discoideum

Description: One mechanism multicellular structures use for controlling cell number [1, 2] involves the secretion and sensing of a factor, such as leptin [3] or myostatin [4], in mammals. Dictyostelium cells secrete autocrine factors for sensing cell density prior to aggregation and multicellular development [5, 6] such as CMF (conditioned-medium factor), which enables starving cells to respond to cAMP pulses [7, 8, 9]. Its actions are mediated by two receptors. CMFR1 activates a G protein-independent signaling pathway regulating gene expression [10]. An unknown Gα1-dependent receptor activates phospholipase C (PLC), which regulates the lifetime of Gα2-GTP [11, 12, 13]. Here, we describe RpkA, an unusual seven-transmembrane receptor that is fused to a C-terminal PIP5 kinase domain and that localizes in membranes of a late endosomal compartment. Loss of RpkA resulted in formation of persistent loose aggregates and altered expression of cAMP-regulated genes. The developmental defect can be rescued by full-length RpkA and the transmembrane domain only. The PIP5 kinase domain is dispensable for the developmental role of RpkA. rpkA− cells secrete and bind CMF but are unable to induce downstream responses. Inactivation of Gα1, a negative regulator of CMF signaling, rescued the developmental defect of the rpkA− cells, suggesting that RpkA actions are mediated by Gα1.Keywords: Comparison of wild type AX2 cells with rpkA minus cellsOverall design: Microarray analysis for the rpkA- mutant.Developmentalstage Microarray Wild type MutantTotal RNA dye Total RNA dyeT0 12874460 Culture A Cy3 Culture A Cy512945001 Culture B Cy3 Culture B Cy512874461 Culture A Cy5 Culture A Cy312945002 Culture B Cy5 Culture B Cy3T8 12882232 Culture A Cy3 Culture A Cy512953218 Culture B Cy3 Culture B Cy512882233 Culture A Cy5 Culture A Cy312953220 Culture B Cy5 Culture B Cy3AX2 cells and rpkA- cells were starved (5 × 10E6 cells/cm2) on phosphate agar plates and total RNA was prepared at 0 and 8 hrs of development. cDNA was prepared from total RNA (10 ug) using the Fairplay Kit (Stratagene Europe, Amsterdam, The Netherlands) as described in the manufacturers protocol. For each microarray experiment, an experimental (mutant, rpkA-) sample was labeled with Cy5 or Cy3 and the reference (wild type, AX2) sample with Cy3 or Cy5, respectively. Fluorescently labeled cDNA of the rpkA- mutant was generated and compared to labeled cDNA from AX2. Microarray production, expression profiling and data analysis were carried out as described in [Farbrother et al., Cell. Microbiology 2006, 3:438-456].

Data type: Transcriptome or Gene expression

Sample scope: Multiisolate

Relevance: ModelOrganism

Last updated: 2007-05-15