Bin/Amphiphysin/Rvs Proteins Research Articles

Plasma membrane nanodeformations promote actin polymerization through CIP4/CDC42 recruitment and regulate type II IFN signaling

In their environment, cells must cope with mechanical stresses constantly. Among these, nanoscale deformations of plasma membrane induced by substrate nanotopography are now largely accepted as a biophysical stimulus influencing cell behavior and function. However, the mechanotransduction cascades involved and their precise molecular effects on cellular physiology are still poorly understood. Here, using homemade fluorescent nanostructured cell culture surfaces, we explored the role of Bin/Amphiphysin/Rvs (BAR) domain proteins as mechanosensors of plasma membrane geometry. Our data reveal that distinct subsets of BAR proteins bind to plasma membrane deformations in a membrane curvature radius–dependent manner. Furthermore, we show that membrane curvature promotes the formation of dynamic actin structures mediated by the Rho GTPase CDC42, the F-BAR protein CIP4, and the presence of PI(4,5)P 2 . In addition, these actin-enriched nanodomains can serve as platforms to regulate receptor signaling as they appear to contain interferon-γ receptor (IFNγ-R) and to lead to the partial inhibition of IFNγ-induced JAK/STAT signaling.

Spatiotemporal Live-Cell Analysis of Photoreceptor Outer Segment Membrane Ingestion by the Retinal Pigment Epithelium Reveals Actin-Regulated Scission.

The photoreceptor outer segment (OS) is the phototransductive organelle in the vertebrate retina. OS tips are regularly ingested and degraded by the adjacent retinal pigment epithelium (RPE), offsetting the addition of new disk membrane at the base of the OS. This catabolic role of the RPE is essential for photoreceptor health, with defects in ingestion or degradation underlying different forms of retinal degeneration and blindness. Although proteins required for OS tip ingestion have been identified, spatiotemporal analysis of the ingestion process in live RPE cells is lacking; hence, the literature reflects no common understanding of the cellular mechanisms that affect ingestion. We imaged live RPE cells from mice (both sexes) to elucidate the ingestion events in real time. Our imaging revealed roles for f-actin dynamics and specific dynamic localizations of two BAR (Bin-Amphiphysin-Rvs) proteins, FBP17 and AMPH1-BAR, in shaping the RPE apical membrane as it surrounds the OS tip. Completion of ingestion was observed to occur by scission of the OS tip from the remainder of the OS, with a transient concentration of f-actin forming around the site of imminent scission. Actin dynamics were also required for regulating the size of the ingested OS tip, and the time course of the overall ingestion process. The size of the ingested tip is consistent with the term "phagocytosis." However, phagocytosis usually refers to engulfment of an entire particle or cell, whereas our observations of OS tip scission indicate a process that is more specifically described as "trogocytosis," in which one cell "nibbles" another cell.SIGNIFICANCE STATEMENT The ingestion of the photoreceptor outer segment (OS) tips by the retinal pigment epithelium (RPE) is a dynamic cellular process that has fascinated scientists for 60 years. Yet its molecular mechanisms had not been addressed in living cells. We developed a live-cell imaging approach to investigate OS tip ingestion, and focused on the dynamic participation of actin filaments and membrane-shaping BAR proteins. We observed scission of OS tips for the first time, and were able to monitor local changes in protein concentration preceding, during, and following scission. Our approach revealed that actin filaments were concentrated at the site of OS scission and were required for regulating the size of the ingested OS tip and the time course of the ingestion process.

Heterologous Expressed NbSWP12 from Microsporidia Nosema bombycis Can Bind with Phosphatidylinositol 3-Phosphate and Affect Vesicle Genesis.

