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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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Type V myosins Myo52 and Myo51 are essential for cell fusion. (A) Fusion efficiency of indicated heterothallic crosses. myoVΔ (=myo51Δ myo52Δ double mutant) is unable to fuse with either fus1Δ or itself. The total number of mating pairs analyzed (three experiments combined) is indicated on the right. (B) Homothallic h90 wild-type, myo51Δ, myo52Δ, and myoVΔ strains expressing Fus1-sfGFP. Images are time-averaged projections over time 15 min at 1 image/min. Type V myosins are important for Fus1 focalization. (C) Quantifications of Fus1-sfGFP zone size in strains as in B; n = 12. (D) Crosses of h+ wild-type (WT) and myoVΔ strains expressing GFP-CHD to h− myo52-tdTomato. Images are time-averaged projections over 15 min at 1 image/min. Type V myosins are important for actin focalization at the fusion site. (E) Asci derived from homothallic h90 myo52Δ and myo51Δ matings. We observed residual cell wall (blue arrowhead) and narrow necks (green arrowhead) in myo52Δ and myo51Δ, respectively. (F) Percentage of asci with residual cell wall and mean neck width in strains as in E. n > 200. (G) Crosses of h− myo51Δtail-3YFP (top) and h− myo52Δtail-tdTomato (bottom) to h+ wild-type cells. Both truncated motors localize correctly to the fusion focus. (H) Fusion efficiency of indicated heterothallic crosses. Note that a lower fusion efficiency is observed for h+ fus1Δ × h− wild type than for h− fus1Δ × h+ wild type in A. n > 100. Bars, 1 µm. Error bars are standard deviations.
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fig4: Type V myosins Myo52 and Myo51 are essential for cell fusion. (A) Fusion efficiency of indicated heterothallic crosses. myoVΔ (=myo51Δ myo52Δ double mutant) is unable to fuse with either fus1Δ or itself. The total number of mating pairs analyzed (three experiments combined) is indicated on the right. (B) Homothallic h90 wild-type, myo51Δ, myo52Δ, and myoVΔ strains expressing Fus1-sfGFP. Images are time-averaged projections over time 15 min at 1 image/min. Type V myosins are important for Fus1 focalization. (C) Quantifications of Fus1-sfGFP zone size in strains as in B; n = 12. (D) Crosses of h+ wild-type (WT) and myoVΔ strains expressing GFP-CHD to h− myo52-tdTomato. Images are time-averaged projections over 15 min at 1 image/min. Type V myosins are important for actin focalization at the fusion site. (E) Asci derived from homothallic h90 myo52Δ and myo51Δ matings. We observed residual cell wall (blue arrowhead) and narrow necks (green arrowhead) in myo52Δ and myo51Δ, respectively. (F) Percentage of asci with residual cell wall and mean neck width in strains as in E. n > 200. (G) Crosses of h− myo51Δtail-3YFP (top) and h− myo52Δtail-tdTomato (bottom) to h+ wild-type cells. Both truncated motors localize correctly to the fusion focus. (H) Fusion efficiency of indicated heterothallic crosses. Note that a lower fusion efficiency is observed for h+ fus1Δ × h− wild type than for h− fus1Δ × h+ wild type in A. n > 100. Bars, 1 µm. Error bars are standard deviations.

Mentions: We tested the function of type V myosins during cell fusion. Myosin V deletion strains (myo51Δ and myo52Δ and the double myo51Δmyo52Δ mutant, noted myoVΔ throughout) fused inefficiently with wild-type cells and were fusion defective when crossed to fus1Δ (Fig. 4 A). We note that myoVΔ cells fused more efficiently with h+ than h− wild-type partners (not depicted), as also observed for fus1Δ × wild type (Fig. 4, compare A and H), though the significance of this observation is currently unknown. Homothallic myoVΔ cells were also fusion defective. However, even myoVΔ double mutant displayed efficient mating pair formation (similar to their ability to polarize during mitotic growth; Motegi et al., 2001; Win et al., 2001; Bendezú and Martin, 2011), suggesting the observed fusion defect is not caused by prior cell polarization defects. The stronger phenotype of the myoVΔ double mutant, compared with each single mutant, indicates that Myo51 and Myo52 contribute at least partially overlapping function to cell fusion. Fus1 displayed a broader localization in myosin V mutants, especially in the double mutant in which it localized over the entire surface at the contact zone (Fig. 4, B and C). Similarly, actin accumulation at the fusion site spread along the contact zone in the double myoVΔ mutant (Fig. 4 D). Thus, type V myosins are required for focalization of the formin Fus1 and of actin filaments in the focus. As we have shown in Fig. 2 (D and E) that Fus1 is required for myosin V focalization, we conclude that actin fusion focus formation relies on positive reinforcement between formin and type V myosins.


