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Rap1-dependent pathways coordinate cytokinesis in Dictyostelium.

Plak K, Keizer-Gunnink I, van Haastert PJ, Kortholt A - Mol. Biol. Cell (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biochemistry, University of Groningen, 9747 AG Groningen, Netherlands.

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Rap1G12V affects 14-3-3 localization in RacE-dependent manner. (A) Images of immunostained 14-3-3 protein in the indicated cell strains. (B) Images showing colocalization of GFP-Rap1G12V and 14-3-3 in fixed Dictyostelium cells. (C) 14-3-3-GFP localization in dividing wild-type cells. (D) Colocalization of 14-3-3 with Aurora-mRFP or α-tubulin. 14-3-3 localization in (E) dividing Rap1G12V and (F) racE-/Rap1G12V cells. Time is indicated in seconds. Scale bars: 10 μm for all panels.
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Figure 5: Rap1G12V affects 14-3-3 localization in RacE-dependent manner. (A) Images of immunostained 14-3-3 protein in the indicated cell strains. (B) Images showing colocalization of GFP-Rap1G12V and 14-3-3 in fixed Dictyostelium cells. (C) 14-3-3-GFP localization in dividing wild-type cells. (D) Colocalization of 14-3-3 with Aurora-mRFP or α-tubulin. 14-3-3 localization in (E) dividing Rap1G12V and (F) racE-/Rap1G12V cells. Time is indicated in seconds. Scale bars: 10 μm for all panels.

Mentions: We showed that Rap1G12V expression affects a number of cellular processes during mitosis progression, including defects in myosin assembly, adhesion, actin polymerization, and microtubule formation. Recently a RacE/14-3-3–dependent pathway was identified in Dictyostelium that links microtubules and cortical myosin to actin dynamics at the cortex (Zhou et al., 2010). Although human 14-3-3 is an interaction partner for Rap proteins (Riou et al., 2013), we were unable to detect any direct binding between activated Dictyostelium Rap1 and 14-3-3. However, because Dictyostelium Rap1G12V cells have increased Rac activation (Plak et al., 2013), we hypothesized that Rap1 may cooperate with RacE and 14-3-3 in regulating Dictyostelium cytokinesis. Staining with 14-3-3–specific antibody, revealed weak cortical localization of 14-3-3 in nonmitotic wild-type cells (Figure 5A). Additionally, 14-3-3 and active Rap1 colocalized at the cell cortex (Figure 5B). A recent proteomic study suggested that Dictyostelium 14-3-3 is associated with centrosome during cell division (Reinders et al., 2006), whereas microscopy data suggest that the protein is enriched at the polar cortex region of the dividing cell in late cytokinesis (Zhou et al., 2010). Our data with both N-terminal or C-terminal GFP-fused 14-3-3 clearly show a dynamic cell cycle–dependent localization: in early mitotic stages, 14-3-3 is localized in the nucleus and subsequently at the centrosome (Figure 5C), where it colocalizes with Aurora kinase and, partially, α-tubulin (Figure 5D). Notably, we did not observe the previously described polar cortical localization of 14-3-3 (Zhou et al., 2010). As the cytokinesis progressed the centrosomal localization disappeared, and at the late stages of cytokinesis, appeared to be lost completely (Figure 5, C and D). This centrosomal localization appears to be specific, as all dividing cells observed(n = 22) showed the same localization pattern. In Rap1G12V-expressing cells, 14-3-3 is mislocalized; vegetative Rap1G12V cells have increased amounts of 14-3-3 at the cortex compared with wild-type cells (Figure 5A). Most importantly, and in contrast to control cells, the cells that expressed low amounts of Rap1G12V and that were capable of dividing did not show nuclear or centrosomal localization of 14-3-3 during their division (Figure 5E).


Rap1-dependent pathways coordinate cytokinesis in Dictyostelium.

Plak K, Keizer-Gunnink I, van Haastert PJ, Kortholt A - Mol. Biol. Cell (2014)

Rap1G12V affects 14-3-3 localization in RacE-dependent manner. (A) Images of immunostained 14-3-3 protein in the indicated cell strains. (B) Images showing colocalization of GFP-Rap1G12V and 14-3-3 in fixed Dictyostelium cells. (C) 14-3-3-GFP localization in dividing wild-type cells. (D) Colocalization of 14-3-3 with Aurora-mRFP or α-tubulin. 14-3-3 localization in (E) dividing Rap1G12V and (F) racE-/Rap1G12V cells. Time is indicated in seconds. Scale bars: 10 μm for all panels.
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Figure 5: Rap1G12V affects 14-3-3 localization in RacE-dependent manner. (A) Images of immunostained 14-3-3 protein in the indicated cell strains. (B) Images showing colocalization of GFP-Rap1G12V and 14-3-3 in fixed Dictyostelium cells. (C) 14-3-3-GFP localization in dividing wild-type cells. (D) Colocalization of 14-3-3 with Aurora-mRFP or α-tubulin. 14-3-3 localization in (E) dividing Rap1G12V and (F) racE-/Rap1G12V cells. Time is indicated in seconds. Scale bars: 10 μm for all panels.
Mentions: We showed that Rap1G12V expression affects a number of cellular processes during mitosis progression, including defects in myosin assembly, adhesion, actin polymerization, and microtubule formation. Recently a RacE/14-3-3–dependent pathway was identified in Dictyostelium that links microtubules and cortical myosin to actin dynamics at the cortex (Zhou et al., 2010). Although human 14-3-3 is an interaction partner for Rap proteins (Riou et al., 2013), we were unable to detect any direct binding between activated Dictyostelium Rap1 and 14-3-3. However, because Dictyostelium Rap1G12V cells have increased Rac activation (Plak et al., 2013), we hypothesized that Rap1 may cooperate with RacE and 14-3-3 in regulating Dictyostelium cytokinesis. Staining with 14-3-3–specific antibody, revealed weak cortical localization of 14-3-3 in nonmitotic wild-type cells (Figure 5A). Additionally, 14-3-3 and active Rap1 colocalized at the cell cortex (Figure 5B). A recent proteomic study suggested that Dictyostelium 14-3-3 is associated with centrosome during cell division (Reinders et al., 2006), whereas microscopy data suggest that the protein is enriched at the polar cortex region of the dividing cell in late cytokinesis (Zhou et al., 2010). Our data with both N-terminal or C-terminal GFP-fused 14-3-3 clearly show a dynamic cell cycle–dependent localization: in early mitotic stages, 14-3-3 is localized in the nucleus and subsequently at the centrosome (Figure 5C), where it colocalizes with Aurora kinase and, partially, α-tubulin (Figure 5D). Notably, we did not observe the previously described polar cortical localization of 14-3-3 (Zhou et al., 2010). As the cytokinesis progressed the centrosomal localization disappeared, and at the late stages of cytokinesis, appeared to be lost completely (Figure 5, C and D). This centrosomal localization appears to be specific, as all dividing cells observed(n = 22) showed the same localization pattern. In Rap1G12V-expressing cells, 14-3-3 is mislocalized; vegetative Rap1G12V cells have increased amounts of 14-3-3 at the cortex compared with wild-type cells (Figure 5A). Most importantly, and in contrast to control cells, the cells that expressed low amounts of Rap1G12V and that were capable of dividing did not show nuclear or centrosomal localization of 14-3-3 during their division (Figure 5E).

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biochemistry, University of Groningen, 9747 AG Groningen, Netherlands.

Show MeSH
Related in: MedlinePlus