Microsporidia are a big group of single-celled obligate intracellular organisms infecting most animals and some protozoans. These minimalist eukaryotes lack numerous genes in metabolism and vesicle trafficking. Here, we demonstrated that the spore wall protein NbSWP12 of microsporidium Nosema bombycis belongs to Bin/Amphiphysin/Rvs (BAR) protein family and can specifically bind with phosphatidylinositol 3-phosphate [Ptdlns(3)P]. Since Ptdlns(3)P is involved in endosomal vesicle biogenesis and trafficking, we heterologous expressed NbSWP12 in yeast Saccharomyces cerevisiae and proved that NbSWP12 can target the cell membrane and endocytic vesicles. Nbswp12 transformed into Gvp36 (a BAR protein of S. cerevisiae) deletion mutant rescued the defect phenotype of vesicular traffic. This study identified a BAR protein function in vesicle genesis and sorting and provided clues for further understanding of how microsporidia internalize nutrients and metabolites during proliferation.

A mechanical-thermodynamic model for understanding endocytosis of COVID-19 virus SARS-CoV-2.

We analyze the endocytosis process of COVID-19 (coronavirus disease 2019) virus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) using a mechanical-thermodynamic model. The virus particle is designed to interface with the cell membrane as a hard sphere. The role of cytoplasmic BAR (Bin/Amphiphysin/RVs) proteins is considered in the endocytosis. Interestingly, the Endophilin N-BAR cytoplasmic proteins show resistance in participating endocytosis, whereas F-BAR, Arfaptin BAR, Amphiphysin N-BAR, and PX-BAR proteins participate in endocytosis. The increase in membrane tension, concentrated force between the cell membrane receptor, and spike glycoprotein present on the surface of virus particle promote the endocytosis. Also, the increase in the bending modulus of membrane leads to the two-phase solution of BAR protein concentration on the interior of cell membrane surface. We observe an unstable region of protein concentration, which may help one to retard the endocytosis process and thus the viral infection. Though the present study is focused on SARS-CoV-2, it can be extended to understand any other viral infections, involving endocytosis process.

Curvature dependence of BAR protein membrane association and dissociation kinetics

BAR (Bin/Amphiphysin/Rvs) domain containing proteins function as lipid bilayer benders and curvature sensors, and they contribute to membrane shaping involved in cell signaling and metabolism. The mechanism for their membrane shape sensing has been investigated by both equilibrium binding and kinetic studies. In prior research, stopped-flow spectroscopy has been used to deduce a positive dependence on membrane curvature for the binding rate constant, kon, of a BAR protein called endophilin. However, the impact of bulk diffusion of endophilin, on the kinetic binding parameters has not been thoroughly considered. Employing similar methods, and using lipid vesicles of multiple sizes, we obtained a linear dependence of kon on vesicle curvature. However, we found that the observed relation can be explained without considering the local curvature sensing ability of endophilin in the membrane association process. In contrast, the diffusion-independent unbinding rate constant (koff) obtained from stopped-flow measurements shows a negative dependence on membrane curvature, which is controlled/mediated by endophilin-membrane interactions. This latter dependency, in addition to protein–protein interactions on the membrane, explains the selective binding of BAR proteins to highly curved membranes in equilibrium binding experiments.

Computational investigation for deformation of lipid membrane by BAR proteins due to electrostatic interaction

Deformation of lipid membrane by peripheral BAR (Bin/Amphiphysin/RVs) proteins due to electrostatic interaction between them has an important implication in field of cell-biological systems. We study the deformation behavior of a lipid membrane sheet subjected to electrostatic traction with the help of a very simple finite element elastic model. The lipid membrane is considered as an elastic sheet subjected to the electrostatic traction derived from the literature for the interaction between the lipid membrane and a single BAR protein. Both the small and large deformation behavior of the lipid membrane is considered. To predict small or infinitesimal deformation of the membrane, we use Hooke’s law and large deformation is predicted by Neo-Hookean strain energy density function. The small deformation behavior obtained from the present finite element model does not support the scaffolding mechanism of a single BAR protein binding with the lipid membrane. However, the large deformation behavior of the membrane predicts very well the scaffolding mechanism for the wide range of BAR proteins involved in cell-biological systems. The present model can be used for understanding deformation of any kind of elastic sheet driven by electrostatic forces.

Revisiting I-BAR Proteins at Central Synapses.

