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Summary: RhoA-inhibitory bacterial toxins, such as Staphylococcus aureus EDIN toxin, induce large transendothelial cell macroaperture (TEM) tunnels that rupture the host endothelium barrier and promote bacterial dissemination. Host cells repair these tunnels by extending actin-rich membrane waves from the TEM edges. We reveal that cyclic-AMP signaling produced by Bacillus anthracis edema toxin (ET) also induces TEM formation, which correlates with increased vascular permeability. We show that ET-induced TEM formation resembles liquid dewetting, a physical process of nucleation and growth of holes within a thin liquid film. We also identify the cellular mechanisms of tunnel closure and reveal that the I-BAR domain protein Missing in Metastasis (MIM) senses de novo membrane curvature generated by the TEM, accumulates at the TEM edge, and triggers Arp2/3-dependent actin polymerization, which induces actin-rich membrane waves that close the TEM. Thus, the balance between ET-induced TEM formation and resealing likely determines the integrity of the host endothelium barrier. [ABSTRACT FROM AUTHOR]

50 years ago, cell biology was a nascent field. Today, it is a vast discipline whose principles and tools are also applied to other disciplines; vice versa, cell biologists are inspired by other fields. So, the question begs: what is cell biology? The answers are as diverse as the people who define it., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The cell membrane is an inhomogeneous system composed of phospholipids, sterols, carbohydrates, and proteins that can be directly attached to underlying cytoskeleton. The protein linkers between the membrane and the cytoskeleton are believed to have a profound effect on the mechanical properties of the cell membrane and its ability to reshape. Here, we investigate the role of membrane-cortex linkers on the extrusion of membrane tubes using computer simulations and experiments. In simulations, we find that the force for tube extrusion has a nonlinear dependence on the density of membrane-cortex attachments: at a range of low and intermediate linker densities, the force is not significantly influenced by the presence of the membrane-cortex attachments and resembles that of the bare membrane. For large concentrations of linkers, however, the force substantially increases compared with the bare membrane. In both cases, the linkers provided membrane tubes with increased stability against coalescence. We then pulled tubes from HEK cells using optical tweezers for varying expression levels of the membrane-cortex attachment protein Ezrin. In line with simulations, we observed that overexpression of Ezrin led to an increased extrusion force, while Ezrin depletion had a negligible effect on the force. Our results shed light on the importance of local protein rearrangements for membrane reshaping at nanoscopic scales., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Collective action by inverse-Bin/Amphiphysin/Rvs (I-BAR) domains drive micron-scale membrane remodeling. The macroscopic curvature sensing and generation behavior of I-BAR domains is well characterized, and computational models have suggested various mechanisms on simplified membrane systems, but there remain missing connections between the complex environment of the cell and the models proposed thus far. Here, we show a connection between the role of protein curvature and lipid clustering in the relaxation of large membrane deformations. When we include phosphatidylinositol 4,5-bisphosphate-like lipids that preferentially interact with the charged ends of an I-BAR domain, we find clustering of phosphatidylinositol 4,5-bisphosphate-like lipids that induce a directional membrane-mediated interaction between membrane-bound I-BAR domains. Lipid clusters mediate I-BAR domain interactions and cause I-BAR domain aggregates that would not arise through membrane fluctuation-based or curvature-based interactions. Inside of membrane protrusions, lipid cluster-mediated interaction draws long side-by-side aggregates together, resulting in more cylindrical protrusions as opposed to bulbous, irregularly shaped protrusions., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Protein-mediated membrane remodeling is a ubiquitous and critical process for proper cellular function. Inverse Bin/Amphiphysin/Rvs (I-BAR) domains drive local membrane deformation as a precursor to large-scale membrane remodeling. We employ a multiscale approach to provide the molecular mechanism of unusual I-BAR domain-driven membrane remodeling at a low protein surface concentration with near-atomistic detail. We generate a bottom-up coarse-grained model that demonstrates similar membrane-bound I-BAR domain aggregation behavior as our recent Mesoscopic Membrane with Explicit Proteins model. Together, these models bridge several length scales and reveal an aggregation behavior of I-BAR domains. We find that at low surface coverage (i.e., low bound protein density), I-BAR domains form transient, tip-to-tip strings on periodic flat membrane sheets. Inside of lipid bilayer tubules, we find linear aggregates parallel to the axis of the tubule. Finally, we find that I-BAR domains form tip-to-tip aggregates around the edges of membrane domes. These results are supported by in vitro experiments showing low curvature bulges surrounded by I-BAR domains on giant unilamellar vesicles. Overall, our models reveal new I-BAR domain aggregation behavior in membrane tubules and on the surface of vesicles at low surface concentration that add insight into how I-BAR domain proteins may contribute to certain aspects of membrane remodeling in