Rap1-dependent pathways coordinate cytokinesis in Dictyostelium.
Bottom Line: Hyperactivation of Rap1 results in severe growth delays and defective spindle formation in adherent cells and cell death in suspension.Furthermore, Rap mutants show aberrant regulation of the actomyosin cytoskeleton, resulting in extended furrow ingression times and asymmetrical cell division.Importantly, mutated forms of Rap also affect cytokinesis in other organisms, suggesting a conserved role for Rap in cell division.
Affiliation: Department of Cell Biochemistry, University of Groningen, 9747 AG Groningen, Netherlands.Show MeSH
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Mentions: Most of the current knowledge about Rap-mediated signaling in Dictyostelium comes from studies using constitutively active Rap1G12V mutants. Cells expressing Rap1G12V are large and flattened (Rebstein et al., 1997), have strongly increased substrate adhesion, and exhibit chemotactic defects (Jeon et al., 2007b). Because of these severe defects, we used here a doxycycline-inducible plasmid system (pDM310) to study the effect of Rap1G12V on cell growth and cell division (Veltman et al., 2009). Seven hours after induction of Rap1G12V expression (Supplemental Figure S1F), cells showed the characteristic Rap1G12V phenotype and became larger and flattened, with an average cell area of 249 ± 247 μm2 (n = 180), compared with 87 ± 26 μm2 (n = 180) for uninduced control cells (Figure 2, A and B). The broad size distribution (Figure 2A) is caused by large variation in Rap1G12V expression levels within the cell population (Supplemental Figure S1G). This is the consequence of the variation in plasmid copy number per cell that is typically observed with the used inducible plasmid system (Veltman et al., 2009; Veltman and van Haastert, 2013).
Affiliation: Department of Cell Biochemistry, University of Groningen, 9747 AG Groningen, Netherlands.