Cell biology is .

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.

Curving Cells Inside and Out: Roles of BAR Domain Proteins in Membrane Shaping and Its Cellular Implications.

Many cellular processes rely on precise and timely deformation of the cell membrane. While many proteins participate in membrane reshaping and scission, usually in highly specialized ways, Bin/amphiphysin/Rvs (BAR) domain proteins play a pervasive role, as they not only participate in many aspects of cell trafficking but also are highly versatile membrane remodelers. Subtle changes in the shape and size of the BAR domain can greatly impact the way in which BAR domain proteins interact with the membrane. Furthermore, the activity of BAR domain proteins can be tuned by external physical parameters, and so they behave differently depending on protein surface density, membrane tension, or membrane shape. These proteins can form 3D structures that mold the membrane and alter its liquid properties, even promoting scission under various circumstances.As such, BAR domain proteins have numerous roles within the cell. Endocytosis is among the most highly studied processes in which BAR domain proteins take on important roles. Over the years, a more complete picture has emerged in which BAR domain proteins are tied to almost all intracellular compartments; examples include endosomal sorting and tubular networks in the endoplasmic reticulum and T-tubules. These proteins also have a role in autophagy, and their activity has been linked with cancer. Here, we briefly review the history of BAR domain protein discovery, discuss the mechanisms by which BAR domain proteins induce curvature, and attempt to settle important controversies in the field. Finally, we review BAR domain proteins in the context of a cell, highlighting their emerging roles in cell signaling and organelle shaping.

Friction Mediates Scission of Tubular Membranes Scaffolded by BAR Proteins.

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.

Tunnelling nanotube formation is driven by Eps8/IRSp53-dependent linear actin polymerization.

Tunnelling nanotubes (TNTs) connect distant cells and mediate cargo transfer for intercellular communication in physiological and pathological contexts. How cells generate these actin-mediated protrusions to span lengths beyond those attainable by canonical filopodia remains unknown. Through a combination of micropatterning, microscopy, and optical tweezer-based approaches, we demonstrate that TNTs formed through the outward extension of actin achieve distances greater than the mean length of filopodia and that branched Arp2/3-dependent pathways attenuate the extent to which actin polymerizes in nanotubes, thus limiting their occurrence. Proteomic analysis using epidermal growth factor receptor kinase substrate 8 (Eps8) as a positive effector of TNTs showed that, upon Arp2/3 inhibition, proteins enhancing filament turnover and depolymerization were reduced and Eps8 instead exhibited heightened interactions with the inverted Bin/Amphiphysin/Rvs (I-BAR) domain protein IRSp53 that provides a direct connection with linear actin polymerases. Our data reveals how common protrusion players (Eps8 and IRSp53) form tunnelling nanotubes, and that when competing pathways overutilizing such proteins and monomeric actin in Arp2/3 networks are inhibited, processes promoting linear actin growth dominate to favour tunnelling nanotube formation.

Building endocytic pits without clathrin.

How endocytic pits are built in clathrin- and caveolin-independent endocytosis still remains poorly understood. Recent insight suggests that different forms of clathrin-independent endocytosis might involve the actin-driven focusing of membrane constituents, the lectin-glycosphingolipid-dependent construction of endocytic nanoenvironments, and Bin-Amphiphysin-Rvs (BAR) domain proteins serving as scaffolding modules. We discuss the need for different types of internalization processes in the context of diverse cellular functions, the existence of clathrin-independent mechanisms of cargo recruitment and membrane bending from a biological and physical perspective, and finally propose a generic scheme for the formation of clathrin-independent endocytic pits.

Actin dynamics drive membrane reorganization and scission in clathrin-independent endocytosis.

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.

Friction-driven membrane scission by the human ESCRT-III proteins CHMP1B and IST1.