cells., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Membrane scission is essential for intracellular trafficking. While BAR domain proteins such as endophilin have been reported in dynamin-independent scission of tubular membrane necks, the cutting mechanism has yet to be deciphered. Here, we combine a theoretical model, in vitro, and in vivo experiments revealing how protein scaffolds may cut tubular membranes. We demonstrate that the protein scaffold bound to the underlying tube creates a frictional barrier for lipid diffusion; tube elongation thus builds local membrane tension until the membrane undergoes scission through lysis. We call this mechanism friction-driven scission (FDS). In cells, motors pull tubes, particularly during endocytosis. Through reconstitution, we show that motors not only can pull out and extend protein-scaffolded tubes but also can cut them by FDS. FDS is generic, operating even in the absence of amphipathic helices in the BAR domain, and could in principle apply to any high-friction protein and membrane assembly., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Mechanical properties of cell membranes are known to be significantly influenced by the underlying cortical cytoskeleton. The technique of pulling membrane tethers from cells is one of the most effective ways of studying the membrane mechanics and the membrane-cortex interaction. In this article, we show that axon membranes make an interesting system to explore as they exhibit both free membrane-like behavior where the tether-membrane junction is movable on the surface of the axons (unlike many other cell membranes) as well as cell-like behavior where there are transient and spontaneous eruptions in the tether force that vanish when F-actin is depolymerized. We analyze the passive and spontaneous responses of axonal membrane tethers and propose theoretical models to explain the observed behavior., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Although membrane shape varies greatly throughout the cell, the contribution of membrane curvature to transmembrane protein targeting is unknown because of the numerous sorting mechanisms that take place concurrently in cells. To isolate the effect of membrane shape, we used cell-sized giant unilamellar vesicles (GUVs) containing either the potassium channel KvAP or the water channel AQP0 to form membrane nanotubes with controlled radii. Whereas the AQP0 concentrations in flat and curved membranes were indistinguishable, KvAP was enriched in the tubes, with greater enrichment in more highly curved membranes. Fluorescence recovery after photobleaching measurements showed that both proteins could freely diffuse through the neck between the tube and GUV, and the effect of each protein on membrane shape and stiffness was characterized using a thermodynamic sorting model. This study establishes the importance of membrane shape for targeting transmembrane proteins and provides a method for determining the effective shape and flexibility of membrane proteins., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In cell mechanics, distinguishing the respective roles of the plasma membrane and of the cytoskeleton is a challenge. The difference in the behavior of cellular and pure lipid membranes is usually attributed to the presence of the cytoskeleton as explored by membrane nanotube extrusion. Here we revisit this prevalent picture by unveiling unexpected force responses of plasma membrane spheres devoid of cytoskeleton and synthetic liposomes. We show that a tiny variation in the content of synthetic membranes does not affect their static mechanical properties, but is enough to reproduce the dynamic behavior of their cellular counterparts. This effect is attributed to an amplified intramembrane friction. Reconstituted actin cortices inside liposomes induce an additional, but not dominant, contribution to the effective membrane friction. Our work underlines the necessity of a careful consideration of the role of membrane proteins on cell membrane rheology in addition to the role of the cytoskeleton., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Boucrot et al. (2012) demonstrate a membrane fission mechanism independent of nucleotide hydrolysis that is based on membrane insertion of amphipathic helices. They show that, for N-BAR domain proteins, which promote membrane curvature but also contain amphipathic helices, fission is opposed by the BAR domain that stabilizes tubular membrane structures., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Nascent transport intermediates detach from donor membranes by scission. This process can take place in the absence of dynamin, notably in clathrin-independent endocytosis, by mechanisms that are yet poorly defined. We show here that in cells scission of Shiga toxin-induced tubular endocytic membrane invaginations is preceded by cholesterol-dependent membrane reorganization and correlates with the formation of membrane domains on model membranes, suggesting that domain boundary forces are driving tubule membrane constriction. Actin triggers scission by inducing such membrane reorganization process. Tubule occurrence is indeed increased upon cellular depletion of the actin nucleator component Arp2, and the formation of a cortical actin shell in liposomes is sufficient to trigger the scission of Shiga toxin-induced tubules in a cholesterol-dependent but dynamin-independent manner. Our study suggests that membranes in tubular Shiga toxin-induced invaginations are poised to undergo actin-triggered reorganization leading to scission by a physical mechanism that may function independently from or in synergy with pinchase activity., (2010 Elsevier Inc. All rights reserved.)

