Bin/Amphiphysin/Rvs Domain Research Articles

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.

Membrane-Deformation Ability of ANKHD1 Is Involved in the Early Endosome Enlargement.

SummaryAnkyrin-repeat domains (ARDs) are conserved in large numbers of proteins. ARDs are composed of various numbers of ankyrin repeats (ANKs). ARDs often adopt curved structures reminiscent of the Bin-Amphiphysin-Rvs (BAR) domain, which is the dimeric scaffold for membrane tubulation. BAR domains sometimes have amphipathic helices for membrane tubulation and vesiculation. However, it is unclear whether ARD-containing proteins exhibit similar membrane deformation properties. We found that the ARD of ANK and KH domain-containing protein 1 (ANKHD1) dimerize and deform membranes into tubules and vesicles. Among 25 ANKs of ANKHD1, the first 15 ANKs can form a dimer and the latter 10 ANKs enable membrane tubulation and vesiculation through an adjacent amphipathic helix and a predicted curved structure with a positively charged surface, analogous to BAR domains. Knockdown and localization of ANKHD1 suggested its involvement in the negative regulation of early endosome enlargement owing to its membrane vesiculation.

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.

BAR domain proteins—a linkage between cellular membranes, signaling pathways, and the actin cytoskeleton

Actin filament assembly typically occurs in association with cellular membranes. A large number of proteins sit at the interface between actin networks and membranes, playing diverse roles such as initiation of actin polymerization, modulation of membrane curvature, and signaling. Bin/Amphiphysin/Rvs (BAR) domain proteins have been implicated in all of these functions. The BAR domain family of proteins comprises a diverse group of multi-functional effectors, characterized by their modular architecture. In addition to the membrane-curvature sensing/inducing BAR domain module, which also mediates antiparallel dimerization, most contain auxiliary domains implicated in protein-protein and/or protein-membrane interactions, including SH3, PX, PH, RhoGEF, and RhoGAP domains. The shape of the BAR domain itself varies, resulting in three major subfamilies: the classical crescent-shaped BAR, the more extended and less curved F-BAR, and the inverse curvature I-BAR subfamilies. Most members of this family have been implicated in cellular functions that require dynamic remodeling of the actin cytoskeleton, such as endocytosis, organelle trafficking, cell motility, and T-tubule biogenesis in muscle cells. Here, we review the structure and function of mammalian BAR domain proteins and the many ways in which they are interconnected with the actin cytoskeleton.

Membrane Deformation Ability of the Ankyrin Repeat and KH Domain-Containing Protein 1 (ANKHD1) and Its Involvement in the Early Endosome Enlargement

Ankyrin-repeat domains (ARDs) are conserved in large numbers of proteins. ARDs are composed of ankyrin repeats (ANKs). The number of repeats varies among ARDs, and the sequence of ANKs often adopt curved structures reminiscent of the Bin-Amphiphysin-Rvs (BAR) domain, which is the dimeric membrane scaffold for membrane tubulation. BAR domains sometimes have amphipathic helices that enable the membrane scission for vesicle formation. Whether ARD-containing proteins exhibit similar membrane deformation and scission had been unclear. We found that the ARD of ankyrin repeat and KH domain-containing protein 1 (ANKHD1) dimerize and efficiently deform membranes into tubules and vesicles. ANKHD1 contains 25 ANKs that are divided into two groups; the first 15 ANKs and the latter 10 ANKs. We found that the first 15 ANKs can form a dimer, and the latter 10 ANKs enable membrane tubulation and vesiculation. These 10 ANKs were shown to have an adjacent amphipathic helix and were predicted to have a curved structure with a positively charged surface, analogous to BAR domains. Mutations of the positively charged amino acid residues and the deletion of the amphipathic helix abolished the membrane vesiculation ability. Interestingly, the dimeric 25 ANKs displayed significantly higher vesiculation ability than the 10 ANKs. Knockdown and localization of ANKHD1 suggested its involvement in the negative regulation of the enlargement of early endosomes. These results indicate that ANKHD1 causes vesiculation of the early endosomal membrane in a manner that is similar to the BAR domain protein.

