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

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

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

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 alpha-actinin and fascin segregate into distinct domains in single actin bundles with a molecular size-dependent competition-based mechanism. Here, by encapsulating actin, alpha-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 alpha-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 alpha-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. By encapsulating proteins in giant unilamellar vesicles, Bashirzadeh et al find that actin crosslinkers, alpha-actinin and fascin, can self-assemble with actin into complex structures that depend on the degree of confinement. Further analysis and modeling show that alpha-actinin and fascin sort to separate domains of these structures. These insights may be generalizable to other biopolymer networks containing crosslinkers