Eukaryote cells have a flexible shape, which dynamically changes according to the function performed by the cell. One mechanism for deforming the cell membrane into the desired shape is through the expression of curved membrane proteins. Furthermore, these curved membrane proteins are often associated with the recruitment of the cytoskeleton, which then applies active forces that deform the membrane. This coupling between curvature and activity was previously explored theoretically in the linear limit of small deformations, and low dimensionality. Here we explore the unrestricted shapes of vesicles that contain active curved membrane proteins, in three-dimensions, using Monte-Carlo numerical simulations. The activity of the proteins is in the form of protrusive forces that push the membrane outwards, as may arise from the cytoskeleton of the cell due to actin or microtubule polymerization occurring near the membrane. For proteins that have an isotropic convex shape, the additional protrusive force enhances their tendency to aggregate and form membrane protrusions (buds). In addition, we find another transition from deformed spheres with necklace type aggregates, to flat pancake-shaped vesicles, where the curved proteins line the outer rim. This second transition is driven by the active forces, coupled to the spontaneous curvature, and the resulting configurations may shed light on the organization of the lamellipodia of adhered and motile cells. † MF and SP provided Monte-Carlo simulations; NG provided the model for active proteins and linear stability analysis; AI, VKI and MD provided the model of selfassembly in equilibrium.

It is believed that in regulated exocytosis the vesicle membrane fuses with the plasma membrane in response to a physiological stimulus. However, in the absence of stimulation, repetitive transient fusion events are also observed, reflecting a stable state. The mechanisms by which the initial fusion pore attains stability are poorly understood. We modelled energetic stability of the fusion pore by taking into account the anisotropic, intrinsic shape of the membrane constituents and their in-plane ordering in the local curvature of the membrane. We used cell-attached membrane capacitance techniques to monitor the appearance and conductance of single fusion pore events in cultured rat lactotrophs. The results revealed a bell-shaped distribution of the fusion pore conductance with a modal value of 25 pS. The experimentally observed increase of the fusion pore stability with decreasing fusion pore radius agrees well with the theoretical predictions. Moreover, the results revealed a correlation between the amplitude of transient capacitance increases and the fusion pore conductance, indicating that larger vesicles may attain a stable fusion pore with larger fusion pore diameters.

Migratory cells use distinct motility modes to navigate different microenvironments, but it is unclear whether these modes rely on the same core set of polarity components. To investigate this, we disrupted actin-related protein 2/3 (Arp2/3) and the WASP-family verprolin homologous protein (WAVE) complex, which assemble branched actin networks that are essential for neutrophil polarity and motility in standard adherent conditions. Surprisingly, confinement rescues polarity and movement of neutrophils lacking these components, revealing a processive bleb-based protrusion program that is mechanistically distinct from the branched actin-based protrusion program but shares some of the same core components and underlying molecular logic. We further find that the restriction of protrusion growth to one site does not always respond to membrane tension directly, as previously thought, but may rely on closely linked properties such as local membrane curvature. Our work reveals a hidden circuit for neutrophil polarity and indicates that cells have distinct molecular mechanisms for polarization that dominate in different microenvironments.

Pores in lipid bilayers are usually not stable; they shrink because of the highly unfavorable line tension of the pore rim. Even in the presence of charged lipids or certain additives such as detergents or isotropic membrane inclusions, membrane pores are generally not expected to be energetically stabilized. We present a theoretical model that predicts the existence of stable pores in a lipid membrane, induced by the presence of anisotropic inclusions. Our model is based on a phenomenological free energy expression that involves three contributions:  the energy associated with the line tension of the pore in the absence of inclusions, the electrostatic energy of the pore for charged membranes, and the interaction energy between the inclusions and the host membrane. We show that the optimal pore size is governed by the shape of the anisotropic inclusions:  saddle-like inclusions favor small pores, whereas more wedgelike inclusions give rise to larger pore sizes. We discuss possible applications of our model and use it to explain the observed dependency of the pore radius in the membrane of red blood cell ghosts on the ionic strength of the surrounding solution.

We model the influence of rigid inclusions on the curvature elasticity of a lipid membrane. Our focus is on conelike transmembrane inclusions that are able to induce long-range deformations in the host bilayer membrane. The elastic properties of the membrane are described in terms of curvature and tilt elasticity. The latter adds an additional degree of freedom that allows the membrane to accommodate an inclusion not only through a curvature deformation but also via changes in lipid tilt. Using a (mean-field level) cell model for homogeneously distributed inclusions in a small membrane segment of prescribed (mesoscopic-scale) spherical shape, we calculate the optimal microscopic-scale deviation of the membrane shape around the intercalated inclusions and the corresponding free energy, analytically. We show that the lipid tilt degree of freedom can lead to local softening of the inclusion-containing lipid bilayer segment. The predicted softening requires a sufficiently small value of the tilt modulus; its origin lies in the reduction of the excess membrane-inclusion interaction energy. We compare our results to the case of suppressed microscopic shape relaxation. Here, too, local softening of the membrane is possible.