Research ArticleBIOPHYSICS

Stratification relieves constraints from steric hindrance in the generation of compact actomyosin asters at the membrane cortex

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Science Advances  11 Mar 2020:
Vol. 6, no. 11, eaay6093
DOI: 10.1126/sciadv.aay6093
  • Fig. 1 Ingredients of agent-based simulations in 2D and in vitro reconstitution setup.

    (A) Schematic of actin filaments (red) and myosin minifilaments (green) used in the agent-based model, with real-space dimensions indicated. One F-actin bead corresponds to 40 G-actin monomers, and one myosin bead corresponds to 3 to 4 heads. (B) Schematic showing a collection of F-actin and myosin minifilaments in 2D, with myosin motors bound to two actin filaments (bound myosin heads are colored blue). (C) Schematic of the in vitro reconstitution system showing the hierarchical assembly of an SLB, linker protein [His-YFP-Ezrin (HYE)], capped actin filaments, and muscle myosin-II. (D to F) Typical simulation snapshots of different aster configurations at steady state, observed in the strictly 2D simulations, as a function of increasing F-actin length la: (D) la = 2.32 μm, (E) la = 4.84 μm, and (F) la = 719 μm. The (+)-ends of the actin filaments are colored black. (G to I) Typical aster configurations at steady state observed in in vitro experiments: isolated asters, connected asters, and aster bundles, as a function of increasing F-actin length: (G) la = 2 ± 1 μm, (H) la = 3 ± 1 .5 μm, and (I) la = 8 ± 3 μm. Scale bars, 5 μm.

  • Fig. 2 Comparison of cluster patterns in the strictly 2D simulations and in vitro experiments.

    (A) Patterning of actomyosin clusters in the dilute limit obtained from agent-based simulations in the strictly 2D geometry. These patterns have been obtained by coarse graining the density of actin (red) and myosin (green) on a 2D grid with spacing 2σ. The relevant parameters are la = 2.32 μm, ca = 1.2 nM, cm/ca = 0.4, fa = 2 pN, and ku/kb = 0.1. Note that there are no overlapping regions (yellow) as a result of strong steric effects. (B) The shapes of most of the clusters observed in the simulations are strongly anisotropic, almost rectangular, and some even with appearance of bi-stripes [marked by white arrows in (A)], as determined from a Fourier analyses of the shape (Materials and Methods). Note the large amplitude of the l = 2 and l = 4 modes compared to the l = 1 mode, averaged over N = 19,718 clusters. (C) Density profiles of myosin and actin along the ⊥ and ∥ directions of the clusters also show strong anisotropy. Data averaged over N = 24,707 clusters. (D) Patterning of actomyosin clusters in the dilute aster limit obtained from the in vitro experiments, with actin (red), myosin (green), and a distinct region of overlap (yellow). Scale bar, 10 μm. (E) In contrast to the strictly 2D simulations, the shapes of most of the clusters are largely circular as shown by the relatively elevated amplitude of the l = 1 mode compared to the higher modes. Data averaged over N = 561 clusters. (F) Normalized intensity profile (I/Imax) extracted from TIRF images of actin and myosin along orthogonal directions (∥ and ⊥) confirming isotropy of the asters. Data averaged over 20 asters across four different experiments. (G) Circularly averaged radial density profiles of actin and myosin show enrichment of the latter at the core (calibration of TIRF images in fig. S4). Data averaged over 49 asters across four different experiments. Error bars denote SD.

  • Fig. 3 Simulations with stratification.

