A formin-nucleated actin aster concentrates cell wall hydrolases for cell fusion in fission yeast.
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.
Affiliation: Department of Fundamental Microbiology, Faculty of Biology and Medicine, University of Lausanne, 1015 Lausanne, Switzerland.Show MeSH
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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.
Affiliation: Department of Fundamental Microbiology, Faculty of Biology and Medicine, University of Lausanne, 1015 Lausanne, Switzerland.