N-BAR Domain Research Articles

A single-particle analysis method for detecting membrane remodelling and curvature sensing.

In biology, shape and function are related. Therefore, it is important to understand how membrane shape is generated, stabilised and sensed by proteins and how this relates to organelle function. Here, we present an assay that can detect curvature preference and membrane remodelling with free-floating liposomes using protein concentrations in physiologically relevant ranges. The assay reproduced known curvature preferences of BAR domains and allowed the discovery of high-curvature preference for the PH domain of AKT and the FYVE domain of HRS (also known as HGS). In addition, our method reproduced the membrane vesiculation activity of the ENTH domain of epsin-1 (EPN1) and showed similar activity for the ANTH domains of PiCALM and Hip1R. Finally, we found that the curvature sensitivity of the N-BAR domain of endophilin inversely correlates to membrane charge and that deletion of its N-terminal amphipathic helix increased its curvature specificity. Thus, our method is a generally applicable qualitative method for assessing membrane curvature sensing and remodelling by proteins.

Distinct Bin/Amphiphysin/Rvs (BAR) family proteins may assemble on the same tubule to regulate membrane organization in vivo

Intracellular membrane tubules play a crucial role in diverse cellular processes, and their regulation is facilitated by Bin-Amphiphysin-Rvs (BAR) domain-containing proteins. This study investigates the roles of Drosophila ICA69 (dICA69) (an N-BAR protein) and Drosophila CIP4 (dCIP4) (an F-BAR protein), focusing on their impact on in vivo membrane tubule organization. In contrast to the prevailing models of BAR-domain protein function, we observed colocalization of endogenous dICA69 with dCIP4-induced tubules, indicating their potential recruitment for tubule formation and maintenance. Moreover, actin-regulatory proteins such as Wasp, SCAR, and Arp2/3 were recruited at the site of CIP4-induced tubule formation. An earlier study indicated that F-BAR proteins spontaneously segregate from the N-BAR domain proteins during membrane tubule formation. In contrast, our observation supports a model in which different BAR-domain family members can associate with the same tubule and cooperate to fine-tune the tubule width, possibly by recruiting actin modulators during the generation of tubules. Our data suggests that cooperative activities of distinct BAR-domain family proteins may determine the length and width of the membrane tubule in vivo.

BIN1 regulates actin-membrane interactions during IRSp53-dependent filopodia formation

Amphiphysin 2 (BIN1) is a membrane and actin remodeling protein mutated in congenital and adult centronuclear myopathies. Here, we report an unexpected function of this N-BAR domain protein BIN1 in filopodia formation. We demonstrated that BIN1 expression is necessary and sufficient to induce filopodia formation. BIN1 is present at the base of forming filopodia and all along filopodia, where it colocalizes with F-actin. We identify that BIN1-mediated filopodia formation requires IRSp53, which allows its localization at negatively-curved membrane topologies. Our results show that BIN1 bundles actin in vitro. Finally, we identify that BIN1 regulates the membrane-to-cortex architecture and functions as a molecular platform to recruit actin-binding proteins, dynamin and ezrin, to promote filopodia formation.

Which Coverages of Arc-Shaped Proteins Are Required for Membrane Tubulation?

Arc-shaped BIN/Amphiphysin/Rvs (BAR) domain proteins generate curvature by binding to membranes and induce membrane tubulation at sufficiently large protein coverages. For the amphiphysin N-BAR domain, Le Roux et al., Nat. Commun. 2021, 12, 6550, measured a threshold coverage of 0.44 ± 0.097 for nanotubules emerging from the supported lipid bilayer. In this article, we systematically investigate membrane tubulation induced by arc-shaped protein-like particles with coarse-grained modeling and simulations and determine the threshold coverages at different particle-particle interaction strengths and membrane spontaneous curvatures. In our simulations, the binding of arc-shaped particles induces a membrane shape transition from spherical vesicles to tubules at a particle threshold coverage of about 0.5, which is rather robust to variations of the direct attractive particle interactions or spontaneous membrane curvature in the coarse-grained model. Our study suggests that threshold coverages of around or slightly below 0.5 are a general requirement for membrane tubulation by arc-shaped BAR domain proteins.

Investigating the mechanical properties and flexibility of N-BAR domains in PICK1 by molecular dynamics simulations.