Dendritic spines, the distinctive postsynaptic feature of central nervous system (CNS) excitatory synapses, have been studied extensively as electrical and chemical compartments, as well as scaffolds for receptor cycling and positioning of signaling molecules. The dynamics of the shape, number, and molecular composition of spines, and how they are regulated by neural activity, are critically important in synaptic efficacy, synaptic plasticity, and ultimately learning and memory. Dendritic spines originate as outward protrusions of the cell membrane, but this aspect of spine formation and stabilization has not been a major focus of investigation compared to studies of membrane protrusions in non-neuronal cells. We review here one family of proteins involved in membrane curvature at synapses, the BAR (Bin-Amphiphysin-Rvs) domain proteins. The subfamily of inverse BAR (I-BAR) proteins sense and introduce outward membrane curvature, and serve as bridges between the cell membrane and the cytoskeleton. We focus on three I-BAR domain proteins that are expressed in the central nervous system: Mtss2, MIM, and IRSp53 that promote negative, concave curvature based on their ability to self-associate. Recent studies suggest that each has distinct functions in synapse formation and synaptic plasticity. The action of I-BARs is also shaped by crosstalk with other signaling components, forming signaling platforms that can function in a circuit-dependent manner. We discuss another potentially important feature—the ability of some BAR domain proteins to impact the function of other family members by heterooligomerization. Understanding the spatiotemporal resolution of synaptic I-BAR protein expression and their interactions should provide insights into the interplay between activity-dependent neural plasticity and network rewiring in the CNS.

CryoEM reveals BIN1 (isoform 8) does not bind to single actin filaments in vitro.

Cells change their appearance by a concerted action of the cytoskeleton and the plasma membrane. The machinery that bends the membrane includes Bin/Amphiphysin/Rvs (BAR) domain proteins. Recently BAR domain proteins garnered attention as actin regulators, either by recruiting actin regulating proteins or through binding to actin directly. BIN1 (an important protein in Alzheimer’s Disease, heart disease and cancer) is one of the few BAR proteins that bind to actin directly. Here, we imaged a complex of BIN1 and actin with cryoEM. Our results reveal that BIN1 cannot be found on single actin filaments.

Filopodium-derived vesicles produced by MIM enhance the migration of recipient cells.

Extracellular vesicles (EVs) are classified as large EVs (l-EVs, or microvesicles) and small EVs (s-EVs, or exosomes). S-EVs are thought to be generated from endosomes through a process that mainly depends on the ESCRT protein complex, including ALG-2 interacting protein X (ALIX). However, the mechanisms of l-EV generation from the plasma membrane have not been identified. Membrane curvatures are generated by the bin-amphiphysin-rvs (BAR) family proteins, among which the inverse BAR (I-BAR) proteins are involved in filopodial protrusions. Here, we show that the I-BAR proteins, including missing in metastasis (MIM), generate l-EVs by scission of filopodia. Interestingly, MIM-containing l-EV production was promoted by invivo equivalent external forces and by the suppression of ALIX, suggesting an alternative mechanism of vesicle formation to s-EVs. The MIM-dependent l-EVs contained lysophospholipids and proteins, including IRS4 and Rac1, which stimulated the migration of recipient cells through lamellipodia formation. Thus, these filopodia-dependent l-EVs, which we named as filopodia-derived vesicles (FDVs), modify cellular behavior.

The Role of BAR Proteins and the Glycocalyx in Brain Endothelium Transcytosis.

Within the brain, endothelial cells lining the blood vessels meticulously coordinate the transport of nutrients, energy metabolites and other macromolecules essential in maintaining an appropriate activity of the brain. While small molecules are pumped across specialised molecular transporters, large macromolecular cargos are shuttled from one side to the other through membrane-bound carriers formed by endocytosis on one side, trafficked to the other side and released by exocytosis. Such a process is collectively known as transcytosis. The brain endothelium is recognised to possess an intricate vesicular endosomal network that mediates the transcellular transport of cargos from blood-to-brain and brain-to-blood. However, mounting evidence suggests that brain endothelial cells (BECs) employ a more direct route via tubular carriers for a fast and efficient transport from the blood to the brain. Here, we compile the mechanism of transcytosis in BECs, in which we highlight intracellular trafficking mediated by tubulation, and emphasise the possible role in transcytosis of the Bin/Amphiphysin/Rvs (BAR) proteins and glycocalyx (GC)—a layer of sugars covering BECs, in transcytosis. Both BAR proteins and the GC are intrinsically associated with cell membranes and involved in the modulation and shaping of these membranes. Hence, we aim to summarise the machinery involved in transcytosis in BECs and highlight an uncovered role of BAR proteins and the GC at the brain endothelium.

