Biomimetic actin cortices shape cell-sized lipid vesicles

Abstract

Animal cells are shaped by a thin layer of actin filaments underneath the plasma membrane known as the actin cortex. This cortex stiffens the cell surface and thus opposes cellular deformation, yet also actively generates membrane protrusions by exerting polymerization forces. It is unclear how the interplay between these two opposing mechanical functions plays out to shape the cell surface. To answer this question, we reconstitute biomimetic actin cortices nucleated by the Arp2/3 complex inside cell-sized lipid vesicles. We show that thin Arp2/3-nucleated actin cortices strongly deform and rigidify the shapes of giant unilamellar vesicles and impart a shape memory on time scales that exceeds the time of actin turnover. In addition, actin cortices can produce finger-like membrane protrusions, showing that Arp2/3-mediated actin polymerization forces alone are sufficient to initiate protrusions in the absence of actin bundling or membrane curving proteins. Combining mathematical modeling and our experimental results reveals that the concentration of actin nucleating proteins, rather than actin polymerization speed, is crucial for protrusion formation. This is because locally concentrated actin polymerization forces can drive a positive feedback loop between recruitment of actin and its nucleators to drive membrane deformation. Our work paints a picture where the actin cortex can either drive or inhibit deformations depending on the local distribution of nucleators.

Exploring Giant Unilamellar Vesicle Production for Artificial Cells — Current Challenges and Future Directions

“…One example is continuous droplet interface crossing encapsulation (cDICE), [88,101] and its recent adaptation termed emulsion cDICE (or eDICE) [102][103][104] which have been successfully implemented for actin cytoskeleton reconstitution experiments by various labs. [88,101,[103][104][105][106][107][108] Other successful examples are the shaking method, which uses dropletstabilized GUVs as intermediates, [109] and droplet-shooting centrifugal formation (DSCF), which makes use of a 3D-printed microcapillary. [110] While other techniques such as microfluidic jetting [111] have seen some success [112][113][114] they have not been widely adopted and further studies are needed to investigate the physicochemical properties of the resulting GUVs.…”

“…[ 88 ] Furthermore, cDICE has successfully been used to encapsulate colloids, red blood cells, [ 101 ] small unilamellar vesicles (SUVs), DNA origami and even live bacteria. [ 88 ] Recently, there has been a clear shift from cDICE toward eDICE, implementing the proposed optimizations for cDICE presented in Van de Cauter et al [ 88 ] eDICE has since been used to reconstitute actin cortices nucleated by the Arp2/3 complex [ 108 ] and VCA [ 104 ] and reconstitution of actomyosin networks. [ 103,159 ] Similar results have been obtained using the shaking method: from encapsulation of F‐actin with SUVs, [ 162 ] a DNA cytoskeleton mimicking actin rings, [ 165 ] and a DNA segregation module, [ 163 ] to cells.…”

“…[171] As an alternative to using sugars, density gradient medium OptiPrep has been successfully used in cDICE and eDICE. [88,89,[102][103][104]108,160,161] In the case of OLA, glycerol is required in both the inner and outer solution, along with the nonionic triblock copolymer surfactant Poloxamer 188 (P188) in the outer solution. It is important to note that these additional additives, especially at the high concentrations typically used, will affect protein and membrane properties during the experiments.…”

CK-666 and CK-869 differentially inhibit Arp2/3 iso-complexes

“…GST-tagged human N-WASP-VCA (392 – 505, UniProt O00401), human SPIN90 C-terminus (267 - 715, UniProt Q9NZQ3-3), were purified following the previously reported protocols (Cao et al ., 2023). Recombinant human Arp2/3 iso-complexes with defined composition (Uniprot P61160, P61158, Q9P1U1, Q92747, O15143, O15144, O15145, P59998, Q9H9F9, Q98PX5) were purified following the previously reported protocol (Baldauf et al , 2023). Skeletal muscle alpha-actin was purified from rabbit muscle atone powder following the published protocol (Spudich & Watt, 1971).…”

Minimal Out-of-Equilibrium Metabolism for Synthetic Cells: A Membrane Perspective

“…By contrast, GUVs have an internal volume sufficient to encapsulate complex molecular mixtures, circumventing stochasticity issues. In fact, cell-free transcription-translation machineries ,− and components of the cytoskeleton − have already been introduced into GUVs. In addition, GUVs also provide a larger membrane surface area to reconstitute membrane proteins once a suitable generic method has been developed.…”