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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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The RE is required for furrow maintenance. (A) GFP-Moesin (green) and Rhodamine-tubulin (red) in WT embryos. From cycle-13 interphase to metaphase, three z sections are shown from 1 to 3 μm below the cortex. (B) Same experiment as in A in nuf embryos. F-actin was progressively lost at the furrow (arrows) as furrows invaginated, and this loss started at the sections closer to the invaginating tips (arrowheads; compare 6:30 to 0:00 at different depths). Spindle fusions (asterisks) occurred where furrows were broken. See Video 3 (available at http://www.jcb.org/cgi/content/full/jcb.200712036/DC1). (C) Time-lapse images of Rab11 cycle-13 furrow progression. Rhodamine-labeled actin was gradually lost at the furrow (arrows). (D) GFP-Dlg (green) and Rhodamine-actin (red) in nuf embryos. Yellow arrowheads indicate loss of membrane at the furrow over time. Loss of F-actin at the furrow generally precedes loss of membrane (arrows). See Video 4 (available at http://www.jcb.org/cgi/content/full/jcb.200712036/DC1). Bars, 10 μm.
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fig4: The RE is required for furrow maintenance. (A) GFP-Moesin (green) and Rhodamine-tubulin (red) in WT embryos. From cycle-13 interphase to metaphase, three z sections are shown from 1 to 3 μm below the cortex. (B) Same experiment as in A in nuf embryos. F-actin was progressively lost at the furrow (arrows) as furrows invaginated, and this loss started at the sections closer to the invaginating tips (arrowheads; compare 6:30 to 0:00 at different depths). Spindle fusions (asterisks) occurred where furrows were broken. See Video 3 (available at http://www.jcb.org/cgi/content/full/jcb.200712036/DC1). (C) Time-lapse images of Rab11 cycle-13 furrow progression. Rhodamine-labeled actin was gradually lost at the furrow (arrows). (D) GFP-Dlg (green) and Rhodamine-actin (red) in nuf embryos. Yellow arrowheads indicate loss of membrane at the furrow over time. Loss of F-actin at the furrow generally precedes loss of membrane (arrows). See Video 4 (available at http://www.jcb.org/cgi/content/full/jcb.200712036/DC1). Bars, 10 μm.

Mentions: Previous studies have shown that the RE component Nuf is required for actin and membrane recruitment to the invaginating furrow (Rothwell et al., 1998; Riggs et al., 2003). To address why nuf mutant embryos exhibit actin as well as membrane defects, we examined furrow formation in living nuf1 (the strongest nuf allele; see Materials and methods) embryos expressing GFP-Moesin. In WT control embryos expressing GFP-Moesin, no breaks were seen at furrows during furrow invagination (Fig. 4 A). In contrast, it appears that the F-actin loss initially occurs basally and progresses apically at the furrows in nuf embryos (Fig. 4 B, arrowheads). Additionally, F-actin loss expands laterally (Fig. 4 B, arrows; and Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200712036/DC1). At metaphase, when furrows have invaginated to their maximal length, breaks were seen throughout the entire length of the furrow resulting in spindle fusions (Fig. 4 B, asterisks; and Video 3). Loss of F-actin stability was also seen in Rab11 embryos during metaphase furrow invagination (Fig. 4 C) and in nuf embryos during cellularization (Fig. S4 A, available at http://www.jcb.org/cgi/content/full/jcb.200712036/DC1). Given that Rab11 is required for RE integrity, these results together indicated that the RE is required to maintain F-actin stability at the invaginating furrows.


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)

The RE is required for furrow maintenance. (A) GFP-Moesin (green) and Rhodamine-tubulin (red) in WT embryos. From cycle-13 interphase to metaphase, three z sections are shown from 1 to 3 μm below the cortex. (B) Same experiment as in A in nuf embryos. F-actin was progressively lost at the furrow (arrows) as furrows invaginated, and this loss started at the sections closer to the invaginating tips (arrowheads; compare 6:30 to 0:00 at different depths). Spindle fusions (asterisks) occurred where furrows were broken. See Video 3 (available at http://www.jcb.org/cgi/content/full/jcb.200712036/DC1). (C) Time-lapse images of Rab11 cycle-13 furrow progression. Rhodamine-labeled actin was gradually lost at the furrow (arrows). (D) GFP-Dlg (green) and Rhodamine-actin (red) in nuf embryos. Yellow arrowheads indicate loss of membrane at the furrow over time. Loss of F-actin at the furrow generally precedes loss of membrane (arrows). See Video 4 (available at http://www.jcb.org/cgi/content/full/jcb.200712036/DC1). Bars, 10 μm.
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fig4: The RE is required for furrow maintenance. (A) GFP-Moesin (green) and Rhodamine-tubulin (red) in WT embryos. From cycle-13 interphase to metaphase, three z sections are shown from 1 to 3 μm below the cortex. (B) Same experiment as in A in nuf embryos. F-actin was progressively lost at the furrow (arrows) as furrows invaginated, and this loss started at the sections closer to the invaginating tips (arrowheads; compare 6:30 to 0:00 at different depths). Spindle fusions (asterisks) occurred where furrows were broken. See Video 3 (available at http://www.jcb.org/cgi/content/full/jcb.200712036/DC1). (C) Time-lapse images of Rab11 cycle-13 furrow progression. Rhodamine-labeled actin was gradually lost at the furrow (arrows). (D) GFP-Dlg (green) and Rhodamine-actin (red) in nuf embryos. Yellow arrowheads indicate loss of membrane at the furrow over time. Loss of F-actin at the furrow generally precedes loss of membrane (arrows). See Video 4 (available at http://www.jcb.org/cgi/content/full/jcb.200712036/DC1). Bars, 10 μm.
Mentions: Previous studies have shown that the RE component Nuf is required for actin and membrane recruitment to the invaginating furrow (Rothwell et al., 1998; Riggs et al., 2003). To address why nuf mutant embryos exhibit actin as well as membrane defects, we examined furrow formation in living nuf1 (the strongest nuf allele; see Materials and methods) embryos expressing GFP-Moesin. In WT control embryos expressing GFP-Moesin, no breaks were seen at furrows during furrow invagination (Fig. 4 A). In contrast, it appears that the F-actin loss initially occurs basally and progresses apically at the furrows in nuf embryos (Fig. 4 B, arrowheads). Additionally, F-actin loss expands laterally (Fig. 4 B, arrows; and Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200712036/DC1). At metaphase, when furrows have invaginated to their maximal length, breaks were seen throughout the entire length of the furrow resulting in spindle fusions (Fig. 4 B, asterisks; and Video 3). Loss of F-actin stability was also seen in Rab11 embryos during metaphase furrow invagination (Fig. 4 C) and in nuf embryos during cellularization (Fig. S4 A, available at http://www.jcb.org/cgi/content/full/jcb.200712036/DC1). Given that Rab11 is required for RE integrity, these results together indicated that the RE is required to maintain F-actin stability at the invaginating furrows.

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