    (A) Patterning of actomyosin clusters in the dilute limit obtained from simulations in the stratified geometry. The parameters are la = 2.32 μm, ca = 1.5 nM, cm/ca = 0.2, fa = 2 pN, and ku/kb = 0.2. In this case, we see a colocalization of myosin and actin when projected in the x-y plane. (B) As in the in vitro study, the clusters are more circular, as shown by the relatively elevated amplitude of the l = 1 mode compared to the higher modes. Data averaged over N = 33,518 clusters. (C) Density of myosin and actin along the ⊥ and ∥ directions of the clusters show isotropic profiles, with myosin enriched at the cores and the relative concentration of actin enriched at the periphery. Data averaged over N = 44,537 clusters. (D) Representative snapshots of the different phases obtained on increasing actin filament length, (i) la = 2.32 μm, (ii) la = 6.02 μm, and (iii) la = 8.37 μm, showing isolated asters, connected asters, and filaments bundles.

  • Fig. 4 Characterization of the phases observed in our simulations of stratified actin filaments and myosin minifilaments.

    (A) Plots of gm–(r) for three different cases depicting three different phases observed. (B) A 2D phase diagram showing three different phases observed in our simulations on the cm/ca versus la plane. The squares correspond to cases where RRc, and triangles correspond to cases with R > Rc, where Rc is a suitable value (0.15) fixed to draw the phase boundaries. The bundle phase is confirmed by calculating the orientational alignment of the bound actin filaments around the myosin core, defined by ϕ (see Materials and Methods), which changes from 0 in a disordered configuration to 1 in a fully aligned configuration. (C) Full phase diagram of the different phases in the space of length of actin (la), concentration of actin (ca), and ratio of myosin to actin concentrations (cm/ca). The phase points have been assigned on the basis of plots as in (B).

  • Fig. 5 Stratified organization of actin and myosin in asters.

    (A to C) 3D STED microscopy of an actomyosin aster showing average intensity projection and XZ/YZ intensity profile of actin (A), myosin (B), and the merge (C) demarcating the core and the periphery used for subsequent measurements. Scale bars, 2 μm (XY) and 1.5 μm (XZ/YZ). (D) Intensity plot showing the relative intensities (I/Imax) of actin and myosin along the z axis in the core (solid line) and periphery (dashed lines). Actin intensity peak in the periphery was arbitrarily chosen as position 0. (E) Distance between actin and myosin intensity peaks [as shown in (D)] in the core and the periphery, quantified across seven different asters.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/11/eaay6093/DC1

    Section S1. Simulation methods

    Section S2. In vitro experiments

    Table S1. Parameter values in real units with dimensionless values used in simulations.

    Fig. S1. Description of agent-based model, active force versus velocity plot for a single myosin-II minifilament, and snapshots from simulation without steric interactions.

    Fig. S2. Time series of aster density and aster strength showing fluctuations about steady state.

    Fig. S3. Relative populations of F-actin (−)-ends near myosin-II heads help determine different aster and bundle phases.

    Fig. S4. Density measurements of actin and myosin in our in vitro experiments.

    Fig. S5. Characterization of the fluorophores on Abberior 775 STED nanoscope.

    Movie S1. A representative time course from the simulation of myosin-II minifilaments and F-actin in the stratified geometry.

    Movie S2. Time-lapse images from steady-state asters in our in vitro experiments.

    References (4850)

  • Supplementary Materials

    The PDF file includes:

    • Section S1. Simulation methods
    • Section S2. In vitro experiments
    • Table S1. Parameter values in real units with dimensionless values used in simulations.
    • Fig. S1. Description of agent-based model, active force versus velocity plot for a single myosin-II minifilament, and snapshots from simulation without steric interactions.
    • Fig. S2. Time series of aster density and aster strength showing fluctuations about steady state.
    • Fig. S3. Relative populations of F-actin (−)-ends near myosin-II heads help determine different aster and bundle phases.
    • Fig. S4. Density measurements of actin and myosin in our in vitro experiments.
    • Fig. S5. Characterization of the fluorophores on Abberior 775 STED nanoscope.
    • Legends for movies S1 and S2.
    • References (4850)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). A representative time course from the simulation of myosin-II minifilaments and F-actin in the stratified geometry.
    • Movie S2 (.avi format). Time-lapse images from steady-state asters in our in vitro experiments.

    Files in this Data Supplement:

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