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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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F-actin is required for maintaining membrane integrity during furrow invagination. (A) In untreated cycle-12 embryos, membrane (GFP-Dlg, green) is closely surrounded by F-actin (red) at the furrow. (B) 2 mM LatA or DMSO was injected at cycle-13 early prophase. GFP-Dlg–marked plasma membrane was progressively lost (arrowheads) as furrows invaginated in LatA-injected embryos (top row). Membrane loss started after most of the F-actin was lost at the furrow (middle row). (C) Furrow integrity was monitored relative to spindle spacing and dynamics (tubulin, red). Spindle fusion (asterisks) occurred where membrane (Dlg, green) was lost at the furrow. See Video 2 (available at http://www.jcb.org/cgi/content/full/jcb.200712036/DC1). Bars, 10 μm.
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fig3: F-actin is required for maintaining membrane integrity during furrow invagination. (A) In untreated cycle-12 embryos, membrane (GFP-Dlg, green) is closely surrounded by F-actin (red) at the furrow. (B) 2 mM LatA or DMSO was injected at cycle-13 early prophase. GFP-Dlg–marked plasma membrane was progressively lost (arrowheads) as furrows invaginated in LatA-injected embryos (top row). Membrane loss started after most of the F-actin was lost at the furrow (middle row). (C) Furrow integrity was monitored relative to spindle spacing and dynamics (tubulin, red). Spindle fusion (asterisks) occurred where membrane (Dlg, green) was lost at the furrow. See Video 2 (available at http://www.jcb.org/cgi/content/full/jcb.200712036/DC1). Bars, 10 μm.

Mentions: To further investigate the relationship between F-actin stability and membrane integrity, we injected LatA into embryos that were previously injected with Rhodamine-labeled actin and also expressed GFP-Dlg. As shown in Fig. 3 A, GFP-Dlg–marked plasma membrane was closely surrounded by F-actin at the furrow in untreated WT embryos. LatA injection caused an immediate loss of F-actin signal intensity at the furrow (Fig. 3 B). Initially, the injection had no apparent effect on GFP-Dlg distribution, but by 3 min after injection, large gaps of GFP-Dlg–labeled membrane were visible (Fig. 3 B, arrowheads). These gaps became more extensive as furrow progression proceeded. The same effect on furrow membrane integrity was seen in older cellularizing embryos after disrupting F-actin at the furrow by LatA injection (Fig. S3 A, available at http://www.jcb.org/cgi/content/full/jcb.200712036/DC1). These studies indicate that in both cycle 13 and cellularization-stage embryos, F-actin loss at the furrow was followed by the loss of membrane integrity. To further verify these results, we analyzed the effect of LatA with a different plasma membrane marker, Spider-GFP, a casein kinase I which is associated with the plasma membrane (Frescas et al., 2006). As with the GFP-Dlg, LatA injection led to breaks in the previously contiguous Spider-GFP signal (Fig. S3 B). These data demonstrate that F-actin is required for maintaining membrane integrity at the invaginating 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)

F-actin is required for maintaining membrane integrity during furrow invagination. (A) In untreated cycle-12 embryos, membrane (GFP-Dlg, green) is closely surrounded by F-actin (red) at the furrow. (B) 2 mM LatA or DMSO was injected at cycle-13 early prophase. GFP-Dlg–marked plasma membrane was progressively lost (arrowheads) as furrows invaginated in LatA-injected embryos (top row). Membrane loss started after most of the F-actin was lost at the furrow (middle row). (C) Furrow integrity was monitored relative to spindle spacing and dynamics (tubulin, red). Spindle fusion (asterisks) occurred where membrane (Dlg, green) was lost at the furrow. See Video 2 (available at http://www.jcb.org/cgi/content/full/jcb.200712036/DC1). Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2483530&req=5

fig3: F-actin is required for maintaining membrane integrity during furrow invagination. (A) In untreated cycle-12 embryos, membrane (GFP-Dlg, green) is closely surrounded by F-actin (red) at the furrow. (B) 2 mM LatA or DMSO was injected at cycle-13 early prophase. GFP-Dlg–marked plasma membrane was progressively lost (arrowheads) as furrows invaginated in LatA-injected embryos (top row). Membrane loss started after most of the F-actin was lost at the furrow (middle row). (C) Furrow integrity was monitored relative to spindle spacing and dynamics (tubulin, red). Spindle fusion (asterisks) occurred where membrane (Dlg, green) was lost at the furrow. See Video 2 (available at http://www.jcb.org/cgi/content/full/jcb.200712036/DC1). Bars, 10 μm.
Mentions: To further investigate the relationship between F-actin stability and membrane integrity, we injected LatA into embryos that were previously injected with Rhodamine-labeled actin and also expressed GFP-Dlg. As shown in Fig. 3 A, GFP-Dlg–marked plasma membrane was closely surrounded by F-actin at the furrow in untreated WT embryos. LatA injection caused an immediate loss of F-actin signal intensity at the furrow (Fig. 3 B). Initially, the injection had no apparent effect on GFP-Dlg distribution, but by 3 min after injection, large gaps of GFP-Dlg–labeled membrane were visible (Fig. 3 B, arrowheads). These gaps became more extensive as furrow progression proceeded. The same effect on furrow membrane integrity was seen in older cellularizing embryos after disrupting F-actin at the furrow by LatA injection (Fig. S3 A, available at http://www.jcb.org/cgi/content/full/jcb.200712036/DC1). These studies indicate that in both cycle 13 and cellularization-stage embryos, F-actin loss at the furrow was followed by the loss of membrane integrity. To further verify these results, we analyzed the effect of LatA with a different plasma membrane marker, Spider-GFP, a casein kinase I which is associated with the plasma membrane (Frescas et al., 2006). As with the GFP-Dlg, LatA injection led to breaks in the previously contiguous Spider-GFP signal (Fig. S3 B). These data demonstrate that F-actin is required for maintaining membrane integrity at the invaginating 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