Super‐Resolution Imaging of Highly Curved Membrane Structures in Giant Vesicles Encapsulating Molecular Condensates

Abstract

Molecular crowding is an inherent feature of cell interiors. Synthetic cells as provided by giant unilamellar vesicles (GUVs) encapsulating macromolecules (poly(ethylene glycol) and dextran) represent an excellent mimetic system to study membrane transformations associated with molecular crowding and protein condensation. Similarly to cells, such GUVs exhibit highly curved structures like nanotubes. Upon liquid–liquid phase separation their membrane deforms into apparent kinks at the contact line of the interface between the two aqueous phases. These structures, nanotubes, and kinks, have dimensions below optical resolution. Here, these are studied with super‐resolution stimulated emission depletion (STED) microscopy facilitated by immobilization in a microfluidic device. The cylindrical nature of the nanotubes based on the superior resolution of STED and automated data analysis is demonstrated. The deduced membrane spontaneous curvature is in excellent agreement with theoretical predictions. Furthermore, the membrane kink‐like structure is resolved as a smoothly curved membrane demonstrating the existence of the intrinsic contact angle, which describes the wettability contrast of the encapsulated phases to the membrane. Resolving these highly curved membrane structures with STED imaging provides important insights in the membrane properties and interactions underlying cellular activities.

Remodeling of Biomembranes and Vesicles by Adhesion of Condensate Droplets

“…It is thus plausible to assume that the kinks in Figure 3 will be replaced by a smoothly curved membrane segment on sufficient small length scales [ 5 ]. This assumption has been recently confirmed by super-resolution STED microscopy [ 52 ], see Figure 14 . In what follows, we will assume that all kinks in Figure 3 will be smoothened out when observed with sufficiently high resolution.…”

“…Therefore, each kink in Figure 3 should be replaced by a smoothly curved membrane segment on sufficiently small length scales [ 5 ]. Such smoothly curved segments have indeed been observed by super-resolution STED microscopy [ 52 ] as described further below.…”

Increased efficiency of charge-mediated fusion in polymer/lipid hybrid membranes

“…11 A ). To this end, we employed rectangular microfluidic traps ( 59 ) with a gap size of approximately 5 μm between the posts. This setup enabled hydrodynamic control over the LUV delivery, fast exchange of the outer solution, and immobilization of the GUVs.…”

Identifying and Manipulating Giant Vesicles: Review of Recent Approaches

“…An automated algorithm for GV structural assessment using confocal laser scanning fluorescence microscopy images was developed by García-Sáez’s group [ 19 ]. More recently, Dimova’s group developed observation techniques for GUVs based on stimulated emission depletion (STED) microscopy, which can provide a high spatial resolution beyond the diffraction limit of light [ 20 , 21 ]. They found that the deduced membrane spontaneous curvature of nanometer-sized vesicular tubes produced from GVs, was in excellent agreement with theoretical predictions.…”