FAM92A1 is a BAR domain protein required for mitochondrial ultrastructure and function.

Mitochondrial function is closely linked to its dynamic membrane ultrastructure. The mitochondrial inner membrane (MIM) can form extensive membrane invaginations known as cristae, which contain the respiratory chain and ATP synthase for oxidative phosphorylation. The molecular mechanisms regulating mitochondrial ultrastructure remain poorly understood. The Bin-Amphiphysin-Rvs (BAR) domain proteins are central regulators of diverse cellular processes related to membrane remodeling and dynamics. Whether BAR domain proteins are involved in sculpting membranes in specific submitochondrial compartments is largely unknown. In this study, we report FAM92A1 as a novel BAR domain protein localizes to the matrix side of the MIM. Loss of FAM92A1 caused a severe disruption to mitochondrial morphology and ultrastructure, impairing organelle bioenergetics. Furthermore, FAM92A1 displayed a membrane-remodeling activity in vitro, inducing a high degree of membrane curvature. Collectively, our findings uncover a role for a BAR domain protein as a critical organizer of the mitochondrial ultrastructure that is indispensable for mitochondrial function.

Phospholipid-flipping activity of P4-ATPase drives membrane curvature.

P4-ATPases are phospholipid flippases that translocate phospholipids from the exoplasmic/luminal to the cytoplasmic leaflet of biological membranes. All P4-ATPases in yeast and some in other organisms are required for membrane trafficking; therefore, changes in the transbilayer lipid composition induced by flippases are thought to be crucial for membrane deformation. However, it is poorly understood whether the phospholipid-flipping activity of P4-ATPases can promote membrane deformation. In this study, we assessed membrane deformation induced by flippase activity via monitoring the extent of membrane tubulation using a system that allows inducible recruitment of Bin/amphiphysin/Rvs (BAR) domains to the plasma membrane (PM). Enhanced phosphatidylcholine-flippase activity at the PM due to expression of ATP10A, a member of the P4-ATPase family, promoted membrane tubulation upon recruitment of BAR domains to the PM This is the important evidence that changes in the transbilayer lipid composition induced by P4-ATPases can deform biological membranes.

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.

A Coincidence Detection Mechanism Controls PX-BAR Domain-Mediated Endocytic Membrane Remodeling via an Allosteric Structural Switch

Clathrin-mediated endocytosis occurs by bending and remodeling of the membrane underneath the coat. Bin-amphiphysin-rvs (BAR) domain proteins are crucial for endocytic membrane remodeling, but how their activity is spatiotemporally controlled is largely unknown. We demonstrate that the membrane remodeling activity of sorting nexin 9 (SNX9), a late-acting endocytic PX-BAR domain protein required for constriction of U-shaped endocytic intermediates, is controlled by an allosteric structural switch involving coincident detection of the clathrin adaptor AP2 and phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2) at endocytic sites. Structural, biochemical, and cell biological data show that SNX9 is autoinhibited in solution. Binding to PI(3,4)P2 via its PX-BAR domain, and concomitant association with AP2 via sequences in the linker region, releases SNX9 autoinhibitory contacts to enable membrane constriction. Our results reveal a mechanism for restricting the latent membrane remodeling activity of BAR domain proteins to allow spatiotemporal coupling of membrane constriction to the progression of the endocytic pathway.

The PICK1 Ca2+ sensor modulates N-methyl-d-aspartate (NMDA) receptor-dependent microRNA-mediated translational repression in neurons