Intracellular transport relies on the action of motor proteins, which work collectively to either carry small vesicles or pull membranes tubes along cytoskeletal filaments. Although the individual properties of kinesin-1 motors have been extensively studied, little is known on how several motors coordinate their action and spatially organize on the microtubule when pulling on fluid membranes. Here we address these questions by studying, both experimentally and numerically, the growth of membrane tubes pulled by molecular motors. Our in vitro setup allows us to simultaneously control the parameters monitoring tube growth and measure its characteristics. We perform numerical simulations of membrane tube growth, using the experimentally measured values of all parameters, and analyze the growth properties of the tube considering various motor cooperation schemes. The comparison of the numerical results and the experimental data shows that motors use simultaneously several protofilaments of a microtubule to pull a single tube, as motors moving along a single protofilament cannot generate the forces required for tube extraction. In our experimental conditions, we estimate the average number of motors pulling the tube to be approximately nine, distributed over three contiguous protofilaments. Our results also indicate that the motors pulling the tube do not step synchronously.

Tethers are nanocylinders of lipid bilayer membrane, arising in situations ranging from micromanipulation experiments on synthetic vesicles to the formation of dynamic tubular networks in the Golgi apparatus. Relying on the extensive theoretical and experimental works aimed to understand the physics of individual tethers formation, we addressed the problem of the interaction between two nanotubes. By using a combination of micropipette manipulation and optical tweezers, we quantitatively studied the process of coalescence that occurred when the separation distance between both vesicle-tether junctions became smaller than a threshold length. Our experiments, which were supported by an original theoretical analysis, demonstrated that the measurements of the tether force and angle between tethers at coalescence directly yield the bending rigidity, kappa, and the membrane tension, sigma, of the vesicles. Contrary to other methods used to probe the bending rigidity of vesicles, the proposed approach permits a direct measurement of kappa without requiring any control of the membrane tension. Finally, after validation of the method and proposal of possible applications, we experimentally investigated the dynamics of the coalescence process.

In this work, we have investigated a new and general method for the reconstitution of membrane proteins into giant unilamellar vesicles (GUVs). We have analyzed systematically the reconstitution of two radically different membrane proteins, the sarcoplasmic reticulum Ca(2+)-ATPase and the H(+) pump bacteriorhodopsin. In a first step, our method involved a detergent-mediated reconstitution of solubilized membrane proteins into proteoliposomes of 0.1-0.2 microm in size. In a second step, these preformed proteoliposomes were partially dried under controlled humidity followed, in a third step, by electroswelling of the partially dried film to give GUVs. The physical characteristics of GUVs were analyzed in terms of morphology, size, and lamellarity using phase-contrast and differential interference contrast microscopy. The reconstitution process was further characterized by analyzing protein incorporation and biological activity. Both membrane proteins could be homogeneously incorporated into GUVs at lipid/protein ratios ranging from 5 to 40 (w/w). After reconstitution, both proteins retained their biological activity as demonstrated by H(+) or Ca(2+) pumping driven by bacteriorhodopsin or Ca(2+)-ATPase, respectively. This constitutes an efficient new method of reconstitution, leading to the production of large unilamellar membrane protein-containing vesicles of more than 20 microm in diameter, which should prove useful for functional and structural studies through the use of optical microscopy, optical tweezers, microelectrodes, or atomic force microscopy.