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A formin-nucleated actin aster concentrates cell wall hydrolases for cell fusion in fission yeast.

Dudin O, Bendezú FO, Groux R, Laroche T, Seitz A, Martin SG - J. Cell Biol. (2015)

Bottom Line: In fission yeast cells, the formin Fus1, which nucleates linear actin filaments, is essential for this process.Structured illumination microscopy and live-cell imaging of Fus1, actin, and type V myosins revealed an aster of actin filaments whose barbed ends are focalized near the plasma membrane.Focalization requires Fus1 and type V myosins and happens asynchronously always in the M cell first.

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Affiliation: Department of Fundamental Microbiology, Faculty of Biology and Medicine, University of Lausanne, 1015 Lausanne, Switzerland.

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Composition and architecture of the actin fusion focus. (A) Homothallic h90 myo52-tdTomato GFP-CHD strain. Myo52 localizes as an intense dot at the cell–cell contact site, at the edge of the actin density. (B) Time-lapse imaging of homothallic h90 fus1-sfGFP pmap3:tdTomato strain. Entry of tdTomato in the h− cell is used as a marker for fusion. Fus1 is detected as an intense dot at the cell–cell contact site. (C) Homothallic h90 myo52-tdTomato fus1-sfGFP strain. Myo52 and Fus1 colocalize at the fusion site. (D) Homothallic h90 wild-type (left) and fus1Δ (right) strains expressing Myo52-GFP. Myo52 localizes as a crescent at the shmoo tip in the absence of Fus1. Cell outlines are shown with dotted lines. (E) Cross of heterothallic h+ myo52-tdTomato and h90 fus1Δ myo52-GFP. Myo52 forms a crescent in the fus1Δ cell and a dot in the wild-type cell. (F) 3D SIM time-lapse of GFP-CHD in homothallic h90 wild-type (WT), for3Δ, fus1Δ, and fus1Δ for3Δ mating pairs. Inverted images are shown. Green arrows point to actin filaments emanating from the fusion focus. No actin cables were detected in fus1Δ for3Δ double mutant, but some mating pairs showed a perinuclear actin ring (asterisks). (G) Mean length of actin filaments emanating from the fusion focus in strains as in F. Filaments are significantly shorter in for3Δ and longer in fus1Δ than wild-type cells (t test, ***, P < 10−6). This indicates that Fus1-dependent filaments are shorter than For3-dependent filaments and that wild-type cells likely contain both types. n = 30 actin filaments measured in three distinct mating pairs. Error bars are standard deviations. (H) Crosses of heterothallic h+ and h− myo52-tdTomato strains coexpressing Cdc12-3GFP, mEGFP-Cdc15, For3-3GFP, Myo51-3YFP, mGFP-Myo1, or Dip1-GFP. Images shown are time-averaged maximum intensity projection of 15 z stacks over 15 min. (I) Venn diagram summarizing the actin binding proteins that we show to be localized or not at the actin fusion focus. Attribution to cables, ring, or patches is adapted from Kovar et al. (2011). Bars, 1 µm.
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fig2: Composition and architecture of the actin fusion focus. (A) Homothallic h90 myo52-tdTomato GFP-CHD strain. Myo52 localizes as an intense dot at the cell–cell contact site, at the edge of the actin density. (B) Time-lapse imaging of homothallic h90 fus1-sfGFP pmap3:tdTomato strain. Entry of tdTomato in the h− cell is used as a marker for fusion. Fus1 is detected as an intense dot at the cell–cell contact site. (C) Homothallic h90 myo52-tdTomato fus1-sfGFP strain. Myo52 and Fus1 colocalize at the fusion site. (D) Homothallic h90 wild-type (left) and fus1Δ (right) strains expressing Myo52-GFP. Myo52 localizes as a crescent at the shmoo tip in the absence of Fus1. Cell outlines are shown with dotted lines. (E) Cross of heterothallic h+ myo52-tdTomato and h90 fus1Δ myo52-GFP. Myo52 forms a crescent in the fus1Δ cell and a dot in the wild-type cell. (F) 3D SIM time-lapse of GFP-CHD in homothallic h90 wild-type (WT), for3Δ, fus1Δ, and fus1Δ for3Δ mating pairs. Inverted images are shown. Green arrows point to actin filaments emanating from the fusion focus. No actin cables were detected in fus1Δ for3Δ double mutant, but some mating pairs showed a perinuclear actin ring (asterisks). (G) Mean length of actin filaments emanating from the fusion focus in strains as in F. Filaments are significantly shorter in for3Δ and longer in fus1Δ than wild-type cells (t test, ***, P < 10−6). This indicates that Fus1-dependent filaments are shorter than For3-dependent filaments and that wild-type cells likely contain both types. n = 30 actin filaments measured in three distinct mating pairs. Error bars are standard deviations. (H) Crosses of heterothallic h+ and h− myo52-tdTomato strains coexpressing Cdc12-3GFP, mEGFP-Cdc15, For3-3GFP, Myo51-3YFP, mGFP-Myo1, or Dip1-GFP. Images shown are time-averaged maximum intensity projection of 15 z stacks over 15 min. (I) Venn diagram summarizing the actin binding proteins that we show to be localized or not at the actin fusion focus. Attribution to cables, ring, or patches is adapted from Kovar et al. (2011). Bars, 1 µm.

