Actomyosin dynamics drive local membrane component organization in an in vitro active composite layer

Abstract

The surface of a living cell provides a platform for receptor signaling, protein sorting, transport, and endocytosis, whose regulation requires the local control of membrane organization. Previous work has revealed a role for dynamic actomyosin in membrane protein and lipid organization, suggesting that the cell surface behaves as an active composite composed of a fluid bilayer and a thin film of active actomyosin. We reconstitute an analogous system in vitro that consists of a fluid lipid bilayer coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors. Upon complete consumption of ATP, this system settles into distinct phases of actin organization, namely bundled filaments, linked apolar asters, and a lattice of polar asters. These depend on actin concentration, filament length, and actin/myosin ratio. During formation of the polar aster phase, advection of the self-organizing actomyosin network drives transient clustering of actin-associated membrane components. Regeneration of ATP supports a constitutively remodeling actomyosin state, which in turn drives active fluctuations of coupled membrane components, resembling those observed at the cell surface. In a multicomponent membrane bilayer, this remodeling actomyosin layer contributes to changes in the extent and dynamics of phase-segregating domains. These results show how local membrane composition can be driven by active processes arising from actomyosin, highlighting the fundamental basis of the active composite model of the cell surface, and indicate its relevance to the study of membrane organization.

Keywords: actin; active mechanics; membrane organization; myosin II.

Fig. 1.

In vitro setup. (A) Schematic of the in vitro system. (B) Schematic of the actin–membrane linker constructs. (C) Representative images of SLB-bound F-actin and plot of the diffusion coefficient HYE (n = 59–147), HYE(R579A) (n = 15–24), and a combination of HKE and HYE(R579A) (n = 10–39) at various F-actin concentrations. (D) Representative images of SLB-bound rhodamine-labeled F-actin with capping protein (CP) and plot of the diffusion constant of HYE (n = 50–80) and a combination of HKE and HYE(R579A) (n = 10–20) with F-actin of decreasing average length (n = 158–749). Small squares depict mean values; box heights, SDs; middle lines, medians; whiskers, 5–95% ranges. (Scale bars: 10 µm.)

Fig. S1.

(A) Graph showing the normalized intensity of SLB-bound HYE after washout at experimental conditions. (B) Area fraction covered by F-actin binding to SLB with increasing concentrations of HYE (black) or HKE (red); plotted are mean and SD, n = 2. (C) Probability distribution of the thickness of a SLB-bound F-actin layer; actin intensity values obtained from line scans were normalized by the average single filament intensity and binned to integer values in a histogram to get an estimation of the actin layer thickness (n = 791). (D) F-actin length probability distribution after actin polymerization with various capping protein concentrations (n = 158–749); Rhodamine-labeled F-actin was added to HYE-containing bilayers at low density, imaged with TIRF microscopy, and length measurements were performed semiautomatic with the ImageJ plugin NeuronJ. (E) Averaged traces from FRAP experiments on SLB-bound His₁₀-GFP (black), HYE alone (blue), or HYE together with 1,000 nM F-actin (light blue); Insets show representative snapshots for recovery of HYE alone (Top) or HYE in the presence of 1,000 nM F-actin (Bottom); each trace shows an average of five experiments; whiskers depict SD; red lines are fitted single-exponential function [derived time constants: b_GFP = 0.142 (±0.002) s⁻¹; b_HYE = 0.113 (±0.002) s⁻¹; b_HYE+actin = 0.076 (±0.001) s⁻¹; the immobile fractions are d_GFP = 7(±1)%, d_HYE = 12(±1)%, d_HYE+actin = 16(±1)%]. (F and G) Immobile fractions (F) and effective diffusion coefficients (G) of membrane-bound proteins derived from individual FRAP experiments. (H) Individual comparison of FCS autocorrelation curves on HYE bound to a SLB under free conditions (blue), in the presence of 1,000 nM F-actin (light blue) or with 1,000 nM short, capping protein bound F-actin (mauve); traces are averages from different scan positions on three to five individual experiments; Inset shows distribution of diffusion time constant obtained by fitting to a model of 2D diffusion. (I) FCS-derived diffusion coefficients of HYE without actin (HYE, n = 22), with bound long (F-actin, n = 23) and short (CP-actin, n = 16) actin. (J) FCS-derived diffusion coefficients of His10-GFP alone (n = 5) or in combination with HKE and no actin (n = 5) or with bound long F-actin (n = 13). (K) Diffusion coefficients of the lipid probe RhoPE derived by FRAP without actin (n = 9) or with 1,000 nM long F-actin bound to the lipid bilayer via HYE (n = 16); diffusion coefficients of HYE in the presence of actin were obtained on the same samples (n = 11). For all box plot diagrams: small squares depict mean values; box heights, the SD; middle lines, the median; and whiskers, the 5–95% range.

