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Nuf, a Rab11 effector, maintains cytokinetic furrow integrity by promoting local actin polymerization.

Cao J, Albertson R, Riggs B, Field CM, Sullivan W - J. Cell Biol. (2008)

Bottom Line: We find that in nuf mutant embryos, an initial loss of F-actin at the furrow is followed by loss of the associated furrow membrane.Drug- or Rho-GTP-induced increase of actin polymerization or genetically mediated decrease of actin depolymerization suppresses the nuf mutant F-actin and membrane defects.We also find that RhoGEF2 does not properly localize at the furrow in nuf mutant embryos and that RhoGEF2-Rho1 pathway components show strong specific genetic interactions with Nuf.

View Article: PubMed Central - PubMed

Affiliation: Sinsheimer Laboratories, Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Cruz, Santa Cruz, CA 95064, USA.

ABSTRACT
Plasma membrane ingression during cytokinesis involves both actin remodeling and vesicle-mediated membrane addition. Vesicle-based membrane delivery from the recycling endosome (RE) has an essential but ill-defined involvement in cytokinesis. In the Drosophila melanogaster early embryo, Nuf (Nuclear fallout), a Rab11 effector which is essential for RE function, is required for F-actin and membrane integrity during furrow ingression. We find that in nuf mutant embryos, an initial loss of F-actin at the furrow is followed by loss of the associated furrow membrane. Wild-type embryos treated with Latrunculin A or Rho inhibitor display similar defects. Drug- or Rho-GTP-induced increase of actin polymerization or genetically mediated decrease of actin depolymerization suppresses the nuf mutant F-actin and membrane defects. We also find that RhoGEF2 does not properly localize at the furrow in nuf mutant embryos and that RhoGEF2-Rho1 pathway components show strong specific genetic interactions with Nuf. We propose a model in which RE-derived vesicles promote furrow integrity by regulating the rate of actin polymerization through the RhoGEF2-Rho1 pathway.

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Reducing actin depolymerization by tsr or promoting actin polymerization by Jasp ameliorates the furrow defects observed in nuf embryos. (A) Green, GFP-Moesin; Red, tubulin. Compared with nuf1 embryos (top row, the same embryo as shown in Fig. 4 A), tsr1/Cy; nuf1 embryos (bottom row) exhibited normal furrow structures from interphase to metaphase during cycle 13. For full movie, see Video 5 (available at http://www.jcb.org/cgi/content/full/jcb.200712036/DC1). (B) Quantifications of results in A and C. At metaphase cycle 13, furrow break index and spindle fusion index in nuf1 (N = 12 and n = 1,378), tsr1/Cy; nuf1 (N = 8 and n = 1,010), and nuf1 + Jasp (N = 7 and n = 846) embryos. N, total embryos examined; n, total nuclei/mitotic spindles analyzed. **, P < 0.01; ***, P < 0.001. Error bars represent SEM. (C and D) nuf1 embryos expressing GFP-Dlg were injected with either 1 mM Jasp or DMSO at cycle-13 early prophase. (C) Jasp-injected nuf embryos show very few furrow breaks at metaphase, whereas DMSO-injected nuf embryos display extensive discontinuous furrows resulting from progressive loss of furrow membrane (arrows). See Videos 6 and 7 (available at http://www.jcb.org/cgi/content/full/jcb.200712036/DC1). (D) The membrane loss before Jasp injection in nuf1 embryos was not compensated after Jasp treatment (arrows). Bars: (A and D) 10 μm; (C) 20 μm.
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fig5: Reducing actin depolymerization by tsr or promoting actin polymerization by Jasp ameliorates the furrow defects observed in nuf embryos. (A) Green, GFP-Moesin; Red, tubulin. Compared with nuf1 embryos (top row, the same embryo as shown in Fig. 4 A), tsr1/Cy; nuf1 embryos (bottom row) exhibited normal furrow structures from interphase to metaphase during cycle 13. For full movie, see Video 5 (available at http://www.jcb.org/cgi/content/full/jcb.200712036/DC1). (B) Quantifications of results in A and C. At metaphase cycle 13, furrow break index and spindle fusion index in nuf1 (N = 12 and n = 1,378), tsr1/Cy; nuf1 (N = 8 and n = 1,010), and nuf1 + Jasp (N = 7 and n = 846) embryos. N, total embryos examined; n, total nuclei/mitotic spindles analyzed. **, P < 0.01; ***, P < 0.001. Error bars represent SEM. (C and D) nuf1 embryos expressing GFP-Dlg were injected with either 1 mM Jasp or DMSO at cycle-13 early prophase. (C) Jasp-injected nuf embryos show very few furrow breaks at metaphase, whereas DMSO-injected nuf embryos display extensive discontinuous furrows resulting from progressive loss of furrow membrane (arrows). See Videos 6 and 7 (available at http://www.jcb.org/cgi/content/full/jcb.200712036/DC1). (D) The membrane loss before Jasp injection in nuf1 embryos was not compensated after Jasp treatment (arrows). Bars: (A and D) 10 μm; (C) 20 μm.