The endosomal sorting complexes required for transport (ESCRT) system is an ancient and ubiquitous membrane scission machinery that catalyzes the budding and scission of membranes. ESCRT-mediated scission events, exemplified by those involved in the budding of HIV-1, are usually directed away from the cytosol ("reverse topology"), but they can also be directed toward the cytosol ("normal topology"). The ESCRT-III subunits CHMP1B and IST1 can coat and constrict positively curved membrane tubes, suggesting that these subunits could catalyze normal topology membrane severing. CHMP1B and IST1 bind and recruit the microtubule-severing AAA+ ATPase spastin, a close relative of VPS4, suggesting that spastin could have a VPS4-like role in normal-topology membrane scission. Here, we reconstituted the process in vitro using membrane nanotubes pulled from giant unilamellar vesicles using an optical trap in order to determine whether CHMP1B and IST1 are capable of membrane severing on their own or in concert with VPS4 or spastin. CHMP1B and IST1 copolymerize on membrane nanotubes, forming stable scaffolds that constrict the tubes, but do not, on their own, lead to scission. However, CHMP1B-IST1 scaffolded tubes were severed when an additional extensional force was applied, consistent with a friction-driven scission mechanism. We found that spastin colocalized with CHMP1B-enriched sites but did not disassemble the CHMP1B-IST1 coat from the membrane. VPS4 resolubilized CHMP1B and IST1 without leading to scission. These observations show that the CHMP1B-IST1 ESCRT-III combination is capable of severing membranes by a friction-driven mechanism that is independent of VPS4 and spastin.

Myosin 1b flattens and prunes branched actin filaments.

Motile and morphological cellular processes require a spatially and temporally coordinated branched actin network that is controlled by the activity of various regulatory proteins, including the Arp2/3 complex, profilin, cofilin and tropomyosin. We have previously reported that myosin 1b regulates the density of the actin network in the growth cone. Here, by performing in vitro F-actin gliding assays and total internal reflection fluorescence (TIRF) microscopy, we show that this molecular motor flattens (reduces the branch angle) in the Arp2/3-dependent actin branches, resulting in them breaking, and reduces the probability of new branches forming. This experiment reveals that myosin 1b can produce force sufficient enough to break up the Arp2/3-mediated actin junction. Together with the former in vivo studies, this work emphasizes the essential role played by myosins in the architecture and dynamics of actin networks in different cellular regions.This article has an associated First Person interview with the first author of the paper.

Regulation of kinesin-1 activity by the Salmonella enterica effectors PipB2 and SifA.

Salmonella enterica is an intracellular bacterial pathogen. The formation of its replication niche, which is composed of a vacuole associated with a network of membrane tubules, depends on the secretion of a set of bacterial effector proteins whose activities deeply modify the functions of the eukaryotic host cell. By recruiting and regulating the activity of the kinesin-1 molecular motor, Salmonella effectors PipB2 and SifA play an essential role in the formation of the bacterial compartments. In particular, they allow the formation of tubules from the vacuole and their extension along the microtubule cytoskeleton, and thus promote membrane exchanges and nutrient supply. We have developed in vitro and in cellulo assays to better understand the specific role played by these two effectors in the recruitment and regulation of kinesin-1. Our results reveal a specific interaction between the two effectors and indicate that, contrary to what studies on infected cells suggested, interaction with PipB2 is sufficient to relieve the autoinhibition of kinesin-1. Finally, they suggest the involvement of other Salmonella effectors in the control of the activity of this molecular motor.This article has an associated First Person interview with the first author of the paper.

The ESCRT-III isoforms CHMP2A and CHMP2B display different effects on membranes upon polymerization.

BACKGROUND: ESCRT-III proteins are involved in many membrane remodeling processes including multivesicular body biogenesis as first discovered in yeast. In humans, ESCRT-III CHMP2 exists as two isoforms, CHMP2A and CHMP2B, but their physical characteristics have not been compared yet. RESULTS: Here, we use a combination of techniques on biomimetic systems and purified proteins to study their affinity and effects on membranes. We establish that CHMP2B binding is enhanced in the presence of PI(4,5)P2 lipids. In contrast, CHMP2A does not display lipid specificity and requires CHMP3 for binding significantly to membranes. On the micrometer scale and at moderate bulk concentrations, CHMP2B forms a reticular structure on membranes whereas CHMP2A (+CHMP3) binds homogeneously. Thus, CHMP2A and CHMP2B unexpectedly induce different mechanical effects to membranes: CHMP2B strongly rigidifies them while CHMP2A (+CHMP3) has no significant effect. CONCLUSIONS: We therefore conclude that CHMP2B and CHMP2A exhibit different mechanical properties and might thus contribute differently to the diverse ESCRT-III-catalyzed membrane remodeling processes.

