Mimicking the mechanical properties of the cell cortex by the self-assembly of an actin cortex in vesicles

Abstract

The composite of the actin cytoskeleton and plasma membrane plays important roles in many biological events. Here, we employed the emulsion method to synthesize artificial cells with biomimetic actin cortex in vesicles and characterized their mechanical properties. We demonstrated that the emulsion method provides the flexibility to adjust the lipid composition and protein concentrations in artificial cells to achieve the desired size distribution, internal microstructure, and mechanical properties. Moreover, comparison of the cortical elasticity measured for reconstituted artificial cells to that of real cells, including those manipulated using genetic depletion and pharmacological inhibition, strongly supports that actin cytoskeletal proteins are dominant over lipid molecules in cortical mechanics. Our study indicates that the assembly of biological systems in artificial cells with purified cellular components provides a powerful way to answer biological questions. The actin cortex is a thin sheet of actin meshwork formed by actin cytoskeletal proteins, including actin, actin crosslinkers (ACs), motor proteins, and other actin binding proteins (Fig. S1). [3][4][5] The plasma membrane is largely a lipid bilayer embedded with transmembrane proteins. The actin cortex and plasma membrane are connected by anchoring proteins. The composite of actin cytoskeleton and plasma membrane plays important roles in sensing mechanical stimuli and subsequently remodeling its own microstructure, which governs many essential cellular events such as migration and morphogenesis. [6][7][8][9] The cell shape changes in these events can be largely considered mechanical processes. Therefore, understanding the mechanical properties of the composite can provide deep insight into the nature of important biological phenomena.Currently, there are two complementary strategies for studying the mechanical properties of cells: the top-down and the bottom-up approaches. The former involves simplifying the cellular systems by the combination of genetic deletion or knockdown of nonessential genes and pharmacological inhibition of the function of certain proteins. 4,[10][11][12] The latter relies on the reconstitution of the actin cytoskeleton with purified or synthesized components in in vitro systems. [13][14][15][16][17][18][19][20][21] Previous measurements conducted with in vivo and in vitro systems suggest that the mechanical properties of the actin cortex depend on the force-dependent affinities of all of these proteins to F-actin as well as their concentrations. Despite the numerous experiments conducted in live cells, only a limited number of genes and proteins can be deleted or inhibited simultaneously, and when expressing genes of interest in cells, it can be difficult to control expression precisely. On the other hand, numerous proteins can be added in precisely controlled concentrations to in vitro reconstitution systems, though this can be labor intensive. One type of in vitro assay is to assemble the actin meshwork into a 2D flat she...

A novel membrane anchor for FtsZ is linked to cell wall hydrolysis in Caulobacter crescentus

“…The precedence of encap-sulating bacterial proteins, including FtsZ membrane tethers, inside giant unilamellar vesicles (GUVs) inspired us to employ this minimal system for assaying FtsZ recruitment to membranes by FzlC (Cabre et al, 2013;Osawa and Erickson, 2013). We used the inverted emulsion method to encapsulate YFP-FzlC and/or FtsZ-CFP 1/-GTP inside GUVs with outer leaflets composed of 4:1 PC:phosphatidylserine (PS) and inner leaflets composed of 1:1 PG:PC (Pautot et al, 2003;Luo et al, 2014). YFP-FzlC alone localized robustly to the membrane while FtsZ-CFP alone remained lumenal under polymerizing (1GTP) and non-polymerizing (-GTP) conditions ( Fig.…”

“…Giant unilamellar vesicles were fabricated using the inverted emulsion technique (Pautot et al, 2003;Luo et al, 2014). For the inside monolayer 1-palmitoyl-2-oleoyl-sn-Novel FtsZ membrane anchor linked to cell wall hydrolysis 277 glycero-3-phosphocholine (POPC) and 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) at a 1:1 (wt:wt) ratio and for the outer layer L-aphosphatidylcholine (Egg, Chicken PC) and 1-palmitoyl-2oleoyl-sn-glycero-3-phospho-L-serine (POPS) at a 4:1 (wt:wt) ratio were aliquoted.…”

Local actin nucleation tunes centrosomal microtubule nucleation during passage through mitosis

“…At the same time, as the anaphase spindle elongates, signals associated with the anaphase chromatin appear to aid relaxation of the polar cortical actomyosin network (Salmon & Wolniak, 1990;Motegi et al, 2006;von Dassow, 2009;Ramkumar & Baum, 2016). This leads to the de-phosphorylation of ERM proteins, which crosslink actin to the plasma membrane (Rodrigues et al, 2015), to the loss of anillin (Kiyomitsu & Cheeseman, 2013) and to activation of SCAR/WAVE and the Arp2/3 complex at opposing cell poles (Zhang & Robinson, 2005;King et al, 2010;Nezis et al, 2010;Bastos et al, 2012;Luo et al, 2014). In some instances, the process of polar relaxation and cell re-spreading is sufficient to drive division in cells that lack an actomyosin ring (Neujahr et al, 1997;Dix et al, 2018).…”

The mechanobiology of actin cytoskeletal proteins during cell–cell fusion

“…where K bend is the bending modulus, θ i,j is the angle between the surface normal to the neighbouring triangular elements i and j and u 0 i is the reference value of θ i at mechanical equilibrium. The dilation modulus of the membrane-cytoskeleton composite is mainly dominated by the contribution of the actin cortex [22,23]. Consequently, the in-plane elastic energy E in-plane has the form…”