Myosin 1b is an actin depolymerase

Figures

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  Fig. 1. Myo1b-Actin gliding assays. a Schematic representation of domain organization of Myo1b. Motor domain (blue); Light Chain Binding Domain (LCBD) (yellow); TH1 domain (red), PH domain (cyan) that binds phosphoinositides. b–e Gliding assays of stabilized actin filaments (b–c) and polymerizing actin filaments (d–e) sliding on Myo1b anchored on coverslip (b–d) or bound to a supported lipid bilayer (c–e)

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  Fig. 2. Sliding on immobilized myosin 1b increases F-actin depolymerization. a Representative kymographs of stabilized F-actin (top) or polymerizing F-actin with 0.6 µM G-actin (bottom), on uncoated glass or sliding on glass coated with Myo1b (2 mM and 0.2 mM ATP (see Supplementary Movies 2 and 3) or MyoII (see Supplementary Movie 6). The sliding distance ΔX and the elongation ΔL of the filaments are indicated by white arrows. Actin fluorescence intensity is represented according to the Fire LUT of Image J. Scale bar, 5μm. 1 image/10 s. b Dot plot representation of the sliding velocities vf of stabilized (top) and polymerizing actin filaments (0.6 µM G-actin) (bottom) on immobilized Myo1b (8000 molecules/μm2) at 2 mM (blue) or 0.2 mM (gray) ATP or sliding on MyoII at 2 mM ATP (orange). The number of analyzed filaments and the mean-values ± s.e.m. are indicated. c Filament elongation ΔL (normalized by the length of the actin subunit (su) equal to 2.7 nm) versus time for filaments shown in A (bottom) in the absence of myosins and in the presence of MyoII or Myo1b at two ATP concentrations. The polymerization rate at the barbed end vp (in su/s) is deduced from the slope. d vp as a function…

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  Fig. 3. Sliding on myosin 1b bound to SLB increases F-actin depolymerization. a Top: Diffusion coefficients of Atto488-DOPE (DOPE*) and Alexa488-labeled Myo1b (Myo1b*) in a SLB with bound Myo1b, with or without 0.3 % methylcellulose (MEC), and in absence or in the presence of a dense F-actin network (n = 30). See Supplementary Fig. 3 for representative FRAP experiments. Bottom: Effective diffusion coefficients of Cherry-CAAX, Cherry-Myo1b, expressed in HEK293T cells (n > 5). Error bars represent s.e.m. b Representative kymographs of non-polymerizing (top) and polymerizing F-actin (bottom) in the presence of 0.6 µM G-actin with Myo1b bound to SLBs (Supplementary Movie 8). Scale bar, 5 μm. 1 image/10 s. c Dot blot representation of the velocities vf of stabilized (top) and polymerizing F-actin (bottom) sliding on immobilized Myo1b (dark blue) or on Myo1b bound to a SLB (cyan). The number of analyzed filaments is indicated. d Model for filament sliding: The effective filament sliding is determined by a balance between the viscous dissipation of the motor moving with a velocity vm in the lipid bilayer with a viscosity ηm and a filament sliding at a velocity \documentclass[12pt]{minimal}…