Lipid-Composition-Mediated Forces Can Stabilize Tubular Assemblies of I-BAR Proteins.

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.

Influence of membrane-cortex linkers on the extrusion of membrane tubes.

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.

Concluding remarks: peptide-membrane interactions.

This article is based on the concluding remarks lecture given at the Faraday Discussion meeting on peptide-membrane interactions, held online, 8-10th September 2021.

Comparing physical mechanisms for membrane curvature-driven sorting of BAR-domain proteins.

Protein enrichment at specific membrane locations in cells is crucial for many cellular functions. It is well-recognized that the ability of some proteins to sense membrane curvature contributes partly to their enrichment in highly curved cellular membranes. In the past, different theoretical models have been developed to reveal the physical mechanisms underlying curvature-driven protein sorting. This review aims to provide a detailed discussion of the two continuous models that are based on the Helfrich elasticity energy, (1) the spontaneous curvature model and (2) the curvature mismatch model. These two models are commonly applied to describe experimental observations of protein sorting. We discuss how they can be used to explain the curvature-induced sorting data of two BAR proteins, amphiphysin and centaurin. We further discuss how membrane rigidity, and consequently the membrane curvature generated by BAR proteins, could influence protein organization on the curved membranes. Finally, we address future directions in extending these models to describe some cellular phenomena involving protein sorting.

Endophilin-A2 functions in membrane scission in clathrin-independent endocytosis.

During endocytosis, energy is invested to narrow the necks of cargo-containing plasma membrane invaginations to radii at which the opposing segments spontaneously coalesce, thereby leading to the detachment by scission of endocytic uptake carriers. In the clathrin pathway, dynamin uses mechanical energy from GTP hydrolysis to this effect, assisted by the BIN/amphiphysin/Rvs (BAR) domain-containing protein endophilin. Clathrin-independent endocytic events are often less reliant on dynamin, and whether in these cases BAR domain proteins such as endophilin contribute to scission has remained unexplored. Here we show, in human and other mammalian cell lines, that endophilin-A2 (endoA2) specifically and functionally associates with very early uptake structures that are induced by the bacterial Shiga and cholera toxins, which are both clathrin-independent endocytic cargoes. In controlled in vitro systems, endoA2 reshapes membranes before scission. Furthermore, we demonstrate that endoA2, dynamin and actin contribute in parallel to the scission of Shiga-toxin-induced tubules. Our results establish a novel function of endoA2 in clathrin-independent endocytosis. They document that distinct scission factors operate in an additive manner, and predict that specificity within a given uptake process arises from defined combinations of universal modules. Our findings highlight a previously unnoticed link between membrane scaffolding by endoA2 and pulling-force-driven dynamic scission.

Lipid packing defects and membrane charge control RAB GTPase recruitment.

Specific intracellular localization of RAB GTPases has been reported to be dependent on protein factors, but the contribution of the membrane physicochemical properties to this process has been poorly described. Here, we show that three RAB proteins (RAB1/RAB5/RAB6) preferentially bind in vitro to disordered and curved membranes, and that this feature is uniquely dependent on their prenyl group. Our results imply that the addition of a prenyl group confers to RAB proteins, and most probably also to other prenylated proteins, the ability to sense lipid packing defects induced by unsaturated conical-shaped lipids and curvature. Consistently, RAB recruitment increases with the amount of lipid packing defects, further indicating that these defects drive RAB membrane targeting. Membrane binding of RAB35 is also modulated by lipid packing defects but primarily dependent on negatively charged lipids. Our results suggest that a balance between hydrophobic insertion of the prenyl group into lipid packing defects and electrostatic interactions of the RAB C-terminal region with charged membranes tunes the specific intracellular localization of RAB proteins.

