Phospholipase Cδ regulates germination of Dictyostelium spores

BACKGROUND: Many eukaryotes, including plants and fungi make spores that resist severe environmental stress. The micro-organism Dictyostelium contains a single phospholipase C gene (PLC); deletion of the gene has no effect on growth, cell movement and differentiation. In this report we show that PLC is essential to sense the environment of food-activated spores. RESULTS: Plc-null spores germinate at alkaline pH, reduced temperature or increased osmolarity, conditions at which the emerging amoebae can not grow. In contrast, food-activated wild-type spores return to dormancy till conditions in the environment allow growth. The analysis of inositol 1,4,5-trisphosphate (IP(3)) levels and the effect of added IP(3) uncover an unexpected mechanism how PLC regulates spore germination: i) deletion of PLC induces the enhanced activity of an IP(5) phosphatase leading to high IP(3) levels in plc-null cells; ii) in wild-type spores unfavourable conditions inhibit PLC leading to a reduction of IP(3) levels; addition of exogenous IP(3) to wild-type spores induces germination at unfavourable conditions; iii) in plc-null spores IP(3) levels remain high, also at unfavourable environmental conditions. CONCLUSIONS: The results imply that environmental conditions regulate PLC activity and that IP(3) induces spore germination; the uncontrolled germination of plc-null spores is not due to a lack of PLC activity but to the constitutive activation of an alternative IP(3)-forming pathway

The local cell curvature guides pseudopodia towards chemoattractants

Many eukaryotic cells use pseudopodia for movement towards chemoattractants. We developed a computer algorithm to identify pseudopodia, and analyzed how pseudopodia of Dictyostelium cells are guided toward cAMP. Surprisingly, the direction of a pseudopod is not actively oriented toward the gradient, but is always perpendicular to the local cell curvature. The gradient induces a bias in the position where the pseudopod emerges: pseudopodia more likely emerge at the side of the cell closer to the gradient where perpendicular pseudopodia are pointed automatically toward the chemoattractant. A mutant lacking the formin dDia2 is not spherical but has many invaginations. Although pseudopodia still emerge at the side closer to the gradient, the surface curvature is so irregular that many pseudopodia are not extended toward cAMP. The results imply that the direction of the pseudopod extension, and therefore also the direction of cell movement, is dominated by two aspects: the position at the cell surface where a pseudopod emerges, and the local curvature of the membrane at that position

Structure of the Roc–COR domain tandem of C. tepidum, a prokaryotic homologue of the human LRRK2 Parkinson kinase

Ras of complex proteins (Roc) belongs to the superfamily of Ras-related small G-proteins that always occurs in tandem with the C-terminal of Roc (COR) domain. This Roc–COR tandem is found in the bacterial and eukaryotic world. Its most prominent member is the leucine-rich repeat kinase LRRK2, which is mutated and activated in Parkinson patients. Here, we investigated biochemically and structurally the Roco protein from Chlorobium tepidum. We show that Roc is highly homologous to Ras, whereas the COR domain is a dimerisation device. The juxtaposition of the G-domains and mutational analysis suggest that the Roc GTPase reaction is stimulated and/or regulated by dimerisation in a nucleotide-dependent manner. The region most conserved between bacteria and man is the interface between Roc and COR, where single-point Parkinson mutations of the Roc and COR domains are in close proximity. The analogous mutations in C. tepidum Roc–COR decrease the GTPase reaction rate, most likely due to a modification of the interaction between the Roc and COR domains