Genetic suppression of defective profilin by attenuated Myosin II reveals a potential role for Myosin II in actin dynamics in vivo in fission yeast

FIGURE 1:. myo2-S1 suppresses the lethality of cdc3-124 at the nonpermissive temperature. (A) Tenfold serial dilutions of wild-type, cdc3-124, myo2-S1, cdc3-124 myo2-S1, myo2-S2, and cdc3-124 myo2-S2 were spotted onto YEA agar plates and grown for 3 d at 24, 34, and 36°C. (B) CW staining was used to visualize the septum of wild-type, cdc3-124, myo2-S1, and cdc3-124 myo2-S1 cells at 34°C. In the first column it is shown the acquired front view image of the septum, while in the second column it is displayed the face-on view of the septum, which was generated with Fiji software. Scale bar 3 µm. (C) Tenfold serial dilutions of wild-type, cdc8-110, cdc12-112, myo2-S1, cdc8-110 myo2-S1, and cdc12-112 myo2-S1 were spotted onto YEA agar plates and grown for 3 d at 24, 34, and 36°C. (D) Cells of the indicated genotypes were grown at 24°C and shifted for 4 h at 34°C and stained with CF633-phalloidin and DAPI. Arrows indicated normal AMRs while asterisks indicated abnormal rings. Scale bar 5 µm. (E) Quantification of CF633-phalloidin and DAPI staining in D of three independent experiments (n = 3). Cells with two nuclei were classified depending on the presence of no ring (2N/no ring), 1 normal ring…

FIGURE 2:. AMR assembly and contraction is partially restored in myo2-S1 and cdc3-124 myo2-S1 at 34°C. (A) Time-lapse series of strains of the indicated genotypes expressing Rlc1-3GFP and mCherry-Atb2. Cells were grown at 24°C and shifted 3 h at 34°C before being imaged at 34°C. More than 20 cells were imaged and quantified for each strain. On the side of each montage it is indicated the percentage of the different cytokinetic behaviors categorized into class I (normal AMR), class II (failed AMR assembly), class III (normal AMR assembly and asymmetrical AMR contraction), and class IV (abnormal AMR structures). Images shown are maximum-intensity projections of Z-stacks. Time indicated in minutes. Scale bars 3 μm. (B) Quantification of the time necessary for AMR assembly of class I and class III cells imaged in A. Statistical significance: Student’s test (****p < 0.0001). Error bars represent SD. (C) Quantification of the time necessary for AMR contraction of class I and class III cells imaged in A. Statistical significance: Student’s test (****p < 0.0001). Error bars represent SD.

FIGURE 3:. Structural basis of S. pombe Myo2-S1 (G515D) mutation. (A) Gly515 in S. pombe Myo2 (equivalent to Gly549 of human myosin 14 in the actomyosin complex structure of PDB ID: 5JLH) is positioned at the boundary of the activation loop at the myosin-actin interface. Inset shows the zoomed view of the interaction between the activation loop and the N-terminus of actin protomer. Gly549 and Arg543, corresponding to Gly515 and Lys510 of S. pombe Myo2, are shown in stick representation. The figure was generated using PyMol. (B) Conservation of Gly515 and Lys510 depicted on a sequence logo representation. The figure was generated using WebLogo (Crooks et al., 2004) using a sequence alignment of 237 nonredundant myosin sequences aligned using ClustalO (Sievers et al., 2011; Sievers and Higgins, 2018). The sequence corresponding to the activation loop was selected using JalView (Waterhouse et al., 2009).

FIGURE 4:. AMR isolated from myo2-S1 and cdc3-124 myo2-S1 cells do not undergo ATP-dependent contraction. (A) AMR were prepared from wild-type (n = 17), myo2-S1 (n = 14), and cdc3-124 myo2-S1 (n = 20) cells grown at 24°C in the presence of 20 µM Jasp. Isolated rings were shifted 15 min at 34°C before proceeding with imaging. Ring contraction experiments were performed at 34°C and contraction was activated by the addition of 0.5 mM ATP (indicated by the red bar). Myosin regulatory light chain (Rlc1-3GFP) was used as a marker protein to visualize AMR. Images shown are maximum intensity projections of Z-stacks. Time indicated in minutes. Scale bar 5 μm. (B) Percentage of rings that either contracted normally or presented slow/no contraction of strains imaged at 34°C illustrated in A. (C) Percentage of rings that either contracted normally or presented slow/no contraction of strains imaged at 24°C illustrated in A.

FIGURE 5:. myo2-S1 suppresses perturbations of actin induced by LatA. (A) Tenfold serial dilutions of wild-type, cdc3-124, myo2-S1, and cdc3-124 myo2-S1 cells were spotted onto YEA agar plates containing different concentrations of LatA and grown for 3 d at 34 and 36°C. (B) Cells were grown at 24°C and shifted for 3 h at 34°C in the presence of 0.375 µM of LatA before PFA fixation. CF633-phalloidin was used to visualize actin structures of wild-type, cdc3-124, myo2-S1, and cdc3-124 myo2-S1 cells. Arrows indicated the presence of actin cables. Scale bar 5 µm. (C) Quantification of the presence of actin cables or AMR structure detected with CF633-phalloidin staining in B of three independent experiments (n = 3).