Branched actin cortices reconstituted in vesicles sense membrane curvature

Abstract

The actin cortex is a complex cytoskeletal machinery that drives and responds to changes in cell shape. It must generate or adapt to plasma membrane curvature to facilitate diverse functions such as cell division, migration, and phagocytosis. Due to the complex molecular makeup of the actin cortex, it remains unclear whether actin networks are inherently able to sense and generate membrane curvature, or whether they rely on their diverse binding partners to accomplish this. Here, we show that curvature sensing is an inherent capability of branched actin networks nucleated by Arp2/3 and VCA. We develop a robust method to encapsulate actin inside giant unilamellar vesicles (GUVs) and assemble an actin cortex at the inner surface of the GUV membrane. We show that actin forms a uniform and thin cortical layer when present at high concentration and distinct patches associated with negative membrane curvature at low concentration. Serendipitously, we find that the GUV production method also produces dumbbell-shaped GUVs, which we explain using mathematical modeling in terms of membrane hemifusion of nested GUVs. We find that branched actin networks preferentially assemble at the neck of the dumbbells, which possess a micrometer-range convex curvature comparable with the curvature of the actin patches found in spherical GUVs. Minimal branched actin networks can thus sense membrane curvature, which may help mammalian cells to robustly recruit actin to curved membranes to facilitate diverse cellular functions such as cytokinesis and migration.

Figure 1

Robust actin encapsulation by eDICE. (A) Schematic of the eDICE process. Pre-formed lipid-stabilized emulsion droplets containing the inner aqueous solution (IAS) with G-actin are injected into a spinning chamber. Centrifugal forces created by rotation of the spinning chamber establish concentric layers of the oil phase and an outer aqueous solution (OAS) and push the droplets through the oil/OAS interface, where they acquire a second lipid leaflet and thus transform into GUVs. (B) Typical widefield image of GUVs formed by eDICE. F-actin is shown in magenta, lipids in cyan. Scale bar, 50 μm. (C) Diameter distributions of GUVs from four independent experiments (left) and the aggregated histogram of GUV sizes (right). N per sample: 98, 149, 137, 110. (D) Actin concentrations in eDICE GUVs measured by quantitative fluorescence microscopy, as a function of the nominal actin concentration in the IAS. Individual data points indicate single GUVs, boxplots indicate medians and quartiles. (E) Histogram of encapsulation efficiency, $c_{\text{rel}}=c_{\text{act}}/c_{\text{nominal}}$, for different nominal actin concentrations. The dashed line represents $c_{\text{rel}}=1$. $N_c=515,468,536,368$, and 447 GUVs for $c=1,2,5,8$, and 12 μM actin, respectively.

Figure 2

Formation of membrane-nucleated actin cortices inside GUVs. (A) Actin filaments (magenta) were nucleated using the Arp2/3 complex, which was activated near the inner leaflet of the GUV membrane (cyan) by membrane-bound VCA (green). (B and C) Top row: at low actin concentration (4.4 μM), actin formed either a continuous cortex (B), or small bright patches (C), or a combination of both. Bottom row: line profiles of actin fluorescence intensity along the yellow lines in the epifluorescence images. (D) Kymograph of a patchy actin cortex from a line drawn along the circumference of a GUV in a time lapse of 50 frames recorded at a frame rate of 1 fps. Scale bars, 10 μm (horizontal), 20 s (vertical). Actin patches could diffuse along the cortex (white rectangle), split (white arrow), or merge (black arrow). (E−H) At high actin concentration (8 μM), we found bright actin foci inside the lumen (E, white arrow), bright deformed actin patches (F, white arrows), extended flat membrane patches (G, white arrow), or a continuous flat cortex (H). All examples show GUVs with 2 μM VCA and 50 nM Arp2/3. Lipids are shown in cyan, actin in magenta. Scale bars, 5 μm. (I) Quantification of cortical phenotypes for 8 μM actin and 50 nM Arp2/3 as a function of the density of VCA activator. Actin patches in the GUV lumen occurred only at low VCA concentrations, deformed patches were most prevalent at intermediate VCA concentrations, and continuous cortices were most prevalent at high VCA concentrations. GUVs were all produced on the same day and statistics are based on $N=72$, 111, 101, and 87 GUVs for VCA concentrations of 0.65, 1.3, 2.6 and 6.5 μM, respectively. (J) Kymograph of an actin cortex (8 μM actin) with large flat patches that were immobile. Scale bars, 5 μm (horizontal), 20 s (vertical).

