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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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Rap1 regulates myosin and actin cytoskeleton dynamics. Images of vegetative induced (+DOX) or control (−DOX) cells expressing (A) Lifeact-GFP (detects filamentous actin) or (B) GFP-MHC. Localization of (C) Lifeact-GFP or (D) GFP-MHC during cytokinesis is shown. Inset: fluorescence intensity was measured at the cell boundary around the circumference of the cell relative to the fluorescence intensity in the cytosol. (E and F) Comparison of representative cell shape and timing of cells during cytokinesis. Shown are myosin II– cells (E) and myosin II- cells with Rap1G12V (F). Scale bars: 10 μm.
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Figure 4: Rap1 regulates myosin and actin cytoskeleton dynamics. Images of vegetative induced (+DOX) or control (−DOX) cells expressing (A) Lifeact-GFP (detects filamentous actin) or (B) GFP-MHC. Localization of (C) Lifeact-GFP or (D) GFP-MHC during cytokinesis is shown. Inset: fluorescence intensity was measured at the cell boundary around the circumference of the cell relative to the fluorescence intensity in the cytosol. (E and F) Comparison of representative cell shape and timing of cells during cytokinesis. Shown are myosin II– cells (E) and myosin II- cells with Rap1G12V (F). Scale bars: 10 μm.

Mentions: Dictyostelium Rap1 is an important regulator of both myosin and actin (Kortholt et al., 2006; Kortholt and van Haastert, 2008; Jeon et al., 2007b). Activation of Rap1 induces actin filaments when exposed to uniform chemoattractant stimulation (Mun and Jeon, 2012), while Rap1-mediated myosin heavy-chain phosphorylation results in myosin II filament disassembly at the leading edge of chemotactic cells (Jeon et al., 2007b). Proper regulation of actin and myosin dynamics is essential for successful closure of the cytokinetic furrow (Faix et al., 1996; O’Connell et al., 2001; Gerisch et al., 2004; Glotzer, 2005). Actin and myosin dynamics were monitored with the filamentous actin marker Lifeact (Riedl et al., 2008) and GFP-MHC (Moores et al., 1996), respectively. Vegetative control cells showed dynamic Lifeact patches at the cell boundary and weak cortical localization of myosin (Figure 4, A and B). After Rap1G12V expression was induced, the cortical myosin localization strongly diminished, and the cells showed a thick but dynamic actin-rich cortex around the whole cell boundary. During cell division, actin became enriched at the poles of both dividing wild-type and Rap1G12V cells, however, Rap1G12V cells showed larger amounts and more intense patches of F-actin at the cell boundary compared with the control cells (Figure 4C, bottom panels). Dividing control cells showed symmetrical localization of myosin at both sides of the cell furrow, which increased with time (Figure 4D, top panels). In contrast, dividing Rap1G12V cells showed an asymmetrical localization of myosin and even at late stages of cytokinesis the amount of myosin at the furrow was much lower than in control cells (Figure 4D, bottom panels). The side of the furrow with less myosin showed slower ingression, which consequently resulted in an asymmetrical cell shape during the division (Figure 4D, bottom panels). These data show that proper Rap1 activation is important for regulating the actomyosin cytoskeleton in response to nuclear division.


Rap1-dependent pathways coordinate cytokinesis in Dictyostelium.

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

Rap1 regulates myosin and actin cytoskeleton dynamics. Images of vegetative induced (+DOX) or control (−DOX) cells expressing (A) Lifeact-GFP (detects filamentous actin) or (B) GFP-MHC. Localization of (C) Lifeact-GFP or (D) GFP-MHC during cytokinesis is shown. Inset: fluorescence intensity was measured at the cell boundary around the circumference of the cell relative to the fluorescence intensity in the cytosol. (E and F) Comparison of representative cell shape and timing of cells during cytokinesis. Shown are myosin II– cells (E) and myosin II- cells with Rap1G12V (F). Scale bars: 10 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4263460&req=5

Figure 4: Rap1 regulates myosin and actin cytoskeleton dynamics. Images of vegetative induced (+DOX) or control (−DOX) cells expressing (A) Lifeact-GFP (detects filamentous actin) or (B) GFP-MHC. Localization of (C) Lifeact-GFP or (D) GFP-MHC during cytokinesis is shown. Inset: fluorescence intensity was measured at the cell boundary around the circumference of the cell relative to the fluorescence intensity in the cytosol. (E and F) Comparison of representative cell shape and timing of cells during cytokinesis. Shown are myosin II– cells (E) and myosin II- cells with Rap1G12V (F). Scale bars: 10 μm.
Mentions: Dictyostelium Rap1 is an important regulator of both myosin and actin (Kortholt et al., 2006; Kortholt and van Haastert, 2008; Jeon et al., 2007b). Activation of Rap1 induces actin filaments when exposed to uniform chemoattractant stimulation (Mun and Jeon, 2012), while Rap1-mediated myosin heavy-chain phosphorylation results in myosin II filament disassembly at the leading edge of chemotactic cells (Jeon et al., 2007b). Proper regulation of actin and myosin dynamics is essential for successful closure of the cytokinetic furrow (Faix et al., 1996; O’Connell et al., 2001; Gerisch et al., 2004; Glotzer, 2005). Actin and myosin dynamics were monitored with the filamentous actin marker Lifeact (Riedl et al., 2008) and GFP-MHC (Moores et al., 1996), respectively. Vegetative control cells showed dynamic Lifeact patches at the cell boundary and weak cortical localization of myosin (Figure 4, A and B). After Rap1G12V expression was induced, the cortical myosin localization strongly diminished, and the cells showed a thick but dynamic actin-rich cortex around the whole cell boundary. During cell division, actin became enriched at the poles of both dividing wild-type and Rap1G12V cells, however, Rap1G12V cells showed larger amounts and more intense patches of F-actin at the cell boundary compared with the control cells (Figure 4C, bottom panels). Dividing control cells showed symmetrical localization of myosin at both sides of the cell furrow, which increased with time (Figure 4D, top panels). In contrast, dividing Rap1G12V cells showed an asymmetrical localization of myosin and even at late stages of cytokinesis the amount of myosin at the furrow was much lower than in control cells (Figure 4D, bottom panels). The side of the furrow with less myosin showed slower ingression, which consequently resulted in an asymmetrical cell shape during the division (Figure 4D, bottom panels). These data show that proper Rap1 activation is important for regulating the actomyosin cytoskeleton in response to nuclear division.

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