Fig. 2.

Myosin-induced formation of polar actin asters. (A) Images of F-actin after myosin II action show three distinct configurations. (Scale bars: 10 µm, 2 µm.) (B) Phase diagram of actomyosin organization obtained by manual classification. (C) Diameter of polar actin asters as a function of mean filament length (n > 20 for each data point) and linear fit showing a linear dependence of aster size to F-actin length (slope, 0.5 ± 0.2); Insets show average projections of image stacks that were created by cropped single actin aster images (n = 12–20) for each indicated F-actin length. (D) Representative images depicting the organization of polar actin asters by imaging rhodamine-actin and Atto-647-myosin II (i), or rhodamine-actin and Alexa 647 capping protein (ii) to obtain a schematic description of a typical actin aster organization (iii). (Scale bar: 2 µm.) (E) X, Y, Z scans of an actomyosin network stabilized with 0.04% glutaraldehyde and imaged with multipoint structural illumination microscopy (SIM) showing the thin membrane-confined actin layer and the local contraction into asters. (Scale bars: in YZ and XZ scans, 2 µm; in XY scan, 5 µm.)

Fig. S2.

(A) Myosin II filament length (in meters) probability distribution (Top) obtained from fluorescence images of Atto-633–labeled myosin II; example image shown below; Inset is a transmission electron microscopy image. [Scale bars: 2 µm and 200 nm (Inset).] Lengths were measured manually with ImageJ. (B) Orientation analysis of bundled filaments and connected apolar asters, and (C) corresponding radial orientation correlations; plotted are mean and SD (n = 10). (D) Density cross-correlation of actin intensities in a disordered lattice of polar asters and connected apolar asters; plotted are mean and SD (n = 10). (E) Exemplar fluorescence image of F-actin asters (Top) and corresponding interaster distance (l) distribution (Bottom) computed from 10 images of the same sample (n = 3,007). (F) Overlay of exemplar STED images of actin (red) and myosin (green) organization in F-actin asters. (Scale bar: 2 µm.) (G) Overlay of actin (red) and capping protein (CP) (cyan) organization in F-actin asters. (Scale bar: 10 µm.) (H) Examples of actin and capping protein organization in connected apolar asters. (Scale bar: 2 µm.) (I) Anecdotal example of a large aster with labeled capping protein showing a second ring of F-actin pointing inward with their barbed ends; plot shows radial intensity of capping protein (cyan) and actin (red); Inset shows the actual fluorescence image. (Scale bar: 10 µm.) (J) Extra-large field view (440 × 420 µm²) of actin asters in a single experiment; images were taken from random positions across the sample and put together in this montage to show homogeneity of aster formation in a standard experiment; image is shown in fire LUT to enhance contrast. (Scale bar: 50 µm.)

Fig. 3.

Actomyosin-induced HYE accumulation during actin aster formation. (A) Snapshots of actin (Left), myosin II (Middle), and HYE (Right) during aster formation. (Scale bar: 10 µm.) (B, Top) View of the outlined region in A. (Scale bar: 2 µm.) (Bottom) Intensity profiles averaged over 40 circular regions cantered on actin asters in A, normalized to [0; 1]; control is computed from 10 circular nonaster regions; images were corrected for photobleaching by a single exponential function. (C) Graph of contractile actin flows computed from PIV data (black; Materials and Methods) and HYE intensity (green) averaged over the same regions as in B at indicated condition. (D) Color-coded time projection of actin (Top) and corresponding HYE-actin cross-correlation (Bottom) of SLBs before (Left) and after addition of myosin II, during contraction (Center) and after its halt (Right). (Scale bar: 10 µm.) (E) Plots of HYE-actin cross-correlation versus the variance of actin density from images in D; mean and SD computed from data within the x-axis whisker’s range.

Fig. S3.

(A) F-actin after addition of myosin II, and a color-coded time projection of F-actin. (Scale bar: 10 µm.) (B) Probability distribution of myosin-induced F-actin contraction velocities (v) obtained from PIV on actin movies. (C) Kymograph of the density–density correlation of the actomyosin contraction shown in A; the arrow indicates the change in the correlation indicating the transition from a rather uniform F-actin distribution to a clustered one, like it is expected for aster formation. (D) Plot of all measured pairs of HYE-actin cross-correlation and variance of actin density corresponding to Fig. 3 D and E. (E) Color-coded time projection of F-actin distribution and HYE-actin cross-correlation of the actin-binding mutant HYE(R579A) during actomyosin contraction bound to the SLB via the nonfluorescent HKE. (Scale bar: 10 µm; duration: 10 min.) (F) Plot of HYE-actin cross-correlation versus the variance of actin density; mean and SD are from data within the x-axis whisker's range. (G) Color-coded time projection of F-actin distribution, HYE-actin and RhoPE-actin cross-correlation during actomyosin contraction. (Scale bar: 10 µm; duration: 20 min.) (H) Plot of HYE-actin (green) and RhoPE-actin (orange) cross-correlation versus the variance of actin density; mean and SD are from data within the x-axis whisker's range.