Mentions: The striking reduction of furrow F-actin in nuf mutant embryos raises the question: does Nuf promote actin polymerization or stabilization at the furrow? If Nuf has such a function, one prediction would be that reducing actin depolymerization should suppress furrow defects in nuf embryos. Cofilin is a well-known actin depolymerizer in a variety of processes that require remodeling of the actin cytoskeleton including cell migration and cytokinesis (Ono, 2007). Homozygous mutations for tsr (twinstar), the D. melanogaster Cofilin homologue, are zygotic lethal and cause cytokinesis defects in contractile ring disassembly (Gunsalus et al., 1995). To test whether tsr can rescue the nuf phenotype, we collected eggs laid by female flies homozygous for nuf and heterozygous for tsr and then examined them for furrow and spindle morphology during cycle 13 in early embryogenesis. When twinstar- alleles tsrN96A and tsrN121 (Ng and Luo, 2004) were crossed into the homozygous nuf1 background (e.g., tsrN96A/Cy; nuf1/nuf1), all the female ovaries had strong membrane defects in the egg chambers that prevented them from undergoing embryonic development (unpublished data). When the hypomorphic tsr1 allele (Gunsalus et al., 1995) was introduced into the nuf1 background, the ovaries exhibited milder membrane defects, and a portion of eggs was able to develop after fertilization: 21% (n = 1,551 eggs) of the total eggs laid by tsr1/Cy; nuf1/nuf1 females hatched and of these larvae, 75% (n = 331 larvae) developed into adult flies, compared with a 0% (n = 547 eggs) hatch rate of eggs laid by nuf1 females. Moreover, these tsr1/Cy; nuf1/nuf1 embryos displayed much less severe defects than nuf1 embryos, with fewer furrow breaks and spindle fusions in metaphase and cellularizing furrows (Fig. 5, A and B; Video 5, available at http://www.jcb.org/cgi/content/full/jcb.200712036/DC1; and not depicted). The suppression of nuf phenotype by reducing Twinstar dosage supports a role for Nuf in promoting actin polymerization at the furrow.


Nuf, a Rab11 effector, maintains cytokinetic furrow integrity by promoting local actin polymerization.

Cao J, Albertson R, Riggs B, Field CM, Sullivan W - J. Cell Biol. (2008)

Reducing actin depolymerization by tsr or promoting actin polymerization by Jasp ameliorates the furrow defects observed in nuf embryos. (A) Green, GFP-Moesin; Red, tubulin. Compared with nuf1 embryos (top row, the same embryo as shown in Fig. 4 A), tsr1/Cy; nuf1 embryos (bottom row) exhibited normal furrow structures from interphase to metaphase during cycle 13. For full movie, see Video 5 (available at http://www.jcb.org/cgi/content/full/jcb.200712036/DC1). (B) Quantifications of results in A and C. At metaphase cycle 13, furrow break index and spindle fusion index in nuf1 (N = 12 and n = 1,378), tsr1/Cy; nuf1 (N = 8 and n = 1,010), and nuf1 + Jasp (N = 7 and n = 846) embryos. N, total embryos examined; n, total nuclei/mitotic spindles analyzed. **, P < 0.01; ***, P < 0.001. Error bars represent SEM. (C and D) nuf1 embryos expressing GFP-Dlg were injected with either 1 mM Jasp or DMSO at cycle-13 early prophase. (C) Jasp-injected nuf embryos show very few furrow breaks at metaphase, whereas DMSO-injected nuf embryos display extensive discontinuous furrows resulting from progressive loss of furrow membrane (arrows). See Videos 6 and 7 (available at http://www.jcb.org/cgi/content/full/jcb.200712036/DC1). (D) The membrane loss before Jasp injection in nuf1 embryos was not compensated after Jasp treatment (arrows). Bars: (A and D) 10 μm; (C) 20 μm.
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Related In: Results  -  Collection