MicroRNAs (miRNAs) are important regulators of localized mRNA translation in neuronal dendrites. The presence of RNA-induced silencing complex proteins in these compartments and the dynamic miRNA expression changes that occur in response to neuronal stimulation highlight their importance in synaptic plasticity. Previously, we demonstrated a novel interaction between the major RNA-induced silencing complex component Argounaute-2 (Ago2) and the BAR (bin/amphiphysin/rvs) domain protein PICK1. PICK1 recruits Ago2 to recycling endosomes in dendrites, where it inhibits miRNA-mediated translational repression. Chemical induction of long-term depression via NMDA receptor activation causes the dissociation of Ago2 from PICK1 and a consequent increase in dendritic miRNA-mediated gene silencing. The mechanism that underlies the regulation of PICK1-Ago2 binding is unknown. In this study, we demonstrate that the PICK1-Ago2 interaction is directly sensitive to Ca2+ ions so that high [Ca2+]free reduces PICK1 binding to Ago2. Mutating a stretch of C-terminal Ca2+-binding residues in PICK1 results in a complete block of NMDA-induced PICK1-Ago2 disassociation in cortical neurons. Furthermore, the same mutant also blocks NMDA-stimulated miRNA-mediated gene silencing. This study defines a novel mechanism whereby elevated [Ca2+] induced by NMDA receptor activation modulates Ago2 and miRNA activity via PICK1. Our work suggests a Ca2+-dependent process to regulate miRNA activity in neurons in response to the induction of long-term depression.

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.

Selenoprotein K regulation of palmitoylation and calpain cleavage of ASAP2 is required for efficient FcγR-mediated phagocytosis.

Effective activation of macrophages through phagocytic Fcγ receptors (FcγR) has been shown to require selenoprotein K (Selk). We set out to determine whether the FcγR-mediated uptake process itself also requires Selk and potential underlying mechanisms. Macrophages from Selk knockout (KO) mice were less efficient compared with wild-type (WT) controls in engulfing IgG-coated fluorescent beads. Using LC-MS/MS to screen for Selk-binding partners involved in FcγR-mediated phagocytosis, we identified Arf-GAP with SH3 domain, ANK repeat, and PH domain-containing protein 2 (ASAP2). Coimmunoprecipitation assays confirmed interactions between Selk and ASAP2. Selk was required for ASAP2 to be cleaved by calpain-2 within the Bin/Amphiphysin/Rvs (BAR) domain of ASAP2. BAR domains promote membrane association, which was consistent with our data showing that Selk deficiency led to retention of ASAP2 within the phagocytic cup. Because Selk was recently identified as a cofactor for the palmitoylation of certain proteins, we investigated whether ASAP2 was palmitoylated and whether this was related to its cleavage by calpain-2. Acyl/biotin exchange assays and MALDI-TOF analysis showed that cysteine-86 in ASAP2 was palmitoylated in WT, but to a much lesser extent in KO, mouse macrophages. Inhibitors of either palmitoylation or calpain-2 cleavage and rescue experiments with different versions of Selk demonstrated that Selk-dependent palmitoylation of ASAP2 leads to cleavage by calpain-2 within the BAR domain, which releases this protein from the maturing phagocytic cup. Overall, these findings identify ASAP2 as a new target of Selk-dependent palmitoylation and reveal a new mechanism regulating the efficiency of FcγR-mediated phagocytosis.

Calmodulin Promotes N-BAR Domain-Mediated Membrane Constriction and Endocytosis

Membrane remodeling by BAR (Bin, Amphiphysin, RVS) domain-containing proteins, such as endophilins and amphiphysins, is integral to the process of endocytosis. However, little is known about the regulation of endocytic BAR domain activity. We have identified an interaction between the yeast Rvs167 N-BAR domain and calmodulin. Calmodulin-binding mutants of Rvs167 exhibited defects in endocytic vesicle release. In vitro, calmodulin enhanced membrane tubulation and constriction by wild-type Rvs167 but not calmodulin-binding-defective mutants. A subset of mammalian N-BAR domains bound calmodulin, and co-expression of calmodulin with endophilin A2 potentiated tubulation in vivo. These studies reveal a conserved role for calmodulin in regulating the intrinsic membrane-sculpting activity of endocytic N-BAR domains.

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.

Dynamics of Membrane Tubes Formed by I-BARS

Membrane deformation is a necessary step in a number of cellular processes such as endocytosis, cell division, filopodia formation and has been shown to involve membrane shaping proteins containing BAR (Bin/Amphiphysin/Rvs) domains. In reconstituted membranes the membrane shaping BAR domains can efficiently deform negatively charged membranes into tubules without any other proteins present. We show how the inverse BAR domain from the filopodia associated ABBA protein dynamically transforms Giant Unilamellar lipid Vesicles (GUVs) into membrane tubes. We characterize their physical properties like persistence length and size and compare the stiffness with protein free tubes of similar diameter. Tube formation can be dynamically reversed by varying the local protein concentration or the membrane tension by using micropipette aspiration or osmotic pressure. At constant protein concentration tubes are found to disappear when membrane tension is increased by aspiration of the GUVs and subsequently reappear when aspiration is reversed.