Mentions: To examine the role of the actin cytoskeleton during cell–cell fusion, we localized F-actin in live cells, using a GFP–calponin homology domain (CHD) reporter construct (Karagiannis et al., 2005; Martin and Chang, 2006). GFP-CHD has been used to study actin structures during mitotic growth, labeling the three actin structures present in these cells: the cytokinetic actin contractile ring nucleated by the formin Cdc12, actin cables assembled by the formin For3, and actin patches, which require Arp2/3 activity (Kovar et al., 2011). Strikingly, during sexual differentiation, we observed an intense accumulation of F-actin at the site of fusion (Fig. 1 A), which appeared distinct from these known actin structures. This structure dynamically formed before cell fusion, which we define as the time of entry in the h− cell of tdTomato driven by an h+ cell-specific promoter (pmap3:tdTomato), and decreased after fusion (Fig. 1, A and D; and Video 1). F-actin accumulation was also observed using LifeAct-GFP in live cells and phalloidin staining on fixed samples (Fig. S1). Disruption of F-actin by treatment with Latrunculin A (LatA), added 4 h after initiation of sexual differentiation upon nitrogen starvation, reduced fusion efficiency in a dose-dependent manner (Fig. 1 B), suggesting that F-actin is essential for cell–cell fusion. Consistent with the molecular function of the pheromone-dependent formin Fus1, F-actin did not accumulate at the site of fusion in fus1Δ pairs, though dynamic actin patches were detected at the shmoo tip of these cells (Fig. 1, C and D; Fig. S1; and Video 2). Similarly, fus1-dependent actin accumulation at the fusion site was previously observed on fixed cells and described as an accumulation of actin patches (Petersen et al., 1998a,b). In contrast, we describe in Figs. 2 and S2 a distinct architecture and composition of this actin structure, which we named the actin fusion focus.


A formin-nucleated actin aster concentrates cell wall hydrolases for cell fusion in fission yeast.

Dudin O, Bendezú FO, Groux R, Laroche T, Seitz A, Martin SG - J. Cell Biol. (2015)

Composition and architecture of the actin fusion focus. (A) Homothallic h90 myo52-tdTomato GFP-CHD strain. Myo52 localizes as an intense dot at the cell–cell contact site, at the edge of the actin density. (B) Time-lapse imaging of homothallic h90 fus1-sfGFP pmap3:tdTomato strain. Entry of tdTomato in the h− cell is used as a marker for fusion. Fus1 is detected as an intense dot at the cell–cell contact site. (C) Homothallic h90 myo52-tdTomato fus1-sfGFP strain. Myo52 and Fus1 colocalize at the fusion site. (D) Homothallic h90 wild-type (left) and fus1Δ (right) strains expressing Myo52-GFP. Myo52 localizes as a crescent at the shmoo tip in the absence of Fus1. Cell outlines are shown with dotted lines. (E) Cross of heterothallic h+ myo52-tdTomato and h90 fus1Δ myo52-GFP. Myo52 forms a crescent in the fus1Δ cell and a dot in the wild-type cell. (F) 3D SIM time-lapse of GFP-CHD in homothallic h90 wild-type (WT), for3Δ, fus1Δ, and fus1Δ for3Δ mating pairs. Inverted images are shown. Green arrows point to actin filaments emanating from the fusion focus. No actin cables were detected in fus1Δ for3Δ double mutant, but some mating pairs showed a perinuclear actin ring (asterisks). (G) Mean length of actin filaments emanating from the fusion focus in strains as in F. Filaments are significantly shorter in for3Δ and longer in fus1Δ than wild-type cells (t test, ***, P < 10−6). This indicates that Fus1-dependent filaments are shorter than For3-dependent filaments and that wild-type cells likely contain both types. n = 30 actin filaments measured in three distinct mating pairs. Error bars are standard deviations. (H) Crosses of heterothallic h+ and h− myo52-tdTomato strains coexpressing Cdc12-3GFP, mEGFP-Cdc15, For3-3GFP, Myo51-3YFP, mGFP-Myo1, or Dip1-GFP. Images shown are time-averaged maximum intensity projection of 15 z stacks over 15 min. (I) Venn diagram summarizing the actin binding proteins that we show to be localized or not at the actin fusion focus. Attribution to cables, ring, or patches is adapted from Kovar et al. (2011). Bars, 1 µm.
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Related In: Results  -  Collection