Bin/Amphiphysin/Rvs167 (BAR) domain superfamily proteins are considered to be able to induce membrane curvature. PICK1 is a unique protein consisting of both a PDZ and a BAR domain that has been associated with many diseases. PICK1 can force membrane curvature during receptor-mediated endocytosis. In addition to understanding how the N-BAR domain can force membrane curvature, it is also of particular interest to understand the hidden connections between structural and mechanical properties of the PICK1 BAR dimers. In this paper, steered molecular dynamics is used to investigate the mechanical properties that are associated with structural changes of the PICK1 BAR domains. Our results suggest that helix kinks may not only help generate curvature of BAR domains but may also provide the extra flexibility necessary to initiate the binding between BAR domains and the membrane. Interestingly, we observe a complicated interaction network both within the BAR monomer and at the binding interface of the two BAR monomers that is key to maintain the mechanical properties of the BAR dimer. Because of such an interaction network, the PICK1 BAR dimer demonstrated different responses to external forces in opposite directions.

The phospho-regulated amphiphysin/endophilin interaction is required for synaptic vesicle endocytosis.

The multidomain adaptor protein amphiphysin-1 (Amph1) is an important coordinator of clathrin-mediated endocytosis in non-neuronal cells and synaptic vesicle (SV) endocytosis at central nerve terminals. Amph1 contains a lipid-binding N-BAR (Bin/Amphiphysin/Rvs) domain, central proline-rich (PRD) and clathrin/AP2 (CLAP) domains, and a C-terminal SH3 domain. Amph1 interacts with both lipids and proteins, with all of these interactions required for SV endocytosis, with the exception of the Amph1 PRD. The Amph1 PRD associates with the endocytosis protein endophilin A1, however, the role of this interaction in SV endocytosis has not been investigated. In this study, we set out to determine whether the Amph1 PRD and its interaction with endophilin A1 was essential for efficient SV endocytosis at typical small central synapses. To achieve this, domain-specific interactions of Amph1 were validated using in vitro GST pull-down assays, with the role of these interactions in SV endocytosis determined in molecular replacement experiments in primary neuronal culture. Using this approach, we confirmed important roles for CLAP and SH3 domain interactions of Amph1 in the control of SV endocytosis. Importantly, we identified the interaction site for endophilin A1 within the Amph1 PRD and exploited specific binding mutants to reveal a key role for this interaction in SV endocytosis. Finally, we determined that the formation of the Amph1-endophilin A1 complex is dependent on the phosphorylation status of Amph1-S293 within the PRD and that the phosphorylation status of this residue is essential for efficient SV regeneration. This work, therefore, reveals a key role for the dephosphorylation-dependent Amph1-endophilin A1 interaction in efficient SV endocytosis.

N-BAR and F-BAR proteins-endophilin-A3 and PSTPIP1-control clathrin-independent endocytosis of L1CAM.

Recent advances in the field demonstrate the high diversity and complexity of endocytic pathways. In the current study, we focus on the endocytosis of L1CAM. This glycoprotein plays a major role in the development of the nervous system, and is involved in cancer development and is associated with metastases and poor prognosis. Two L1CAM isoforms are subject to endocytosis: isoform 1, described as a clathrin-mediated cargo; isoform 2, whose endocytosis has never been studied. Deciphering the molecular machinery of isoform 2 internalisation should contribute to a better understanding of its pathophysiological role. First, we demonstrated in our cellular context that both isoforms of L1CAM are mainly a clathrin-independent cargo, which was not expected for isoform 1. Second, the mechanism of L1CAM endocytosis is specifically mediated by the N-BAR domain protein endophilin-A3. Third, we discovered PSTPIP1, an F-BAR domain protein, as a novel actor in this endocytic process. Finally, we identified galectins as endocytic partners and negative regulators of L1CAM endocytosis. In summary, the interplay of the BAR proteins endophilin-A3 and PSTPIP1, and galectins fine tune the clathrin-independent endocytosis of L1CAM.

Purification of Recombinant Human Amphiphysin 1 and its N-BAR Domain.

Bin/Amphiphysin/Rvs (BAR) proteins are known as classical membrane curvature generators during endocytosis. Amphiphysin, a member of the N-BAR sub-family of proteins that contain a characteristic amphipathic sequence at the N-terminus of the BAR domain, is involved in clathrin-mediated endocytosis. Full-length amphiphysin contains a ~ 400 amino acid long disordered linker connecting the N-BAR domain and a C-terminal Src homology 3 (SH3) domain. We express and purify recombinant amphiphysin and its N-BAR domain along with an N-terminal glutathione-S-transferase (GST) tag. The GST tag allows extraction of the protein of interest using affinity chromatography and is removed in the subsequent protease treatment and ion-exchange chromatography steps. In the case of the N-BAR domain, cleavage of the GST tag was found to cause precipitation. This issue can be minimized by adding glycerol to the protein purification buffers. In the final step, size exclusion chromatography removes any potential oligomeric species. This protocol has also been successfully used to purify other N-BAR proteins, such as endophilin, Bin1, and their corresponding BAR domains. Graphical overview.

