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: Supplemental Figure S1A shows that N-terminal green fluorescent protein (GFP)-fused Rap1 is localized uniformly at the cell membrane during both growth and cytokinesis of Dictyostelium cells. To monitor spatial activation of the protein, rather than its localization, we used the previously described molecular probe for active Rap1, RalGDS-GFP (Jeon et al., 2007b; Kortholt and van Haastert, 2008). In unstimulated vegetative cells, Rap1 activation was observed at the cell cortex and in patches at the cell membrane that may correspond to macropinosomes (Supplemental Figure S1B). At the moment cells rounded up and entered cytokinesis, these RalGDS patches disappeared and Rap1 became uniformly activated at the cell cortex. Once the cell started to elongate, Rap1 activation was restricted to the sites that later became the poles of the dividing cell (Figure 1A). In dumbbell-shaped cells, the RalGDS marker was depleted from the furrow region, and strongly localized to the poles (Figure 1, A and B). RalGDS-GFP intensity at the membrane was measured around the circumference of cells (Figure 1B). The average fluorescence of RalGDS-GFP at the poles was 1.48 ± 0.12 (n = 10) times the cytosolic fluorescence, while the fluorescence at the furrow region (1.01 ± 0.12, n = 10) was similar to that in the cytosol (Figure 1, A and B). This asymmetric Rap1 activation persisted until the moment the two daughter cells separated from each other.
Affiliation: Department of Cell Biochemistry, University of Groningen, 9747 AG Groningen, Netherlands.