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fig5: Reducing actin depolymerization by tsr or promoting actin polymerization by Jasp ameliorates the furrow defects observed in nuf embryos. (A) Green, GFP-Moesin; Red, tubulin. Compared with nuf1 embryos (top row, the same embryo as shown in Fig. 4 A), tsr1/Cy; nuf1 embryos (bottom row) exhibited normal furrow structures from interphase to metaphase during cycle 13. For full movie, see Video 5 (available at http://www.jcb.org/cgi/content/full/jcb.200712036/DC1). (B) Quantifications of results in A and C. At metaphase cycle 13, furrow break index and spindle fusion index in nuf1 (N = 12 and n = 1,378), tsr1/Cy; nuf1 (N = 8 and n = 1,010), and nuf1 + Jasp (N = 7 and n = 846) embryos. N, total embryos examined; n, total nuclei/mitotic spindles analyzed. **, P < 0.01; ***, P < 0.001. Error bars represent SEM. (C and D) nuf1 embryos expressing GFP-Dlg were injected with either 1 mM Jasp or DMSO at cycle-13 early prophase. (C) Jasp-injected nuf embryos show very few furrow breaks at metaphase, whereas DMSO-injected nuf embryos display extensive discontinuous furrows resulting from progressive loss of furrow membrane (arrows). See Videos 6 and 7 (available at http://www.jcb.org/cgi/content/full/jcb.200712036/DC1). (D) The membrane loss before Jasp injection in nuf1 embryos was not compensated after Jasp treatment (arrows). Bars: (A and D) 10 μm; (C) 20 μm.
Mentions: The striking reduction of furrow F-actin in nuf mutant embryos raises the question: does Nuf promote actin polymerization or stabilization at the furrow? If Nuf has such a function, one prediction would be that reducing actin depolymerization should suppress furrow defects in nuf embryos. Cofilin is a well-known actin depolymerizer in a variety of processes that require remodeling of the actin cytoskeleton including cell migration and cytokinesis (Ono, 2007). Homozygous mutations for tsr (twinstar), the D. melanogaster Cofilin homologue, are zygotic lethal and cause cytokinesis defects in contractile ring disassembly (Gunsalus et al., 1995). To test whether tsr can rescue the nuf phenotype, we collected eggs laid by female flies homozygous for nuf and heterozygous for tsr and then examined them for furrow and spindle morphology during cycle 13 in early embryogenesis. When twinstar- alleles tsrN96A and tsrN121 (Ng and Luo, 2004) were crossed into the homozygous nuf1 background (e.g., tsrN96A/Cy; nuf1/nuf1), all the female ovaries had strong membrane defects in the egg chambers that prevented them from undergoing embryonic development (unpublished data). When the hypomorphic tsr1 allele (Gunsalus et al., 1995) was introduced into the nuf1 background, the ovaries exhibited milder membrane defects, and a portion of eggs was able to develop after fertilization: 21% (n = 1,551 eggs) of the total eggs laid by tsr1/Cy; nuf1/nuf1 females hatched and of these larvae, 75% (n = 331 larvae) developed into adult flies, compared with a 0% (n = 547 eggs) hatch rate of eggs laid by nuf1 females. Moreover, these tsr1/Cy; nuf1/nuf1 embryos displayed much less severe defects than nuf1 embryos, with fewer furrow breaks and spindle fusions in metaphase and cellularizing furrows (Fig. 5, A and B; Video 5, available at http://www.jcb.org/cgi/content/full/jcb.200712036/DC1; and not depicted). The suppression of nuf phenotype by reducing Twinstar dosage supports a role for Nuf in promoting actin polymerization at the furrow.

Bottom Line: We find that in nuf mutant embryos, an initial loss of F-actin at the furrow is followed by loss of the associated furrow membrane.Drug- or Rho-GTP-induced increase of actin polymerization or genetically mediated decrease of actin depolymerization suppresses the nuf mutant F-actin and membrane defects.We also find that RhoGEF2 does not properly localize at the furrow in nuf mutant embryos and that RhoGEF2-Rho1 pathway components show strong specific genetic interactions with Nuf.

View Article: PubMed Central - PubMed

Affiliation: Sinsheimer Laboratories, Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Cruz, Santa Cruz, CA 95064, USA.

ABSTRACT
Plasma membrane ingression during cytokinesis involves both actin remodeling and vesicle-mediated membrane addition. Vesicle-based membrane delivery from the recycling endosome (RE) has an essential but ill-defined involvement in cytokinesis. In the Drosophila melanogaster early embryo, Nuf (Nuclear fallout), a Rab11 effector which is essential for RE function, is required for F-actin and membrane integrity during furrow ingression. We find that in nuf mutant embryos, an initial loss of F-actin at the furrow is followed by loss of the associated furrow membrane. Wild-type embryos treated with Latrunculin A or Rho inhibitor display similar defects. Drug- or Rho-GTP-induced increase of actin polymerization or genetically mediated decrease of actin depolymerization suppresses the nuf mutant F-actin and membrane defects. We also find that RhoGEF2 does not properly localize at the furrow in nuf mutant embryos and that RhoGEF2-Rho1 pathway components show strong specific genetic interactions with Nuf. We propose a model in which RE-derived vesicles promote furrow integrity by regulating the rate of actin polymerization through the RhoGEF2-Rho1 pathway.

Show MeSH
Related in: MedlinePlus