Estimation of anisotropic bending rigidities and spontaneous curvatures of crescent curvature-inducing proteins from tethered-vesicle experimental data.

The Bin/amphiphysin/Rvs (BAR) superfamily proteins have a crescent binding domain and bend biomembranes along the domain axis. However, their anisotropic bending rigidities and spontaneous curvatures have not been experimentally determined. Here, we estimated these values from the bound protein densities on tethered vesicles using a mean-field theory of anisotropic bending energy and orientation-dependent excluded volume. The dependence curves of the protein density on the membrane curvature are fitted to the experimental data for the I-BAR and N-BAR domains reported by C. Prévost et al. Nat. Commun., 2015, 6, 8529 and F.-C. Tsai et al. Soft Matter, 2021, 17, 4254-4265, respectively. For the I-BAR domain, all three density curves of different chemical potentials exhibit excellent fits with a single parameter set of anisotropic bending energy. When the classical isotropic bending energy is used instead, one of the curves can be fitted well, but the others exhibit large deviations. In contrast, for the N-BAR domain, two curves are not well fitted simultaneously the anisotropic model, although it is significantly improved compared to the isotropic model. This deviation likely suggests a cluster formation of the N-BAR domains.

A lysine-rich cluster in the N-BAR domain of ARF GTPase-activating protein ASAP1 is necessary for binding and bundling actin filaments

Actin filament maintenance is critical for both normal cell homeostasis and events associated with malignant transformation. The ADP-ribosylation factor GTPase-activating protein ASAP1 regulates the dynamics of filamentous actin-based structures, including stress fibers, focal adhesions, and circular dorsal ruffles. Here, we have examined the molecular basis for ASAP1 association with actin. Using a combination of structural modeling, mutagenesis, and in vitro and cell-based assays, we identify a putative-binding interface between the N-Bin-Amphiphysin-Rvs (BAR) domain of ASAP1 and actin filaments. We found that neutralization of charges and charge reversal at positions 75, 76, and 79 of ASAP1 reduced the binding of ASAP1 BAR-pleckstrin homology tandem to actin filaments and abrogated actin bundle formation in vitro. In addition, overexpression of actin-binding defective ASAP1 BAR-pleckstrin homology [K75, K76, K79] mutants prevented cellular actin remodeling in U2OS cells. Exogenous expression of [K75E, K76E, K79E] mutant of full-length ASAP1 did not rescue the reduction of cellular actin fibers consequent to knockdown of endogenous ASAP1. Taken together, our results support the hypothesis that the lysine-rich cluster in the N-BAR domain of ASAP1 is important for regulating actin filament organization.

In vitro Fluorescence Imaging-based Actin Bundling Assay.

Understanding the molecular and structural mechanisms that govern the assembly and organization of higher-order actin architecture requires the use of in vitro actin binding and bundling assays. Crosslinking of actin filaments into bundles can be monitored in vitro via several techniques, including negative staining/electron microscopy, low-speed co-sedimentation assay/SDS-PAGE, and fluorescence staining/confocal microscopy. We and others have previously characterized the N-BAR domain of ASAP1, an ADP-ribosylation factor GTPase-activating protein, as an actin-bundling module; we further identified key lysine residues responsible for actin cross-linking. Here, we use the ASAP1 BAR domain as an example and describe a detailed procedure for observing the actin bundle formation by confocal microscopy. This protocol requires small reaction volumes and takes advantage of bright commercially available fluorescent phalloidins, making it an ideal choice for medium-throughput screening of mutants or domain truncations in their ability to bundle actin. Graphical abstract.

Unfolding Mechanisms and Conformational Stability of the Dimeric Endophilin N-BAR Domain

Endophilin, which is a member of the Bin-amphiphysin-Rvs (BAR) domain protein superfamily, contains a homodimeric N-BAR domain of a characteristic crescent shape. The N-BAR domain comprises a six-helix bundle and is known to sense and generate membrane curvature. Here, we characterize aspects of the unfolding mechanism of the endophilin A1 N-BAR domain during thermal denaturation and examine factors that influence the thermal stability of this domain. Far-UV circular dichroism (CD) spectroscopy was applied to monitor changes in the secondary structure above room temperature. The protein’s conformational changes were further characterized through Foerster resonance energy transfer and cross-linking experiments at varying temperatures. Our results indicate that thermal unfolding of the endophilin N-BAR is (minimally) a two-step process, with a dimeric intermediate that displays partial helicity loss. Furthermore, a thermal shift assay and temperature-dependent CD were applied to compare the unfolding processes of several truncated versions of endophilin. The melting temperature of the N-BAR domain decreased when we deleted either the N-terminal H0 helix or the unstructured linker of endophilin. This result suggests that these intrinsically disordered domains may play a role in structurally stabilizing the functional N-BAR domain in vivo. Finally, we show that single-site mutations can also compromise endophilin’s thermal stability.