Development of a Membrane Curvature-Sensing Peptide Based on a Structure-Activity Correlation Study.

Membrane curvature formation is important for various biological processes such as cell motility, intracellular signal transmission, and cellular uptake of foreign substances. However, it remains still a challenging topic to visualize the membrane curvature formation on the cell membranes in real-time imaging. To develop and design membrane curvature-sensors, we focused on amphipathic helical peptides of proteins belonging to the Bin/Amphiphysin/Rvs (BAR) family as the starting point. BAR proteins individually have various characteristic structures that recognize different curvatures, and the derived peptides possess the potential to function as curvature sensors with a variety of recognition abilities. Peptide-based curvature sensors can have wide applications in biological research fields due to their small size, easy modification, and large production capability in comparison to protein-based sensors. In the present study, we found that an amphipathic peptide derived from sorting nexin1 (SNX1) has a curvature-recognition ability. The mutation studies of the initial peptide revealed a close correlation between the α-helicity and lipid binding ability of the peptides. In particular, the amino acids located on the hydrophobic face played a vital role in curvature recognition. The α-helix formation of the peptides was thought to serve to accommodate lipid-packing defects on the membrane surface and to maintain their binding to lipid vesicles. The structure-activity correlation found in this study have the potential to contribute to the design of peptide-based curvature sensors that will enable the capture of various life phenomena in cells.

ACAP1 assembles into an unusual protein lattice for membrane deformation through multiple stages.

Studies on the Bin-Amphiphysin-Rvs (BAR) domain have advanced a fundamental understanding of how proteins deform membrane. We previously showed that a BAR domain in tandem with a Pleckstrin Homology (PH domain) underlies the assembly of ACAP1 (Arfgap with Coil-coil, Ankryin repeat, and PH domain I) into an unusual lattice structure that also uncovers a new paradigm for how a BAR protein deforms membrane. Here, we initially pursued computation-based refinement of the ACAP1 lattice to identify its critical protein contacts. Simulation studies then revealed how ACAP1, which dimerizes into a symmetrical structure in solution, is recruited asymmetrically to the membrane through dynamic behavior. We also pursued electron microscopy (EM)-based structural studies, which shed further insight into the dynamic nature of the ACAP1 lattice assembly. As ACAP1 is an unconventional BAR protein, our findings broaden the understanding of the mechanistic spectrum by which proteins assemble into higher-ordered structures to achieve membrane deformation.

Mechanistic Studies of Membrane Remodeling in Receptor Mediated Endocytosis

Receptor mediated endocytosis requires the generation of membrane curvature. Followed by external stimulation, various G-protein coupled receptors and epidermal growth factor receptors are internalized and recycled by this crucial membrane trafficking pathway. Bin/Amphiphysin/RVS (BAR) superfamily proteins have emerged as key effectors in membrane reshaping during the endocytic events. In addition to a crescent shaped BAR domain, many of these proteins contain a Src homology 3 (SH3) domain. BAR proteins sense and generate membrane curvature with their membrane binding domain whereas the SH3 domain regulates their interaction with other protein binding partners. Receptors containing proline rich domains (PRD) have been found to interact with different classes of SH3 domain containing proteins. While it has been hypothesized that the SH3 domain-PRD interaction plays an important role in BAR protein mediated receptor internalization, the exact mechanism has thus far remained elusive. We mimic SH3 domain-PRD interactions in artificial lipid bilayers and investigate their effects on the characteristic membrane curvature generation properties of BAR proteins. PRDs covalently linked to the lipid bilayer are designed to recruit the BAR proteins. The associated membrane shape changes are monitored by both optical and electron microscopy. Insights into BAR protein mediated membrane remodeling in receptor internalization processes from these biophysical studies will be discussed in this contribution.