Fig. 4.

The remodeling actomyosin–membrane system shows features of an active composite. (A) Aster disassembly after addition of ATP to polar actin asters. (Scale bar: 10 µm.) (B) Color-coded time projection of actin (Left) and corresponding HYE-actin cross-correlation (Right) of a remodeling actomyosin network. (Scale bar: 2 µm; duration, 15 min.) (C) Plot of HYE-actin cross-correlation versus the variance of actin density from images in B; mean and SD computed from data within the x-axis whisker's range. (D) Individual HYE intensity probability distributions for SLBs containing HYE alone, jammed (actin plus myosin II), or remodeling actomyosin (high ATP); dashed lines depict Gaussian fits of data from each condition. (E) Number fluctuations of HYE ($\mathrm{\Delta }N\sim b^{\alpha }$) under the indicated conditions; squares depict mean values; box heights, SDs; middle lines, medians; dots, individual experiments (n = 7–12). (F) Number fluctuations of HYE as a function of interrogation box size (A_box) obtained from one sample at conditions as indicated.

Fig. S4.

(A) Time series of constitutively remodeling actin networks at high ATP concentrations (1 mM) and with an ATP regeneration system ([ATP] = 0.5 mM). (Scale bar: 2 µm.) (B) Kymograph of the density cross-correlation of a disordered lattice of polar asters after addition of ATP; the dissociation of asters is marked in the slower decay of correlation, and steady state is reached after 2 min. (C) Color-coded time projection of actomyosin remodeling in the presence of constant ATP concentrations and the corresponding HYE-actin density cross-correlation. (Scale bar: 10 µm; duration: 20 min.) (D) Plot of all measured pairs of HYE-actin cross-correlation and variance of actin density corresponding to Fig. 4 B and C. (E) SD of the HYE intensity distributions at various conditions; small squares depict mean values; box heights, SDs; middle lines, medians; and dots, individual experiments (n = 7–12). (F) L-skewness of HYE intensity distributions at various conditions (n = 7–12); small squares depict mean values; box heights, the SD; middle lines, the median; and whiskers, the 5–95% range. (G) Plot of obtained from calculating the slopes of the number fluctuation data shown in Fig. 4F; data depict mean values and SDs. (H) Individual traces for number fluctuations analysis (shown in Fig. 4E) of HYE intensities (Top) and slope values (Bottom) calculated from the above traces that correspond to the value (α − 1), where α is the exponent in the relation <N²>^1/2 ∼ <N>^α.

Fig. 5.

Consequences of the remodeling actomyosin on a phase-separating membrane. (A) Schematic showing the setup for SLBs with ternary lipid mixture. (B) Example fluorescent images of SLBs containing RhoPE, HYE, and binding actin with regions devoid of domains (Top) or showing lipid segregation (dark regions) (Bottom). (Scale bar: 10 µm.) T = 28 °C. (C) Examples of lo domains (dark regions) in SLBs containing HYE only (Left), bound F-actin (Center), or a remodeling actomyosin (Right). (Scale bar: 10 µm.) (D) Averaged lo domain sizes at different conditions; lines represent averages for each condition (n = 4–6, with 15–158 domains per experiment), shaded area is SD, and t₀ indicates start of imaging. (E) Relative change in net lo domain area between t₀ and 750 s later for indicated conditions; squares depict mean values; box heights, SDs; dots, individual experiments. (F) Color-coded time projection of actomyosin remodeling (from Fig. S5B) and the corresponding HYE-actin density cross-correlation. (Scale bar: 20 µm; duration, 2 min.) (G) Plot of HYE-actin cross-correlation versus the variance of actin density; mean and SD are from data within the x-axis whisker's range.

Fig. S5.

(A) Snapshots of a time series showing the effect of lo domains (dark regions) on actin filament (cyan) and HYE (magenta) distribution. (Scale bar: 20 µm.) (B) Snapshots of a time series showing the effect of a remodeling actomyosin network on a phase-separating SLB with HYE as linker protein. (Scale bar: 10 µm.) (C) Graphs depicting averaged lo domain radii of individual experiments at different conditions (with 15–158 domains in each experiment; t₀ indicates start of imaging). (D) Plot of total lo domain area at the start of imaging (A₀) of individual experiments used in Fig. 5E.