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 BAR domain of the Arf GTPase-activating protein ASAP1 directly binds actin filaments

The Arf GTPase-activating protein (Arf GAP) with SH3 domain, ankyrin repeat and PH domain 1 (ASAP1) establishes a connection between the cell membrane and the cortical actin cytoskeleton. The formation, maintenance, and turnover of actin filaments and bundles in the actin cortex are important for cell adhesion, invasion, and migration. Here, using actin cosedimentation, polymerization, and depolymerization assays, along with total internal reflection fluorescence (TIRF), confocal, and EM analyses, we show that the N-terminal N-BAR domain of ASAP1 directly binds to F-actin. We found that ASAP1 homodimerization aligns F-actin in predominantly unipolar bundles and stabilizes them against depolymerization. Furthermore, the ASAP1 N-BAR domain moderately reduced the spontaneous polymerization of G-actin. The overexpression of the ASAP1 BAR-PH tandem domain in fibroblasts induced the formation of actin-filled projections more effectively than did full-length ASAP1. An ASAP1 construct that lacked the N-BAR domain failed to induce cellular projections. Our results suggest that ASAP1 regulates the dynamics and the formation of higher-order actin structures, possibly through direct binding to F-actin via its N-BAR domain. We propose that ASAP1 is a hub protein for dynamic protein-protein interactions in mechanosensitive structures, such as focal adhesions, invadopodia, and podosomes, that are directly implicated in oncogenic events. The effect of ASAP1 on actin dynamics puts a spotlight on its function as a central signaling molecule that regulates the dynamics of the actin cytoskeleton by transmitting signals from the plasma membrane.

BIN1 protein isoforms are differentially expressed in astrocytes, neurons, and microglia: neuronal and astrocyte BIN1 are implicated in tau pathology

BackgroundIdentified as an Alzheimer’s disease (AD) susceptibility gene by genome wide-association studies, BIN1 has 10 isoforms that are expressed in the Central Nervous System (CNS). The distribution of these isoforms in different cell types, as well as their role in AD pathology still remains unclear.MethodsUtilizing antibodies targeting specific BIN1 epitopes in human post-mortem tissue and analyzing mRNA expression data from purified microglia, we identified three isoforms expressed in neurons and astrocytes (isoforms 1, 2 and 3) and four isoforms expressed in microglia (isoforms 6, 9, 10 and 12). The abundance of selected peptides, which correspond to groups of BIN1 protein isoforms, was measured in dorsolateral prefrontal cortex, and their relation to neuropathological features of AD was assessed.ResultsPeptides contained in exon 7 of BIN1’s N-BAR domain were found to be significantly associated with AD-related traits and, particularly, tau tangles. Decreased expression of BIN1 isoforms containing exon 7 is associated with greater accumulation of tangles and subsequent cognitive decline, with astrocytic rather than neuronal BIN1 being the more likely culprit. These effects are independent of the BIN1 AD risk variant.ConclusionsExploring the molecular mechanisms of specific BIN1 isoforms expressed by astrocytes may open new avenues for modulating the accumulation of Tau pathology in AD.

Lipid-Dependent Interaction of Human N-BAR Domain Proteins with Sarcolemma Mono- and Bilayers.

The N-BAR domain of the human Bin1 protein is indispensable for T-tubule biogenesis in skeletal muscles. It binds to lipid mono- and bilayers that mimic the sarcolemma membrane composition, and it transforms vesicles into uniform tubules by generating a decorating protein scaffold. We found that Δ(1-33)BAR, lacking the N-terminal amphipathic helix (H0), and H0 alone bind to sarcolemma monolayers, although both proteins are not able to tubulate sarcolemma vesicles. By variation of the lipid composition, we elucidated the role of PI(4,5)P2, cholesterol, and an asymmetric sarcolemma composition for Bin1-N-BAR binding and sarcolemma tubulation. Our results indicate that Bin1-N-BAR binding is low in the absence of PI(4,5)P2 and it is affected by additional changes in the negative headgroup charge and lipid acyl chain composition. However, it is not dependent on the cholesterol content. The results from Langmuir monolayer experiments are complementary to lipid bilayer studies using electron microscopy that provides information on membrane curvature generation.