Membrane re-modelling by BAR domain superfamily proteins via molecular and non-molecular factors.

Lipid membranes are structural components of cell surfaces and intracellular organelles. Alterations in lipid membrane shape are accompanied by numerous cellular functions, including endocytosis, intracellular transport, and cell migration. Proteins containing Bin-Amphiphysin-Rvs (BAR) domains (BAR proteins) are unique, because their structures correspond to the membrane curvature, that is, the shape of the lipid membrane. BAR proteins present at high concentration determine the shape of the membrane, because BAR domain oligomers function as scaffolds that mould the membrane. BAR proteins co-operate with various molecular and non-molecular factors. The molecular factors include cytoskeletal proteins such as the regulators of actin filaments and the membrane scission protein dynamin. Lipid composition, including saturated or unsaturated fatty acid tails of phospholipids, also affects the ability of BAR proteins to mould the membrane. Non-molecular factors include the external physical forces applied to the membrane, such as tension and friction. In this mini-review, we will discuss how the BAR proteins orchestrate membrane dynamics together with various molecular and non-molecular factors.

BAR Domain Proteins Regulate Rho GTPase Signaling.

The Bin-Amphiphysin-Rvs (BAR) domain is a membrane lipid binding domain present in a wide variety of proteins, often proteins with a role in Rho-regulated signaling pathways. BAR domains do not only confer binding to lipid bilayers, they also possess a membrane sculpturing ability and thereby directly control the topology of biomembranes. BAR domain-containing proteins participate in a plethora of physiological processes but the common denominator is their capacity to link membrane dynamics to actin dynamics and thereby integrate processes such as endocytosis, exocytosis, vesicle trafficking, cell morphogenesis and cell migration. The Rho family of small GTPases constitutes an important bridging theme for many BAR domain-containing proteins. This review article will focus predominantly on the role of BAR proteins as regulators or effectors of Rho GTPases and it will only briefly discuss the structural and biophysical function of the BAR domains.

Mtss1 promotes maturation and maintenance of cerebellar neurons via splice variant-specific effects.

Efficient coupling of the actin cytoskeleton to the cell membrane is crucial for histogenesis and maintenance of the nervous system. At this critical interface, BAR (Bin-Amphiphysin-Rvs) proteins regulate membrane bending, shown to be instrumental for mobility and morphogenesis of individual cells. Yet, the systemic significance of these proteins remains largely unexplored. Here, we probe the role of a prominent member of this protein family, the inverse-BAR protein Mtss1, for the development and function of a paradigmatic neuronal circuit, the cerebellar cortex. Mtss1-null mice show granule cell ectopias, dysmorphic Purkinje cells, malformed axons, and a protracted neurodegeneration entailing age-dependent motor deficits. In postmitotic granule cells, which transiently express Mtss1 while they migrate and form neurites, Mtss1 impinges on directional persistence and neuritogenesis. The latter effect can be specifically attributed to its exon 12a splice variant. Targeted re-expression of Mtss1 in Mtss1-null animals indicated that these pathologies were largely due to cell type-specific and intrinsic effects. Together, our results provide a mechanistic perspective on Mtss1 function for brain development and degeneration and relate it to structural features of this protein.

How curvature-generating proteins build scaffolds on membrane nanotubes

Bin/Amphiphysin/Rvs (BAR) domain proteins control the curvature of lipid membranes in endocytosis, trafficking, cell motility, the formation of complex subcellular structures, and many other cellular phenomena. They form 3D assemblies that act as molecular scaffolds to reshape the membrane and alter its mechanical properties. It is unknown, however, how a protein scaffold forms and how BAR domains interact in these assemblies at protein densities relevant for a cell. In this work, we use various experimental, theoretical, and simulation approaches to explore how BAR proteins organize to form a scaffold on a membrane nanotube. By combining quantitative microscopy with analytical modeling, we demonstrate that a highly curving BAR protein endophilin nucleates its scaffolds at the ends of a membrane tube, contrary to a weaker curving protein centaurin, which binds evenly along the tube's length. Our work implies that the nature of local protein-membrane interactions can affect the specific localization of proteins on membrane-remodeling sites. Furthermore, we show that amphipathic helices are dispensable in forming protein scaffolds. Finally, we explore a possible molecular structure of a BAR-domain scaffold using coarse-grained molecular dynamics simulations. Together with fluorescence microscopy, the simulations show that proteins need only to cover 30-40% of a tube's surface to form a rigid assembly. Our work provides mechanical and structural insights into the way BAR proteins may sculpt the membrane as a high-order cooperative assembly in important biological processes.

