Reconstituted branched actin networks sense and generate micron-scale membrane curvature

Abstract

The actin cortex is a complex cytoskeletal machinery which 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 and generation 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 that generate negative membrane curvature at low concentration. Serendipitously, we find that the GUV production method also produces dumbbell-shaped GUVs, which we explain using mathematical modelling in terms of membrane hemifusion of nested GUVs. We find that dendritic actin networks preferentially assemble at the neck of the dumbbells, which possess a micron-range convex curvature that matches the curvature generated by actin patches in spherical GUVs. Minimal dendritic actin networks can thus both generate and sense membrane curvatures, which may help mammalian cells to robustly recruit actin to curved membranes in order to facilitate diverse cellular functions such as cytokinesis and migration.

Biomimetic actin cortices shape cell-sized lipid vesicles

“…1A). The method produces a high yield of GUVs with a broad size distribution centered reproducibly around 9 µm and extending up to 40 µm (25), which covers the lower end of the 10-100 μm size range of mammalian cells (27). Actin was strongly localized to the membrane in almost 90% of the GUVs (Fig.…”

“…1B, yellow arrow) or lacked any actin signal (Fig. 1B, white arrow), likely because of some variability in the encapsulation efficiency (25). In the following, all analysis refers to GUVs in which we observed membrane-localized actin signal.…”

“…Lyophilized rabbit skeletal muscle actin was purchased from Hypermol EK and stored as a 23.8 µM solution in G-buffer at -80°C (see ref. (25) for details). Monomeric (G)-actin was fluorescently labeled with AlexaFluor 488 carboxylic acid succimidyl ester (Invitrogen) (62).…”

“…GUVs were prepared by eDICE as described in (25), encapsulating 8 μM actin of which 10 % was fluorescently labeled, 50 nM Arp2/3 (human ArpC1B/C5L unless otherwise specified), and varying concentrations of VCA (6.5 μM unless otherwise specified). The inner buffer contained 20 mM Tris(hydroxy-methyl)aminomethane hydrochloride (Tris-HCl) pH 7.4, 50 mM KCl, 2 mM MgCl 2 , 1 mM DTT and 0.5 mM MgATP, and was supplemented with 6.5 % (v/v) Optiprep (Cat.…”

Out With a Bang: Celebrating Global Chemical Biology

“…20−22 In the second part of her presentation, she focused on the progress of her laboratory in rebuilding cell division using a novel method to generate giant unilamellar vesicles (GUVs) encapsulating actin to form a cortex at the inner surface of the vesicle, which is sufficient to create curved membrane structures. 23,24 Prof. Tomas Kirchhausen (Harvard Medical School, USA) started with a retrospective to the time 20 years ago before the NCCR started, when they discovered the compound Dynasore, a very potent Dynamin inhibitor. 25 He then presented his inspiring latest research using large scale data visualization to achieve automated recognition of organelles from FIB-SEM data for both cultured cells and tissue using deep learning-aided image processing.…”