PMC

The biochemical cycle of the myosin heads with rates k₁–k₆ assumed in the model and the simulations. The rate k₅ is ATP-dependent.
DOI: http://dx.doi.org/10.7554/eLife.00116.016

The fractions of myosin heads in states 1–6, and in the actin-bound state (sum of states 3–6) in dependence on the ATP concentration. The values are calculated from the model of the myosin head cycle in .
DOI: http://dx.doi.org/10.7554/eLife.00116.017

Fluctuations of the compression force (upper row), the actin curvature (middle row) and the fraction of attached myosin heads (lower row) during 20 s of the simulation, for three different ATP concentrations (p_st=0).
DOI: http://dx.doi.org/10.7554/eLife.00116.015

Dependence of the mean velocity and mean number of attached myosin heads on the ATP concentration obtained from the simulations of myofilament–actin filament interaction. (A) Mean velocity (points and solid line: p_st=0, dashed line: p_st=0.1). (B) Mean fraction of attached myosin heads.
DOI: http://dx.doi.org/10.7554/eLife.00116.014

Trajectories of individual myofilaments moving along actin filaments. Left panel displays the trajectories of myofilaments at low (1 µM) ATP concentration (see also the corresponding ). Right panel shows trajectories of myofilaments at high (4 mM) ATP concentration (see also the corresponding ). Myofilaments were tracked for one minute and those who stayed less than 900 ms attached to the actin filament were filtered out ().
DOI: http://dx.doi.org/10.7554/eLife.00116.012

Simulation of the interaction between myofilaments and an actin filament. (A) Mean tension force F within the myofilament when bending of actin is not allowed; dependence on ATP concentration for several different numbers of interacting myosin heads n_m. The forces when the myosin heads of the trailing end are not performing steps (p_st=0, points connected by a solid line) are slightly higher than the forces when the steps occur with the probability p_st=0.1 (dashed lines). (B) Mean tension force when the actin filament is allowed to bend at the threshold force of 23 pN (points and solid line: p_st=0, dashed line: p_st=0.1). (C) Actin filament curvature fluctuations during 20 s of the simulation at 0.0025 mM ATP concentration, showing that the critical curvature of 5.6 µm⁻¹ needed for actin filament breakage is often reached, while at higher ATP concentration (0.01 mM), the critical curvature is never reached (D) (p_st=0 in (C) and (D)).
DOI: http://dx.doi.org/10.7554/eLife.00116.011

Single molecule analysis of the myofilament movement and actin fragmentation. (A) Dual-color TIRFM time-lapse sequence of a Alexa-647 labeled myofilament (red) moving along an Alexa-488-phalloidin labeled actin filament (green). White asterisks mark the position of the myofilament. Yellow arrowheads point to actin filament deformations. White arrowheads indicate an increase in fluorescence intensity. Scale bars, 5 µm. (B) x (grey curve) and y (red curve) positions of the myofilament movement shown in (A) as a function of time. Inset depicts the trajectory (green curve). (C) Myofilament velocity (red curve) calculated from the xy positions in (B) and actin filament intensity (blue [raw data] and black [smoothed] curves) over time. Red arrowheads denote acceleration events. Black arrows point to fluorescence intensity increases. Red arrowheads in (A)–(C) mark corresponding time points in (A). (D) Proposed model for myofilament driven actin fragmentation and compaction (details in text).
DOI: http://dx.doi.org/10.7554/eLife.00116.009

MAC composition and actin pattern formation by myofilaments. (A) Scheme of the MAC. Biotinylated actin filaments are coupled to a supported lipid bilayer (Egg PC) containing biotinylated lipids (DSPE-PEG(2000)-Biotin) via Neutravidin. (B) TIRFM images of MACs containing Alexa-488-phalloidin labeled actin filaments. The increase of actin filament densities (left to right) corresponds to an increase in the amount of DSPE-PEG200-Biotin (low = 0.01 mol%, medium = 0.1 mol%, high = 1 mol%) in the membrane. Scale bars, 10 µm. (C) Length distribution of myofilaments. The median length (L_m) and the 25th and 75th percentile (brackets) are indicated in µm. Inset shows a topographical AFM image of a myofilament. Height, 12 nm; scale bar 200 nm. (D) Dual-color TIRFM time-lapse images of a medium actin density MAC with Alexa-488-phalloidin labeled actin filaments (green) and myofilaments (0.3 µM unlabeled myosin II doped with Alexa 647 myosin II (red)) before (left image) and during actin pattern formation. Scale bars, 10 µm.
DOI: http://dx.doi.org/10.7554/eLife.00116.003

Actin filament shortening and compaction by myofilaments. (A) TIRFM time-lapse images of a low actin density MAC with Alexa-488-phalloidin labeled actin filaments before (left image) and after addition of (non-labeled) myofilaments (0.3 µM). Scale bars, 10 µm. (B) Actin filament length distribution at 0, 20 and 53 min after myofilament addition. The median length (L_m) and the 25th and 75th percentile (brackets) are indicated in µm. (C) TIRFM time-lapse sequence of an Alexa-488-phalloidin labeled actin filament in the presence of myofilaments (0.3 µM). Yellow arrowheads point at deformation and breakage events. White arrowheads indicate an increase in fluorescence intensity. Scale bar, 5 µm. (D) and (E) image and the corresponding intensity profile (blue curve) of the actin filament. The intensity was measured along the yellow dashed line shown in (D). The line started and ended outside the actin filament to indicate the background level. Asteriks in (C) and (D) mark the image taken for the intensity profile measurement.
DOI: http://dx.doi.org/10.7554/eLife.00116.006