Drp1 polymerization stabilizes curved tubular membranes similar to those of constricted mitochondria.

Dynamin-related protein 1 (Drp1), an 80 kDa mechanochemical GTPase of the dynamin superfamily, is required for mitochondrial division in mammals. Despite the role of Drp1 dysfunction in human disease, its molecular mechanism remains poorly understood. Here, we examined the effect of Drp1 on membrane curvature using tubes pulled from giant unilamellar vesicles (GUVs). We found that GTP promoted rapid rearrangement of Drp1 from a uniform distribution to discrete foci, in line with the assembly of Drp1 scaffolds at multiple nucleation sites around the lipid tube. Polymerized Drp1 preserved the membrane tube below the protein coat, also in the absence of pulling forces, but did not induce spontaneous membrane fission. Strikingly, Drp1 polymers stabilized membrane curvatures similar to those of constricted mitochondria against pressure changes. Our findings support a new model for mitochondrial division whereby Drp1 mainly acts as a scaffold for membrane curvature stabilization, which sets it apart from other dynamin homologs.

The ESCRT protein CHMP2B acts as a diffusion barrier on reconstituted membrane necks.

Endosomal sorting complexes required for transport (ESCRT)-III family proteins catalyze membrane remodeling processes that stabilize and constrict membrane structures. It has been proposed that stable ESCRT-III complexes containing CHMP2B could establish diffusion barriers at the post-synaptic spine neck. In order to better understand this process, we developed a novel method based on fusion of giant unilamellar vesicles to reconstitute ESCRT-III proteins inside GUVs, from which membrane nanotubes are pulled. The new assay ensures that ESCRT-III proteins polymerize only when they become exposed to physiologically relevant membrane topology mimicking the complex geometry of post-synaptic spines. We establish that CHMP2B, both full-length and with a C-terminal deletion (ΔC), preferentially binds to membranes containing phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Moreover, we show that CHMP2B preferentially accumulates at the neck of membrane nanotubes, and provide evidence that CHMP2B-ΔC prevents the diffusion of PI(4,5)P2 lipids and membrane-bound proteins across the tube neck. This indicates that CHMP2B polymers formed at a membrane neck may function as a diffusion barrier, highlighting a potential important function of CHMP2B in maintaining synaptic spine structures.

Whole-GUV patch-clamping.

Studying how the membrane modulates ion channel and transporter activity is challenging because cells actively regulate membrane properties, whereas existing in vitro systems have limitations, such as residual solvent and unphysiologically high membrane tension. Cell-sized giant unilamellar vesicles (GUVs) would be ideal for in vitro electrophysiology, but efforts to measure the membrane current of intact GUVs have been unsuccessful. In this work, two challenges for obtaining the "whole-GUV" patch-clamp configuration were identified and resolved. First, unless the patch pipette and GUV pressures are precisely matched in the GUV-attached configuration, breaking the patch membrane also ruptures the GUV. Second, GUVs shrink irreversibly because the membrane/glass adhesion creating the high-resistance seal (>1 GΩ) continuously pulls membrane into the pipette. In contrast, for cell-derived giant plasma membrane vesicles (GPMVs), breaking the patch membrane allows the GPMV contents to passivate the pipette surface, thereby dynamically blocking membrane spreading in the whole-GMPV mode. To mimic this dynamic passivation mechanism, beta-casein was encapsulated into GUVs, yielding a stable, high-resistance, whole-GUV configuration for a range of membrane compositions. Specific membrane capacitance measurements confirmed that the membranes were truly solvent-free and that membrane tension could be controlled over a physiological range. Finally, the potential for ion transport studies was tested using the model ion channel, gramicidin, and voltage-clamp fluorometry measurements were performed with a voltage-dependent fluorophore/quencher pair. Whole-GUV patch-clamping allows ion transport and other voltage-dependent processes to be studied while controlling membrane composition, tension, and shape.

Unusual Organization of I-BAR Proteins on Tubular and Vesicular Membranes.

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.