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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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Fus1-dependent actin accumulation at the prospective fusion site. (A) Homothallic h90 pmap3:tdTomato GFP-CHD strain. Arrowheads show the fusion site where actin gradually accumulates. Fusion between partner cells occurs at 100 min as shown by appearance of the tdTomato signal in the h− cell. (B) LatA treatment reduces fusion efficiency of wild-type homothallic h90 strain. Mating cells were starved in MSL−N for 4 h, to allow pheromone response and shmooing, before addition of increasing concentrations of LatA (0, 50, and 200 µg/µl). Cells were immediately spotted on MSL−N 2% agarose pads (not containing LatA and thus diluting the LatA concentration) and incubated overnight at 25°C before imaging for fusion efficiency quantification. n > 200. (C) Homothallic h90 fus1Δ GFP-CHD strain. Cells grow toward each other but are unable to fuse. Though actin patches are present, no actin focus is detected. (D) Quantification of GFP-CHD intensity at the zone of cell contact and of pmap3-driven tdTomato intensity in the h− partner cell in homothallic h90 wild-type mating pairs expressing both markers (as in A). Individual curves were aligned to fusion time and averaged. GFP-CHD intensity at the zone of cell contact in fus1Δ is also indicated, though no alignment could be performed as a result of fusion failure. Error bars are standard deviations. WT, wild type. Bars, 1 µm.
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fig1: Fus1-dependent actin accumulation at the prospective fusion site. (A) Homothallic h90 pmap3:tdTomato GFP-CHD strain. Arrowheads show the fusion site where actin gradually accumulates. Fusion between partner cells occurs at 100 min as shown by appearance of the tdTomato signal in the h− cell. (B) LatA treatment reduces fusion efficiency of wild-type homothallic h90 strain. Mating cells were starved in MSL−N for 4 h, to allow pheromone response and shmooing, before addition of increasing concentrations of LatA (0, 50, and 200 µg/µl). Cells were immediately spotted on MSL−N 2% agarose pads (not containing LatA and thus diluting the LatA concentration) and incubated overnight at 25°C before imaging for fusion efficiency quantification. n > 200. (C) Homothallic h90 fus1Δ GFP-CHD strain. Cells grow toward each other but are unable to fuse. Though actin patches are present, no actin focus is detected. (D) Quantification of GFP-CHD intensity at the zone of cell contact and of pmap3-driven tdTomato intensity in the h− partner cell in homothallic h90 wild-type mating pairs expressing both markers (as in A). Individual curves were aligned to fusion time and averaged. GFP-CHD intensity at the zone of cell contact in fus1Δ is also indicated, though no alignment could be performed as a result of fusion failure. Error bars are standard deviations. WT, wild type. 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)

Fus1-dependent actin accumulation at the prospective fusion site. (A) Homothallic h90 pmap3:tdTomato GFP-CHD strain. Arrowheads show the fusion site where actin gradually accumulates. Fusion between partner cells occurs at 100 min as shown by appearance of the tdTomato signal in the h− cell. (B) LatA treatment reduces fusion efficiency of wild-type homothallic h90 strain. Mating cells were starved in MSL−N for 4 h, to allow pheromone response and shmooing, before addition of increasing concentrations of LatA (0, 50, and 200 µg/µl). Cells were immediately spotted on MSL−N 2% agarose pads (not containing LatA and thus diluting the LatA concentration) and incubated overnight at 25°C before imaging for fusion efficiency quantification. n > 200. (C) Homothallic h90 fus1Δ GFP-CHD strain. Cells grow toward each other but are unable to fuse. Though actin patches are present, no actin focus is detected. (D) Quantification of GFP-CHD intensity at the zone of cell contact and of pmap3-driven tdTomato intensity in the h− partner cell in homothallic h90 wild-type mating pairs expressing both markers (as in A). Individual curves were aligned to fusion time and averaged. GFP-CHD intensity at the zone of cell contact in fus1Δ is also indicated, though no alignment could be performed as a result of fusion failure. Error bars are standard deviations. WT, wild type. Bars, 1 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4384723&req=5

fig1: Fus1-dependent actin accumulation at the prospective fusion site. (A) Homothallic h90 pmap3:tdTomato GFP-CHD strain. Arrowheads show the fusion site where actin gradually accumulates. Fusion between partner cells occurs at 100 min as shown by appearance of the tdTomato signal in the h− cell. (B) LatA treatment reduces fusion efficiency of wild-type homothallic h90 strain. Mating cells were starved in MSL−N for 4 h, to allow pheromone response and shmooing, before addition of increasing concentrations of LatA (0, 50, and 200 µg/µl). Cells were immediately spotted on MSL−N 2% agarose pads (not containing LatA and thus diluting the LatA concentration) and incubated overnight at 25°C before imaging for fusion efficiency quantification. n > 200. (C) Homothallic h90 fus1Δ GFP-CHD strain. Cells grow toward each other but are unable to fuse. Though actin patches are present, no actin focus is detected. (D) Quantification of GFP-CHD intensity at the zone of cell contact and of pmap3-driven tdTomato intensity in the h− partner cell in homothallic h90 wild-type mating pairs expressing both markers (as in A). Individual curves were aligned to fusion time and averaged. GFP-CHD intensity at the zone of cell contact in fus1Δ is also indicated, though no alignment could be performed as a result of fusion failure. Error bars are standard deviations. WT, wild type. 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