Extracellular Cues Govern Shape and Cytoskeletal Organization in Giant Unilamellar Lipid Vesicles

Abstract

Spontaneous and induced front-rear polarization and a subsequent asymmetric actin cytoskeleton is a crucial event leading to cell migration, a key process involved in a variety of physiological and pathological conditions such as tissue development, wound healing, and cancer. Migration of adherent cells relies on the balance between adhesion to the underlying matrix and cytoskeleton-driven front protrusion and rear retraction. A current challenge is to uncouple the effect of adhesion and shape from the contribution of the cytoskeleton in regulating the onset of front-rear polarization. Here, we present a minimal model system that introduces an asymmetric actin cytoskeleton in synthetic cells, which are resembled by giant unilamellar lipid vesicles (GUVs) adhering onto symmetric and asymmetric micropatterned surfaces. Surface micropatterning of streptavidin-coated regions with varying adhesion shape and area was achieved by maskless UV photopatterning. To further study the effects of GUV shape on the cytoskeletal organization, actin filaments were polymerized together with bundling proteins inside the GUVs. The micropatterns induce synthetic cell deformation upon adhesion to the surface, with the cell shape adapting to the pattern shape and size. As expected, asymmetric patterns induce an asymmetric deformation in adherent synthetic cells. Actin filaments orient along the long axis of the deformed GUV, when having a length similar to the size of the major axis, whereas short filaments exhibit random orientation. With this bottom-up approach we have laid the first steps to identify the relationship between cell front-rear polarization and cytoskeleton organization in the future. Such a minimal system will allow us to further study the major components needed to create a polarized cytoskeleton at the onset of migration.

Bottom-up Assembled Synthetic SARS-CoV-2 Miniviruses Reveal Lipid Membrane Affinity of Omicron Variant Spike Glycoprotein

“…GUVs were prepared using the emulsion transfer method, following previously described protocols. − In short, a lipid-in-oil solution was prepared by mixing the desired phospholipids with mineral oil, 400 μL of which was placed on top of the outside aqueous phase. The remaining lipid-in-oil solution was mixed with the inner aqueous phase of the vesicle to form a water-in-oil emulsion.…”

How cytoskeletal crosstalk makes cells move: Bridging cell-free and cell studies

“…Surface micropatterning provides an interesting approach to impose polarized shapes to synthetic cells by forcing them to adapt to the pattern shape and size. 269 In addition, one can use light-induced dimerization to induce spatial patterning and symmetry breaking of cytoskeletal networks. Light-inducible dimers (LIDs) come from photoactivatable systems naturally occurring in plants and allow for reversible photoactivation.…”

GeoV: An Open‐Source Software Package for Quantitative Image Analysis of 3D Vesicle Morphologies

“…The simplest example is a plain spherical giant unilamellar vesicle (GUV) 1). In bottom-up synthetic biology, different strategies have been developed to control the morphology of a GUV, including the attachment of DNA origami to the GUV surface [15,19] 2), or the adhesion of GUVs on patterned surfaces [23] 3). GeoV reconstructs both cases well.…”

“…[1][2][3][4] Shape remodeling of GUVs has been achieved by exploiting physico-chemical effects like osmosis, [5][6][7] by reconstituting active membrane-remodeling components like actomyosin, [8][9][10][11][12][13] or by creating synthetic mimics thereof, for instance with DNA nanotechnology. [14][15][16][17][18][19] This led to the realization of several important function such as GUV adhesion, [20][21][22][23] division, [24,25] or morphogenesis. [26] In particular, for relatively easy systems like these with only few components a quantitative analysis of the resulting shapes can provide important information to guide precision engineering.…”