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)

Type V myosins Myo52 and Myo51 are essential for cell fusion. (A) Fusion efficiency of indicated heterothallic crosses. myoVΔ (=myo51Δ myo52Δ double mutant) is unable to fuse with either fus1Δ or itself. The total number of mating pairs analyzed (three experiments combined) is indicated on the right. (B) Homothallic h90 wild-type, myo51Δ, myo52Δ, and myoVΔ strains expressing Fus1-sfGFP. Images are time-averaged projections over time 15 min at 1 image/min. Type V myosins are important for Fus1 focalization. (C) Quantifications of Fus1-sfGFP zone size in strains as in B; n = 12. (D) Crosses of h+ wild-type (WT) and myoVΔ strains expressing GFP-CHD to h− myo52-tdTomato. Images are time-averaged projections over 15 min at 1 image/min. Type V myosins are important for actin focalization at the fusion site. (E) Asci derived from homothallic h90 myo52Δ and myo51Δ matings. We observed residual cell wall (blue arrowhead) and narrow necks (green arrowhead) in myo52Δ and myo51Δ, respectively. (F) Percentage of asci with residual cell wall and mean neck width in strains as in E. n > 200. (G) Crosses of h− myo51Δtail-3YFP (top) and h− myo52Δtail-tdTomato (bottom) to h+ wild-type cells. Both truncated motors localize correctly to the fusion focus. (H) Fusion efficiency of indicated heterothallic crosses. Note that a lower fusion efficiency is observed for h+ fus1Δ × h− wild type than for h− fus1Δ × h+ wild type in A. n > 100. Bars, 1 µm. Error bars are standard deviations.
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fig4: Type V myosins Myo52 and Myo51 are essential for cell fusion. (A) Fusion efficiency of indicated heterothallic crosses. myoVΔ (=myo51Δ myo52Δ double mutant) is unable to fuse with either fus1Δ or itself. The total number of mating pairs analyzed (three experiments combined) is indicated on the right. (B) Homothallic h90 wild-type, myo51Δ, myo52Δ, and myoVΔ strains expressing Fus1-sfGFP. Images are time-averaged projections over time 15 min at 1 image/min. Type V myosins are important for Fus1 focalization. (C) Quantifications of Fus1-sfGFP zone size in strains as in B; n = 12. (D) Crosses of h+ wild-type (WT) and myoVΔ strains expressing GFP-CHD to h− myo52-tdTomato. Images are time-averaged projections over 15 min at 1 image/min. Type V myosins are important for actin focalization at the fusion site. (E) Asci derived from homothallic h90 myo52Δ and myo51Δ matings. We observed residual cell wall (blue arrowhead) and narrow necks (green arrowhead) in myo52Δ and myo51Δ, respectively. (F) Percentage of asci with residual cell wall and mean neck width in strains as in E. n > 200. (G) Crosses of h− myo51Δtail-3YFP (top) and h− myo52Δtail-tdTomato (bottom) to h+ wild-type cells. Both truncated motors localize correctly to the fusion focus. (H) Fusion efficiency of indicated heterothallic crosses. Note that a lower fusion efficiency is observed for h+ fus1Δ × h− wild type than for h− fus1Δ × h+ wild type in A. n > 100. Bars, 1 µm. Error bars are standard deviations.
Mentions: We tested the function of type V myosins during cell fusion. Myosin V deletion strains (myo51Δ and myo52Δ and the double myo51Δmyo52Δ mutant, noted myoVΔ throughout) fused inefficiently with wild-type cells and were fusion defective when crossed to fus1Δ (Fig. 4 A). We note that myoVΔ cells fused more efficiently with h+ than h− wild-type partners (not depicted), as also observed for fus1Δ × wild type (Fig. 4, compare A and H), though the significance of this observation is currently unknown. Homothallic myoVΔ cells were also fusion defective. However, even myoVΔ double mutant displayed efficient mating pair formation (similar to their ability to polarize during mitotic growth; Motegi et al., 2001; Win et al., 2001; Bendezú and Martin, 2011), suggesting the observed fusion defect is not caused by prior cell polarization defects. The stronger phenotype of the myoVΔ double mutant, compared with each single mutant, indicates that Myo51 and Myo52 contribute at least partially overlapping function to cell fusion. Fus1 displayed a broader localization in myosin V mutants, especially in the double mutant in which it localized over the entire surface at the contact zone (Fig. 4, B and C). Similarly, actin accumulation at the fusion site spread along the contact zone in the double myoVΔ mutant (Fig. 4 D). Thus, type V myosins are required for focalization of the formin Fus1 and of actin filaments in the focus. As we have shown in Fig. 2 (D and E) that Fus1 is required for myosin V focalization, we conclude that actin fusion focus formation relies on positive reinforcement between formin and type V myosins.

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