Shaping Giant Membrane Vesicles in 3D-Printed Protein Hydrogel Cages

Giant unilamellar phospholipid vesicles are attractive starting points for constructing minimal living cells from the bottom-up. Their membranes are compatible with many physiologically functional modules and act as selective barriers, while retaining a high morphological flexibility. However, their spherical shape renders them rather inappropriate to study phenomena that are based on distinct cell shape and polarity, such as cell division. Here, a microscale device based on 3D printed protein hydrogel is introduced to induce pH-stimulated reversible shape changes in trapped vesicles without compromising their free-standing membranes. Deformations of spheres to at least twice their aspect ratio, but also toward unusual quadratic or triangular shapes can be accomplished. Mechanical force induced by the cages to phase-separated membrane vesicles can lead to spontaneous shape deformations, from the recurrent formation of dumbbells with curved necks between domains to full budding of membrane domains as separate vesicles. Moreover, shape-tunable vesicles are particularly desirable when reconstituting geometry-sensitive protein networks, such as reaction-diffusion systems. In particular, vesicle shape changes allow to switch between different modes of self-organized protein oscillations within, and thus, to influence reaction networks directly by external mechanical cues

Communication

The geometry of reaction compartments can affect the local outcome of interface-restricted reactions. Giant unilamellar vesicles (GUVs) are commonly used to generate cell-sized, membrane-bound reaction compartments, which are, however, always spherical. Herein, we report the development of a microfluidic chip to trap and reversibly deform GUVs into cigar-like shapes. When trapping and elongating GUVs that contain the primary protein of the bacterial Z ring, FtsZ, we find that membrane-bound FtsZ filaments align preferentially with the short GUV axis. When GUVs are released from this confinement and membrane tension is relaxed, FtsZ reorganizes reversibly from filaments into dynamic rings that stabilize membrane protrusions; a process that allows reversible GUV deformation. We conclude that microfluidic traps are useful for manipulating both geometry and tension of GUVs, and for investigating how both affect the outcome of spatially-sensitive reactions inside them, such as that of protein self-organization.We acknowledge the MPIB Biochemistry Core Facility for assistance in protein purification

Actin crosslinker competition and sorting drive emergent GUV size-dependent actin network architecture

The proteins that make up the actin cytoskeleton can self-assemble into a variety of structures. In vitro experiments and coarse-grained simulations have shown that the actin crosslinking proteins α-actinin and fascin segregate into distinct domains in single actin bundles with a molecular size-dependent competition-based mechanism. Here, by encapsulating actin, α-actinin, and fascin in giant unilamellar vesicles (GUVs), we show that physical confinement can cause these proteins to form much more complex structures, including rings and asters at GUV peripheries and centers; the prevalence of different structures depends on GUV size. Strikingly, we found that α-actinin and fascin self-sort into separate domains in the aster structures with actin bundles whose apparent stiffness depends on the ratio of the relative concentrations of α-actinin and fascin. The observed boundary-imposed effect on protein sorting may be a general mechanism for creating emergent structures in biopolymer networks with multiple crosslinkers

Protein Reconstitution Inside Giant Unilamellar Vesicles

Giant unilamellar vesicles (GUVs) have gained great popularity as mimicries for cellular membranes. As their sizes are comfortably above the optical resolution limit, and their lipid composition is easily controlled, they are ideal for quantitative light microscopic investigation of dynamic processes in and on membranes. However, reconstitution of functional proteins into the lumen or the GUV membrane itself has proven technically challenging. In recent years, a selection of techniques has been introduced that tremendously improve GUV-assay development and enable the precise investigation of protein-membrane interactions under well-controlled conditions. Moreover, due to these methodological advances, GUVs are considered important candidates as protocells in bottom-up synthetic biology. In this review, we discuss the state of the art of the most important vesicle production and protein encapsulation methods and highlight some key protein systems whose functional reconstitution has advanced the field

Active shape oscillations of giant vesicles with cyclic closure and opening of membrane necks