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.

Shape Transformaiton of Biomembrane Induced by Banana-Shaped Protein Rods: Tubulation and Formation of Polyhedral Vesicles

In living cells, morphology of biomembranes is regulated by various proteins. Many of these proteins contain a banana-shaped binding module called BAR (Bin-Amphiphysin-Rvs) domain. We have studied how anisotropic spontaneous curvatures of banana-shaped protein rod induce effective interaction between the proteins and change membrane shapes by using implicit-solvent meshless membrane simulations [1-3].We found that the self-assembly of the rods is divided to two directional assemblies at the low rod density [1] and polyhedral vesicles and polygonal tubes are formed at the high density [2]. We also revealed that a small spontaneous curvature perpendicular to the rod can remarkably alter the tubulation dynamics at high rod density whereas minor effects are only obtained at low density [3]. A percolated network, which suppresses tubule protrusion, is intermediately formed for negative perpendicular curvatures.[1] Noguchi, H. (2014) EPL 108, 48001.[2] Noguchi, H. (2015) J. Chem. Phys. 143, 243109.[3] Noguchi, H. arXiv:1503.00973.

Human autoantibodies to amphiphysin induce defective presynaptic vesicle dynamics and composition

Stiff-person syndrome is the prototype of a central nervous system disorder with autoantibodies targeting presynaptic antigens. Patients with paraneoplastic stiff-person syndrome may harbour autoantibodies to the BAR (Bin/Amphiphysin/Rvs) domain protein amphiphysin, which target its SH3 domain. These patients have neurophysiological signs of compromised central inhibition and respond to symptomatic treatment with medication enhancing GABAergic transmission. High frequency neurotransmission as observed in tonic GABAergic interneurons relies on fast exocytosis of neurotransmitters based on compensatory endocytosis. As amphiphysin is involved in clathrin-mediated endocytosis, patient autoantibodies are supposed to interfere with this function, leading to disinhibition by reduction of GABAergic neurotransmission. We here investigated the effects of human anti-amphiphysin autoantibodies on structural components of presynaptic boutons ex vivo and in vitro using electron microscopy and super-resolution direct stochastic optical reconstruction microscopy. Ultrastructural analysis of spinal cord presynaptic boutons was performed after in vivo intrathecal passive transfer of affinity-purified human anti-amphiphysin autoantibodies in rats and revealed signs of markedly disabled clathrin-mediated endocytosis. This was unmasked at high synaptic activity and characterized by a reduction of the presynaptic vesicle pool, clathrin coated intermediates, and endosome-like structures. Super-resolution microscopy of inhibitory GABAergic presynaptic boutons in primary neurons revealed that specific human anti-amphiphysin immunoglobulin G induced an increase of the essential vesicular protein synaptobrevin 2 and a reduction of synaptobrevin 7. This constellation suggests depletion of resting pool vesicles and trapping of releasable pool vesicular proteins at the plasma membrane. Similar effects were found in amphiphysin-deficient neurons from knockout mice. Application of specific patient antibodies did not show additional effects. Blocking alternative pathways of clathrin-independent endocytosis with brefeldin A reversed the autoantibody induced effects on molecular vesicle composition. Endophilin as an interaction partner of amphiphysin showed reduced clustering within presynaptic terminals. Collectively, these results point towards an autoantibody-induced structural disorganization in GABAergic synapses with profound changes in presynaptic vesicle pools, activation of alternative endocytic pathways, and potentially compensatory rearrangement of proteins involved in clathrin-mediated endocytosis. Our findings provide novel insights into synaptic pathomechanisms in a prototypic antibody-mediated central nervous system disease, which may serve as a proof-of-principle example in this evolving group of autoimmune disorders associated with autoantibodies to synaptic antigens.