Figure 3

Dumbbell-shaped GUVs formed by eDICE. (A) Dumbbell-shaped GUV with a bright and a dim spherical cap connected by an open membrane neck. (B) GUV doublets clearly look different, exhibiting a membrane septum between the two lobes that each have equal membrane intensity. (C) Some dumbbells showed two clearly separate membranes in (parts of) the bright half of the dumbbell with a narrow (left) or wide (right) gap. (D) Membrane signal in a 5-pixel-wide line, starting at the neck and following clockwise along the GUV membrane shown in (A). Light cyan data points denote the bright half of the GUV; dark cyan data points show the dimmer half. The average signal is 1.8-fold higher (2.1-fold after background subtraction) in the bright than in the dim lobe (black lines). (E) Boxplot of membrane intensity ratios after background subtraction. The average ratio is 1.9 ($N=25$ dumbbells from five independent experiments). (F) We measured morphological features of GUV dumbbells by fitting circles to their bright and dim lobes (orange circles) and measuring the neck diameter as the distance between the maxima in membrane fluorescence in a line profile drawn through the neck. (G) Histogram of the size ratio between bright and dim dumbbell lobes ($N=85$). (H) Corresponding histogram of neck-to-lobe size ratios ($N=85$). Scale bars, 5 μm.

Figure 4

Dumbbell shapes result from hemifusion and are determined by osmotic pressure and line tension. (A) Confocal image of an eDICE GUV encapsulating another GUV (scale bar, 5 μm). Such nested GUVs were rare ($\sim$5%–10$\%$ of GUVs) but present at frequencies comparable with dumbbells. (B) Proposed mechanism for dumbbell formation. (i) One vesicle with radius $R_{\text{tot}}$ encapsulates another vesicle with radius $R_{\mathrm{v}}$. (ii) The inner GUV bursts and hemifuses with the membrane of the outer GUV. The shape of the resulting dumbbell is determined by membrane tension and line tension along the hemifusion line. (C) Proposed microscopic configuration of the dumbbell neck (gray rectangle in B), where one inner and one outer leaflet (dim lobe, top) join four concentric leaflets in the bright lobe (bottom). (D) FRAP measurement of the bright lobe of a dumbbell reveals a recovery of the normalized fluorescence intensity from $\sim$2 (pre-bleach, $t<0$) to $\sim$1 (post-bleach, $t>0$) within seconds. (E and F) The model predicted dumbbell shapes (relative lobe sizes, E, and neck diameters relative to the average lobe diameters, F) that quantitatively match the experimental data (G and H). Experimental and simulated data represent $N=85$ and $N={10}^4$ dumbbell GUVs, respectively. Lines display linear fits (see legend for fit equations).

Figure 5

Cortical actin networks preferentially localize to dumbbell necks, indicating curvature recognition. (A–D) Dumbbell GUVs containing cortical actin showed four phenotypes: actin was evenly distributed (A), enriched in a single patch at the neck (B), enriched in several distinct patches around the neck (C), or enriched in the entire neck region (D). Columns show a single confocal slice, a maximum intensity projection, a side view of the 3D-reconstructed z stack, and a view through the neck section in the z stack. Actin intensity is shown in false color (magma) for clarity. (E) Bar plot of actin enrichment patterns in dumbbell-shaped GUVs, showing an even distribution over the four groups (A–D) (N = 27 dumbbell GUVs). (F) Bar plot shows that 75% of dumbbell-shaped GUVs show actin enrichment at the neck, both for GUVs with and without a continuous actin cortex (N = 23 and 4, respectively). (G) Bar plot shows that actin enrichment at the neck was slightly more common in GUVs with 2 μM VCA compared with 6.5 μM VCA (N = 8 and 19, respectively). (H) We quantified the amount of actin enrichment at the neck by comparing line intensity profiles across the neck versus the dumbbell’s symmetry axis (yellow lines). (I) Line profiles of membrane (cyan) and actin (magenta) intensity along the neck and symmetry axis of the dumbbell GUV shown in (H), normalized to the maximum pixel value in the neck profiles. Peaks in actin and membrane intensity coincide at the neck, but not at the poles. (J) Boxplot of the degree of actin enrichment at dumbbell necks in GUVs where actin nucleated spontaneously in the GUV lumen (−Arp, black dots, N = 21) or at the membrane with the help of Arp2/3 (+Arp, magenta dots, N = 27). (K) Confocal image of a dumbbell where actin polymerized spontaneously in the GUV lumen and does not localize to the neck region. Scale bars, 5 μm.