Reconstitution of an actin cortex inside a liposome

Abstract

The composite and versatile structure of the cytoskeleton confers complex mechanical properties on cells. Actin filaments sustain the cell membrane and their dynamics insure cell shape changes. For example, the lamellipodium moves by actin polymerization, a mechanism that has been studied using simplified experimental systems. Much less is known about the actin cortex, a shell-like structure underneath the membrane that contracts for cell movement. We have designed an experimental system that mimicks the cell cortex by allowing actin polymerization to nucleate and assemble at the inner membrane of a liposome. Actin shell growth can be triggered inside the liposome, which offers a useful system for a controlled study. The observed actin shell thickness and estimated mesh size of the actin structure are in good agreement with cellular data. Such a system paves the way for a thorough characterization of cortical dynamics and mechanics.

Figure 1

Schematic illustration of liposome preparation. (A) Assembly of the outer layer. (B) The inner layer, created by emulsion that was sedimented through the monolayer. (C) The liposome obtained by the assembly described in A and B, with different solutions inside and outside. (D) Polymerization was triggered when the pores were added, thus allowing salt and ATP to flow into the liposome. This figure is adapted from Pautot et al. (16).

Figure 2

Triggering polymerization at the membrane. (A–D) Phase contrast microscopy (A and D) and epifluorescence microscopy (B and E) of actin. Without pores (A), the inside of the liposomes appeared denser and correlated to mass fluorescence of the actin (B). (C) Fluorescence profile along the yellow line drawn in B. In the presence of pores (D), sucrose left the liposome, and the contrast observed in phase contrast vanished, whereas actin fluorescence was localized at the membrane (E), which is shown by peaks (F) on the profile along the line shown in E. A fraction of 10% of actin was marked with Alexa Fluor 488 (G) and actin filaments were revealed by rhodamine-phalloidin staining (H) that colocalized with the actin shell (I). Scale bars: (A and B) 10 μm; (D and E) 5 μm, (G–I) 10 μm.

Figure 3

Effect of the presence of cholesterol, the Arp2/3 complex, and VVCA-His or drugs on cortex formation in the absence (A) and presence (B) of cholesterol (x = 0.37), in the lipid composition. Plain bars represent the percentage of liposomes displaying a fluorescent shell (actin contrast, C > 0.01), and hatched bars refer to the contrast. Solid bars, the complete system of proteins (see Materials and Methods); dark shaded bars, VVCA-His is omitted; light shaded bars, both VVCA and Arp2/3 complex are omitted; open bar, in the presence of latrunculin A. For each condition, we give the total number, n, of observed liposomes. The shell fluorescence percentages in the different conditions either in the absence or in the presence of cholesterol were proven to differ significantly using the χ² statistic.

Figure 4

Evolution of the gel thickness as a function of liposome size. Solid squares or open circles correspond to one single liposome measurement. Solid-square points were obtained under concentration conditions of 0.12 μM for the Arp2/3 complex, 50 nM for gelsolin, 2 μM for ADF-cofilin, 1 μM for profilin, 6.5 μM for G-actin (including 20% fluorescently labeled actin), and 0.64 μM VVCA-His. Solid “×”symbols are thickness values averaged over each 0.5-μm radius increase. Shaded crosses are thickness values averaged over each 0.5-μm radius increase in the case of twice the protein concentration. Open circles are shell thicknesses for liposomes in the presence of cholesterol in the same protein conditions as solid points. Most points lie between the slopes 0.08 and 0.2 (dashed lines). (Inset) Scheme of notations used in the text.