Higher-order assemblies of BAR domain proteins for shaping membranes.

Most cellular organelles contain lipid bilayer membranes. The earliest characterization of cellular organelles was performed by electron microscopy observation of such membranes. However, the precise mechanisms for shaping the membrane in particular subcellular organelles is poorly understood. Classically, the overall cellular shape, i.e. the shape of the plasma membrane, was thought to be governed by the reorganization of cytoskeletal components such as actin and microtubules. The plasma membrane contains various submicron structures such as clathrin-coated pits, caveolae, filopodia and lamellipodia. These subcellular structures are either invaginations or protrusions and are associated with the cytoskeleton. Therefore, it could be hypothesized that there are membrane-binding proteins that cooperates with cytoskeleton in shaping of plasma membrane organelles. Proteins with the Bin-Amphiphysin-Rvs (BAR) domain connect a variety of membrane shapes to actin filaments. The BAR domains themselves bend the membranes by their rigidity and then mold the membranes into tubules through their assembly as spiral polymers, which are thought to be involved in the various submicron structures. Membrane tubulation by polymeric assembly of the BAR domains is supposed to be regulated by binding proteins, binding lipids and the mechanical properties of the membrane. This review gives an overview of BAR protein assembly, describes the significance of the assembly and discusses how to study the assembly in the context of membrane and cellular morphology. The technical problems encountered in microscopic observation of BAR domain assembly are also discussed.

Membrane tubule formation by banana-shaped proteins with or without transient network structure.

In living cells, membrane morphology is regulated by various proteins. Many membrane reshaping proteins contain a Bin/Amphiphysin/Rvs (BAR) domain, which consists of a banana-shaped rod. The BAR domain bends the biomembrane along the rod axis and the features of this anisotropic bending have recently been studied. Here, we report on the role of the BAR protein rods in inducing membrane tubulation, using large-scale coarse-grained simulations. We reveal that a small spontaneous side curvature perpendicular to the rod can drastically alter the tubulation dynamics at high protein density, whereas no significant difference is obtained at low density. A percolated network is intermediately formed depending on the side curvature. This network suppresses tubule protrusion, leading to the slow formation of fewer tubules. Thus, the side curvature, which is generated by protein–protein and membrane–protein interactions, plays a significant role in tubulation dynamics. We also find that positive surface tensions and the vesicle membrane curvature can stabilize this network structure by suppressing the tubulation.

Bar Domain Proteins can Differ Substantially in their Capacity to Generate Membrane Curvature

BAR (Bin/amphiphysin/Rvs) domains bind to and reshape cell membranes. Numerous BAR proteins are known to participate in endocytosis pathways. Whether their function is redundant or specifically different has remained unclear. Here we first compared the membrane curvature generation abilities of three N-BAR domains involved in endocytosis: endophilin, amphiphysin and SNX9. For this purpose, we used a membrane shape stability assay recently developed in our lab that allows determination of shape stability diagrams obtained via pipette aspirated giant unilamellar vesicles (GUVs). Significantly different membrane stability phase diagrams were observed for these three proteins, suggesting their functional non-redundant. We further compared the membrane binding and curvature generating of endophilin with an F-BAR protein: FCHo2, which arrives at clathrin coated pits (CCPs) earlier than endophilin. We observed interesting competitive membrane binding between FCHo2 and endophilin, as well as stronger curvature induction abilities of FCHo2 when compared with endophilin. These observations suggest a membrane binding regulation mechanism by the BAR domains, and promising to explain why FCHo2 act as the nucleator of the CCPs.