Myosin 1b flattens and prunes branched actin filaments

Figures

• 

  Fig. 1.. Sliding on Myo1b reduces branching in stabilized F-actin. (A) Myo1b domain organization. (B) Sketch illustrating the gliding assays for phalloidin-stabilized branched (I), and polymerizing and branching (II) F-actin sliding on Myo1b anchored to a coverslip. (C) Representative time-lapse images of phalloidin-stabilized branched F-actin sliding or not on Myo1b (from Movie 1). Yellow arrowheads point to a single branch. Scale bars: 5 µm. (D) Distribution of F-actin (mother and daughter) lengths at t=160 s in one field corresponding to Movie 1 (with no myosin and Myo1b). A two-tailed t-test (P=2.08×10−5) shows a significant difference between the conditions. (E) Mean±s.e.m. relative number of branches along F-actin filaments (see Materials and Methods) normalized by N0, the branch number at t=0, over time in the absence of Myo1b (N0=60, from two movies) or when sliding on Myo1b (N0=38, two movies). (F) Dot plot of sliding velocities vf of stabilized F-actin in the presence of Arp2/3 showing or not showing branches, analyzed from the same movies (five movies). Number of analyzed filaments and mean±s.e.m. are indicated. A two-tailed t-test (P=2.89×10−27) shows a significant difference…

• 

  Fig. 2.. Myo1b reduces stabilized F-actin angles in branched structures. (A) Time-lapse images of stabilized branched F-actin, sliding on Myo1b, showing a change of the angle between mother and daughter filaments (Movie 3). Crosses indicate barbed ends and white arrows the sliding direction. Scale bar: 5 µm. (B) Representation of the θ angle between mother and daughter filaments. (C) Dot plot of θ angle for filaments without myosin (12 filaments, 168 images) or sliding on Myo1b (19 filaments, 168 images). The blue color scale indicates the time since acquisition started. A two-tailed t-test (P=6.16×10−51) shows a significant difference between the conditions. (D) STORM images of stabilized branched F-actin in 2D (top) or 3D (bottom) without or with Myo1b. These images correspond to the squares in Fig. S1A. Color code indicates the height (z). Without Myo1b, branch and mother filaments are in the same plane and attached. On Myo1b, branches #1 and #2 exhibit a θ angle that is much lower than 70°, but are physically connected to the mother filament. Note that branch #2 is in a lower plane. (E) Electron microcopy images of stabilized F-actin, branched or not, recorded after gliding for 10 min…

• 

  Fig. 3.. Sliding on Myo1b decreases branching of polymerizing actin filaments. (A) Effect of Myo1b on branched F-actin polymerization as determined using a pyrene assay. (B) Time-lapse images of polymerizing and branching F-actin in the absence of Myo1b or when sliding on Myo1b with 2 mM ATP (Movie 4). Scale bars: 5 µm. White, cyan and orange arrows point to a mother filament with debranching and branching events, respectively. (C) Numbers of branches per µm of F-actin, without myosin or when sliding on Myo1b. Data are obtained from the total number of branches detected at different times (Fig. S2A) normalized by the corresponding total length of F-actin filaments (Fig. S2C) (three movies). A two-tailed t-test (P=1.47×10−13) shows a significant difference between the conditions. (D) Numbers of debranching events per µm of F-actin, without or with Myo1b, obtained from the same normalization as in C, using data shown in Fig. S2B,C. A two-tailed t-test (P=2.61×10−8) shows a significant difference between the conditions. ****P<0.0001.

• 

  Fig. 4.. Scheme of the forces exerted on a daughter filament. Mother filament sliding (red arrow) induces a friction force (Ffric) on the daughter filament. Ffric combined with the force generated by the motors on the branch (Fmot) results in a total force F at the extremity of the filament, and thus in a torque leading to the reduction of the angle between both filaments (magenta arrow).