Reconstitution of contractile actomyosin rings in vesicles

Abstract

One of the grand challenges of bottom-up synthetic biology is the development of minimal machineries for cell division. The mechanical transformation of large-scale compartments, such as Giant Unilamellar Vesicles (GUVs), requires the geometry-specific coordination of active elements, several orders of magnitude larger than the molecular scale. Of all cytoskeletal structures, large-scale actomyosin rings appear to be the most promising cellular elements to accomplish this task. Here, we have adopted advanced encapsulation methods to study bundled actin filaments in GUVs and compare our results with theoretical modeling. By changing few key parameters, actin polymerization can be differentiated to resemble various types of networks in living cells. Importantly, we find membrane binding to be crucial for the robust condensation into a single actin ring in spherical vesicles, as predicted by theoretical considerations. Upon force generation by ATP-driven myosin motors, these ring-like actin structures contract and locally constrict the vesicle, forming furrow-like deformations. On the other hand, cortex-like actin networks are shown to induce and stabilize deformations from spherical shapes.

Fig. 1. Encapsulation of bundled actin in giant unilamellar vesicles.

a Schematic depiction of the vesicle generation process. The aqueous protein solution is injected into a rotating chamber through a glass capillary. Droplets form at the capillary tip in the oil phase, which contains lipids. The droplets then pass through a water–oil interphase lined with a second lipid monolayer, forming the giant unilamellar vesicles (GUVs). b Field of view image (Z-projections of confocal stacks) with many cytoskeletal vesicles. Actin in green. See Supplementary Movie 1 for 3D effect. c Comparison of cytoskeletal vesicles with actin bundled by four different types of bundling proteins. We used 2 µM actin in all cases, but due to differences in bundling activity, different concentrations of bundling protein: 0.3 µM fascin, 0.9 µM VASP, 1 µM α-actinin, 2 µM talin, and 2 µM vinculin. d Automated tracing of bundles by analysis script. Confocal z-stacks are converted into a three-dimensional “skeleton” model. Supplementary Movie 2 shows a 3D view of both representations of these vesicles.

Fig. 2. Membrane attachment affects curvature of actin bundles.

a Each image shows a section of the membrane of a GUV with actin bundles cross-linked by fascin (2 µM actin and 0.2 µM fascin). Actin is in green. Unattached bundles have long sections with low curvature, while membrane-bound bundles follow the curvature of the membrane. b Distribution of curvatures for actin bundles in vesicles with and without membrane binding. n = 4 GUVs for each condition. Confocal z-stacks were converted into 3D information (see Fig. 1d). Bundles were then divided into small segments and their curvature was measured. Curvature is normalized to membrane curvature, so that a curvature of 1.0 equals the curvature of the membrane. Data are shown as mean values with individual histogram counts and SEM. More details about the analysis can be found in the Supplementary Method.

Fig. 3. Actin organization in dependence on bundler to actin ratio and membrane binding.

Actin is positioned close to the membrane regardless of membrane-anchoring. a Overview of conditions with varying actin concentration (2 µM and 6 µM), fascin to actin ratios (3.3:100–15:100) and with and without actin–membrane binding. Supplementary Movie 3 shows a 3D version of this figure panel. b Simulations for 6 µM conditions as in a using radius = 6 μm. c Average membrane proximity of actin signal for vesicles with 2 µM actin. Normalized range from −1 (all actin in the center of the vesicle) to +1 (all actin on the membrane). d Membrane proximity of actin signal for vesicles with 6 µM actin. Data in c and d are presented as mean values with individual data points and SEM. n = 10 vesicles for each condition (except n ≥ 3 for controls with 0% fascin).

Fig. 4. Formation of membrane-anchored actin rings.

a Membrane-binding promotes ring formation. Shown is the probability of the formation of single actin rings in GUVs (i.e., GUVs with one single unbranched actin bundle connected into a ring) in the absence or presence of membrane-anchoring. Data are shown as mean values with individual fractions of experimental runs. Three hundred ninety two vesicles between 15 and 20 µm were analyzed in n = 2 (fascin) or n = 3 experimental runs per condition. We use 2 µM actin in all cases, but due to differences in bundling activity different concentrations of bundling protein: 0.3 µM fascin, 1 µM α-actinin, 2 µM talin, and 2 µM vinculin. b Probability of ring formation for simulations with different initial parameters (R: vesicle radius, L: filament lengths). In simulations, we classified rings with small gaps or closed rings with additional side branches as “ring like” (see Supplementary Method). Snapshots from all simulation shown in Supplementary Fig. 11. c Condition with particularly robust ring formation: actin bundled by talin with vinculin and bound to the membrane. Supplementary Movie 4 shows a 3D view of this image. Supplementary Figure 8 shows a DIC image of this field of view, and Supplementary Figs. 9 and 10 show rings formed by other bundling proteins.

Fig. 5. Actomyosin contraction leads to membrane deformations.

a Time series of a contracting ring-like structure in a GUV. Same conditions as in Fig. 4c but with 0.1 µM myosin II. Only the midsection of the vesicle is shown, top and bottom are missing, but the yellow dotted lines in the first frame show the approximate position of the bundles (see also Supplementary Fig. 13). Cyan dotted lines in the following frames indicate the outline of the vesicle. Orange arrows indicate membrane deformations (vesicle constriction). The partially visible vesicle on the left can be seen to undergo a similar transition from a large actin network (t = 0) to myosin-constricted cluster (t = 60 min). Supplementary Movie 6 shows the same field of view. b Additional example of large-scale vesicle deformation through actomyosin contraction. Even though no furrow-like deformation was observed, the contraction of actin bundles still causes large-scale vesicle deformations (orange arrows).

Fig. 6. Actin network governs vesicle shape in osmotically deflated vesicles.

a Variety of vesicle shapes produced by different morphologies of encapsulated actin networks, some with stabilizing cortices and others with filopodia-like membrane protrusions. b Upon exposure to photodamage through increased laser power, cytoskeletal vesicles lose their stabilizing actin cortex, and often take on a flat and round shape. All three examples are shown in Supplementary Movie 7. c Vesicles with a stabilizing artificial actin cortex can be dried, frozen, and imaged using cryo-scanning electron microscopy.