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fig2: Composition and architecture of the actin fusion focus. (A) Homothallic h90 myo52-tdTomato GFP-CHD strain. Myo52 localizes as an intense dot at the cell–cell contact site, at the edge of the actin density. (B) Time-lapse imaging of homothallic h90 fus1-sfGFP pmap3:tdTomato strain. Entry of tdTomato in the h− cell is used as a marker for fusion. Fus1 is detected as an intense dot at the cell–cell contact site. (C) Homothallic h90 myo52-tdTomato fus1-sfGFP strain. Myo52 and Fus1 colocalize at the fusion site. (D) Homothallic h90 wild-type (left) and fus1Δ (right) strains expressing Myo52-GFP. Myo52 localizes as a crescent at the shmoo tip in the absence of Fus1. Cell outlines are shown with dotted lines. (E) Cross of heterothallic h+ myo52-tdTomato and h90 fus1Δ myo52-GFP. Myo52 forms a crescent in the fus1Δ cell and a dot in the wild-type cell. (F) 3D SIM time-lapse of GFP-CHD in homothallic h90 wild-type (WT), for3Δ, fus1Δ, and fus1Δ for3Δ mating pairs. Inverted images are shown. Green arrows point to actin filaments emanating from the fusion focus. No actin cables were detected in fus1Δ for3Δ double mutant, but some mating pairs showed a perinuclear actin ring (asterisks). (G) Mean length of actin filaments emanating from the fusion focus in strains as in F. Filaments are significantly shorter in for3Δ and longer in fus1Δ than wild-type cells (t test, ***, P < 10−6). This indicates that Fus1-dependent filaments are shorter than For3-dependent filaments and that wild-type cells likely contain both types. n = 30 actin filaments measured in three distinct mating pairs. Error bars are standard deviations. (H) Crosses of heterothallic h+ and h− myo52-tdTomato strains coexpressing Cdc12-3GFP, mEGFP-Cdc15, For3-3GFP, Myo51-3YFP, mGFP-Myo1, or Dip1-GFP. Images shown are time-averaged maximum intensity projection of 15 z stacks over 15 min. (I) Venn diagram summarizing the actin binding proteins that we show to be localized or not at the actin fusion focus. Attribution to cables, ring, or patches is adapted from Kovar et al. (2011). Bars, 1 µm.
Mentions: To examine the role of the actin cytoskeleton during cell–cell fusion, we localized F-actin in live cells, using a GFP–calponin homology domain (CHD) reporter construct (Karagiannis et al., 2005; Martin and Chang, 2006). GFP-CHD has been used to study actin structures during mitotic growth, labeling the three actin structures present in these cells: the cytokinetic actin contractile ring nucleated by the formin Cdc12, actin cables assembled by the formin For3, and actin patches, which require Arp2/3 activity (Kovar et al., 2011). Strikingly, during sexual differentiation, we observed an intense accumulation of F-actin at the site of fusion (Fig. 1 A), which appeared distinct from these known actin structures. This structure dynamically formed before cell fusion, which we define as the time of entry in the h− cell of tdTomato driven by an h+ cell-specific promoter (pmap3:tdTomato), and decreased after fusion (Fig. 1, A and D; and Video 1). F-actin accumulation was also observed using LifeAct-GFP in live cells and phalloidin staining on fixed samples (Fig. S1). Disruption of F-actin by treatment with Latrunculin A (LatA), added 4 h after initiation of sexual differentiation upon nitrogen starvation, reduced fusion efficiency in a dose-dependent manner (Fig. 1 B), suggesting that F-actin is essential for cell–cell fusion. Consistent with the molecular function of the pheromone-dependent formin Fus1, F-actin did not accumulate at the site of fusion in fus1Δ pairs, though dynamic actin patches were detected at the shmoo tip of these cells (Fig. 1, C and D; Fig. S1; and Video 2). Similarly, fus1-dependent actin accumulation at the fusion site was previously observed on fixed cells and described as an accumulation of actin patches (Petersen et al., 1998a,b). In contrast, we describe in Figs. 2 and S2 a distinct architecture and composition of this actin structure, which we named the actin fusion focus.

Bottom Line: In fission yeast cells, the formin Fus1, which nucleates linear actin filaments, is essential for this process.Structured illumination microscopy and live-cell imaging of Fus1, actin, and type V myosins revealed an aster of actin filaments whose barbed ends are focalized near the plasma membrane.Focalization requires Fus1 and type V myosins and happens asynchronously always in the M cell first.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Fundamental Microbiology, Faculty of Biology and Medicine, University of Lausanne, 1015 Lausanne, Switzerland.

Show MeSH
Related in: MedlinePlus