Quantitative Nano-Platforms for Interrogation of Curvature Sensitive Proteins

Membrane curvature is a defining parameter which mediates many critical cellular processes like endocytosis and exocytosis. This parameter is mainly mediated by several types of curvature-sensing domains, including N-BAR, F-BAR, I-BAR, and ENTH domain. Previous studies of these curvature-sensing domains lacking precise quantification of curvature sensitivity while maintaining the ease of use the assay. Herein, we design a concise platform to quantitatively determine the curvature-sensing range for several curvature-sensing proteins, which implies the role of these proteins in several membrane-associated processes. The core of our technology consists of simple nanofabrication and microfluidics, integrated with quick lipid bilayer engineering and protein lysate preparation. This design provides us with the flexibility to switch lipid compositions, manipulate lysate components, and engineer complex curvatures, which serves as a powerful tool to investigate curvature-dependent cellular processes.

The ArfGAP ASAP1 Controls Actin Stress Fiber Organization via Its N-BAR Domain.

SummaryASAP1 is a multi-domain ArfGAP that controls cell migration, spreading, and focal adhesion dynamics. Although its GAP activity contributes to remodeling of the actin cytoskeleton, it does not fully explain all cellular functions of ASAP1. Here we find that ASAP1 regulates actin filament assembly directly through its N-BAR domain and controls stress fiber maintenance. ASAP1 depletion caused defects in stress fiber organization. Conversely, overexpression of ASAP1 enhanced actin remodeling. The BAR-PH fragment was sufficient to affect actin. ASAP1 with the BAR domain replaced with the BAR domain of the related ACAP1 did not affect actin. The BAR-PH tandem of ASAP1 bound and bundled actin filaments directly, whereas the presence of the ArfGAP and the C-terminal linker/SH3 domain reduced binding and bundling of filaments by BAR-PH. Together these data provide evidence that ASAP1 may regulate the actin cytoskeleton through direct interaction of the BAR-PH domain with actin filaments.

A putative N-BAR-domain protein is crucially required for the development of hyphae tip appressorium-like structure and its plant infection in Magnaporthe oryzae

Membrane remodeling modulates many biological processes. The binding of peripheral proteins to lipid membranes results in membrane invaginations and protrusions, which regulate essential intra-cellular membrane and extra-cellular trafficking events. Proteins that bind and re-shape bio-membranes have been identified and extensively investigated. The Bin/Amphiphysin/Rvs (BAR) domain proteins are crescent-shape and play a conserved role in tubulation and sculpturing of cell membranes. We deployed targeted gene replacement technique to functionally characterize two hypothetical proteins (MoBar-A and MoBar-B) containing unitary N-BAR domain in Magnaporthe oryzae. The results obtained from phenotypic examinations showed that MoBAR-A deletion exerted a significant reduction in the growth of the defective ∆Mobar-A strain. Also, MoBAR-A disruption exclusively compromised hyphae-mediated infection. Additionally, the targeted replacement of MoBAR-A suppressed the expression of genes associated with the formation of hyphae tip appressorium-like structure in M. oryzae. Furthermore, single as well as combined deletion of MoBAR-A and MoBAR-B down-regulated the expression of nine different membrane-associated genes. From these results, we inferred that MoBAR-A plays a key and unique role in the pathogenesis of M. oryzae through direct or indirect regulation of the development of appressorium-like structures developed by hyphae tip. Taken together, these results provide unique insights into the direct contribution of the N-BAR domain proteins to morphological, reproduction, and infectious development of M. oryzae.

Cellular Blebs and Membrane Invaginations Are Coupled through Membrane Tension Buffering

Bleb-type cellular protrusions play key roles in a range of biological processes. It was recently found that bleb growth is facilitated by a local supply of membrane from tubular invaginations, but the interplay between the expanding bleb and the membrane tubes remains poorly understood. On the one hand, the membrane area stored in tubes may serve as a reservoir for bleb expansion. On the other hand, the sequestering of excess membrane in stabilized invaginations may effectively increase the cell membrane tension, which suppresses spontaneous protrusions. Here, we investigate this duality through physical modeling and in vivo experiments. In agreement with observations, our model describes the transition into a tube-flattening mode of bleb expansion while also predicting that the blebbing rate is impaired by elevating the concentration of the curved membrane proteins that form the tubes. We show both theoretically and experimentally that the stabilizing effect of tubes could be counterbalanced by the cortical myosin contractility. Our results largely suggest that proteins able to induce membrane tubulation, such as those containing N-BAR domains, can buffer the effective membrane tension—a master regulator of all cell deformations.