Reaction-diffusion systems encapsulated within giant unilamellar vesicles (GUVs) can lead to shape oscillations of these vesicles as recently observed for the bacterial Min protein system. This system contains two Min proteins, MinD and MinE, which periodically attach to and detach from the GUV membranes, with the detachment being driven by ATP hydrolysis. Here, we address these shape oscillations within the theoretical framework of curvature elasticity and show that they can be understood in terms of a spontaneous curvature that changes periodically with time. We focus on the simplest case provided by a attachment–detachment kinetics that is laterally uniform along the membrane. During each oscillation cycle, the vesicle shape is transformed from a symmetric dumbbell with two subcompartments of equal size to an asymmetric dumbbell with two subcompartments of different size, followed by the reverse, symmetry-restoring transformation. This sequence of shapes is first analyzed within the spontaneous curvature model which is then extended to the area-difference-elasticity model by decomposing the spontaneous curvature into a local and nonlocal component. For both symmetric and asymmetric dumbbells, the two subcompartments are connected by a narrow membrane neck with a circular waistline. The radius of this waistline undergoes periodic oscillations, the time dependence of which can be reasonably well fitted by a single Fourier mode with an average time period of 56 s

Rapid Encapsulation of Reconstituted Cytoskeleton inside Giant Unilamellar Vesicles

Giant unilamellar vesicles (GUVs) are frequently used as models of biological membranes and thus are a great tool to study membrane-related cellular processes in vitro. In recent years, encapsulation within GUVs has proven to be a helpful approach for reconstitution experiments in cell biology and related fields. It better mimics confinement conditions inside living cells, as opposed to conventional biochemical reconstitution. Methods for encapsulation inside GUVs are often not easy to implement, and success rates can differ significantly from lab to lab. One technique that has proven to be successful for encapsulating more complex protein systems is called continuous droplet interface crossing encapsulation (cDICE). Here, a cDICE-based method is presented for rapidly encapsulating cytoskeletal proteins in GUVs with high encapsulation efficiency. In this method, first, lipid-monolayer droplets are generated by emulsifying a protein solution of interest in a lipid/oil mixture. After being added into a rotating 3D-printed chamber, these lipid-monolayered droplets then pass through a second lipid monolayer at a water/oil interface inside the chamber to form GUVs that contain the protein system. This method simplifies the overall procedure of encapsulation within GUVs and speeds up the process, and thus allows us to confine and observe the dynamic evolution of network assembly inside lipid bilayer vesicles. This platform is handy for studying the mechanics of cytoskeleton-membrane interactions in confinement

Beating vesicles: Encapsulated protein oscillations cause dynamic membrane deformations

The bacterial Min protein system was encapsulated in giant unilamellar vesicles (GUVs). Using confocal fluorescence microscopy, we identified several distinct modes of spatiotemporal patterns inside spherical GUVs. For osmotically deflated GUVs, the vesicle shape actively changed in concert with the Min oscillations. The periodic relocation of Min proteins from the vesicle lumen to the membrane and back is accompanied by drastic changes in the mechanical properties of the lipid bilayer. In particular, two types of oscillating membrane‐shape changes are highlighted: 1) GUVs that repeatedly undergo fission into two connected compartments and fusion of these compartments back into a dumbbell shape and 2) GUVs that show periodic budding and subsequent merging of the buds with the mother vesicle, accompanied by an overall shape change of the vesicle reminiscent of a bouncing ball. These findings demonstrate how reaction–diffusion‐based protein self‐organization can directly yield visible mechanical effects on membrane compartments, even up to autonomous division, without the need for coupling to cytoskeletal elements

Phosphoinositides regulate force-independent interactions between talin, vinculin, and actin

Focal adhesions (FA) are large macromolecular assemblies which help transmit mechanical forces and regulatory signals between the extracellular matrix and an interacting cell. Two key proteins talin and vinculin connecting integrin to actomyosin networks in the cell. Both proteins bind to F-actin and each other, providing a foundation for network formation within FAs. However, the underlying mechanisms regulating their engagement remain unclear. Here, we report on the results of in vitro reconstitution of talin-vinculin-actin assemblies using synthetic membrane systems. We find that neither talin nor vinculin alone recruit actin filaments to the membrane. In contrast, phosphoinositide-rich membranes recruit and activate talin, and the membrane-bound talin then activates vinculin. Together, the two proteins then link actin to the membrane. Encapsulation of these components within vesicles reorganized actin into higher-order networks. Notably, these observations were made in the absence of applied force, whereby we infer that the initial assembly stage of FAs is force independent. Our findings demonstrate that the local membrane composition plays a key role in controlling the stepwise recruitment, activation, and engagement of proteins within FAs