Figure 1. Quantitative analysis of actin network flow and turnover.

Bulk contracting actomyosin networks are formed by encapsulating Xenopus extract in water-in-oil droplets. The network is labeled using a low concentration of GFP-Lifeact, which does not significantly alter the network (see Methods), and imaged by time-lapse spinning-disk confocal microscopy. Images are acquired at the mid-plane of the droplets, where the network flow is approximately planar due to the symmetry of the set up. The system reaches a dynamic steady-state characterized by a radially-symmetric inward flow. (a) Schematic of the actin network inside a water-in-oil droplet, illustrating the actin turnover dynamics and myosin-driven contraction. (b) Top: Spinning disk confocal image of the equatorial cross section of a network labeled with GFP-Lifeact. Bottom: Bright-field images of a droplet showing the aggregate of particulates that forms an exclusion zone around the contraction center. (c) The network velocity field for the droplet shown in (b), as determined by correlation analysis of the time-lapse movie (Movie 1). (d) The actin network density as a function of distance from the contraction center. The thin lines depict data from individual droplets, and the thick line is the average density profile. The density is normalized to have a peak intensity =1. (e) The radial velocity as a function of distance from the contraction center. The inward velocity increases linearly with distance. (f-h) Analysis of net actin network turnover. (f) Schematic illustration showing that at steady-state, the divergence of the actin network flux is equal to the net network turnover rate. The network flux is equal to the product of the local network density and velocity J(r) = ρ(r)V(r). (g) The divergence of the network flux, $\nabla \cdot \overrightarrow{J}\left(r\right)$ (see Methods), is plotted as a function of distance from the contraction center, showing the spatial distribution of the net turnover. Negative values (at smaller r) correspond to net disassembly, while positive values (near the droplet’s periphery) indicate net assembly. (h) The divergence of the network flux is plotted as a function of the local actin network density. The net turnover decreases roughly linearly with actin network density.