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.     

I-BAR domains,IRSp53 and filopodium formation

Filopodia and lamellipodia are dynamic actin-based structures that determine cell shape and migration. Filopodia are thought to sense the environment and direct processes such as axon guidance and neurite outgrowth. Cdc42 is a small GTP-binding protein and member of the RhoGTPase family. Cdc42 and its effector IRSp53 (insulin receptor phosphotyrosine 53 kDa substrate) have been shown to be strong inducers of filopodium formation. IRSp53 consists of an I-BAR (inverse-Bin-Amphiphysin-Rvs) domain, a Cdc42-binding domain and an SH3 domain. The I-BAR domain of IRSp53 induces membrane tubulation of vesicles and dynamic membrane protrusions lacking actin in cells. The IRSp53 SH3 domain interacts with proteins that regulate actin filament formation e.g. Mena, N-WASP, mDia1 and Eps8. In this review we suggest that the mechanism for Cdc42-driven filopodium formation involves coupling I-BAR domain-induced membrane protrusion with SH3 domain-mediated actin dynamics through IRSp53.     

BAR domain competition during directional cellular migration

While directed cellular migration facilitates the coordinated movement of cells during development and tissue repair, the precise mechanisms regulating the interplay between the extracellular environment, the actin cytoskeleton and the overlying plasma membrane remain inadequately understood. The BAR domain family of lipid binding, actin cytoskeletal regulators are gaining greater appreciation for their role in these critical processes. BAR domain proteins are involved as both positive and negative regulators of endocytosis, membrane plasticity and directional cell migration. This review focuses on the functional relationship between different classes of BAR domain proteins and their role in guiding cell migration through regulation of the endocytic machinery. Competition for key signaling substrates by positive and negative BAR domain endocytic regulators appears to mediate control of directional cell migration, and may have wider applicability to other trafficking functions associated with development and carcinogenesis.Key words: BAR domain, MIM, directed cell migration, competition, membrane dynamics, actin cytoskeleton, endocytosis     

BAR domain competition during directional cellular migration

While directed cellular migration facilitates the coordinated movement of cells during development and tissue repair, the precise mechanisms regulating the interplay between the extracellular environment, the actin cytoskeleton, and the overlying plasma membrane remain inadequately understood. The BAR domain family of lipid binding, actin cytoskeletal regulators are gaining greater appreciation for their role in these critical processes. BAR domain proteins are involved as both positive and negative regulators of endocytosis, membrane plasticity, and directional cell migration. This review focuses on the functional relationship between different classes of BAR domain proteins and their role in guiding cell migration through regulation of the endocytic machinery. Competition for key signaling substrates by positive and negative BAR domain endocytic regulators appears to mediate control of directional cell migration, and may have wider applicability to other trafficking functions associated with development and carcinogenesis.     

Subcellular membrane curvature mediated by the BAR domain superfamily proteins

The Bin-Amphiphysin-Rvs167 (BAR) domain superfamily consists of proteins containing the BAR domain, the extended FCH (EFC)/FCH-BAR (F-BAR) domain, or the IRSp53-MIM homology domain (IMD)/inverse BAR (I-BAR) domain. These domains bind membranes through electrostatic interactions between the negative charges of the membranes and the positive charges on the structural surface of homo-dimeric BAR domain superfamily members. Some BAR superfamily members have membrane-penetrating insertion loops, which also contribute to the membrane binding by the proteins. The membrane-binding surface of each BAR domain superfamily member has its own unique curvature that governs or senses the curvature of the membrane for BAR-domain binding. The wide range of BAR-domain surface curvatures correlates with the various invaginations and protrusions of cells. Therefore, each BAR domain superfamily member may generate and recognize the curvature of the membrane of each subcellular structure, such as clathrin-coated pits or filopodia. The BAR domain superfamily proteins may regulate their own catalytic activity or that of their binding proteins, depending on the membrane curvature of their corresponding subcellular structures.     

Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins

Cell membranes undergo continuous curvature changes as a result of membrane trafficking and cell motility. Deformations are achieved both by forces extrinsic to the membrane as well as by structural modifications in the bilayer or at the bilayer surface that favor the acquisition of curvature. We report here that a family of proteins previously implicated in the regulation of the actin cytoskeleton also have powerful lipid bilayer-deforming properties via an N-terminal module (F-BAR) similar to the BAR domain. Several such proteins, like a subset of BAR domain proteins, bind to dynamin, a GTPase implicated in endocytosis and actin dynamics, via SH3 domains. The ability of BAR and F-BAR domain proteins to induce tubular invaginations of the plasma membrane is enhanced by disruption of the actin cytoskeleton and is antagonized by dynamin. These results suggest a close interplay between the mechanisms that control actin dynamics and those that mediate plasma membrane invagination and fission.     

The Unique Mechanism of SNX9 BAR Domain for Inducing Membrane Tubulation

Sorting nexin 9 (SNX9) is a member of the sorting nexin family of proteins and plays a critical role in clathrin-mediated endocytosis. It has a Bin-Amphiphysin-Rvs (BAR) domain which can form a crescent-shaped homodimer structure that induces deformation of the plasma membrane. While other BAR-domain containing proteins such as amphiphysin and endophilin have an amphiphatic helix in front of the BAR domain which plays a critical role in membrane penetration, SNX9 does not. Thus, whether and how SNX9 BAR domain could induce the deformation of the plasma membrane is not clear. The present study identified the internal putative amphiphatic stretch in the 1^st α-helix of the SNX9 BAR domain and proved that together with the N-terminal helix (H₀) region, this internal putative amphiphatic stretch is critical for inducing membrane tubulation. Therefore, our study shows that SNX9 uses a unique mechanism to induce the tubulation of the plasma membrane which mediates proper membrane deformation during clathrin-mediated endocytosis.     

Dimerization is necessary for MIM-mediated membrane deformation and endocytosis

MIM [missing in metastasis; also called MTSS1 (metastasis suppressor 1)] is an intracellular protein that binds to actin and cortactin and has an intrinsic capacity to sense and facilitate the formation of protruded membranous curvatures implicated in cell-ular polarization, mobilization and endocytosis. The N-terminal 250 amino acids of MIM undergo homodimerization and form a structural module with the characteristic of an I-BAR [inverse BAR (Bin/amphiphysin/Rvs)] domain. To discern the role of the dimeric configuration in the function of MIM, we designed several peptides able to interfere with MIM dimerization in a manner dependent upon their lengths. Overexpression of one of the peptides effectively abolished MIM-mediated membrane protrusions and transferrin uptake. However, a peptide with a high potency inhibiting MIM dimerization failed to affect its binding to actin and cortactin. Thus the results of the present study indicate that the dimeric configuration is essential for MIM-mediated membrane remodelling and serves as a proper target to develop antagonists specifically against an I-BAR-domain-containing protein.     

Biochemical and functional significance of F-BAR domain proteins interaction with WASP/N-WASP

The Bin-Amphiphysin-Rvs (BAR) domain family of proteins includes groups which promote positive (classical BAR, N-BAR, and F-BAR) and negative (I-BAR) membrane deformation. Of these groups, the F-BAR subfamily is the most diverse in its biochemical properties. F-BAR domain proteins dimerize to form a tight scaffold about the membrane. The F-BAR domain provides a banana-shaped, alpha-helical structure that senses membrane curvature. Different types of F-BAR domain proteins contain tyrosine kinase or GTPase activities; some interact with phosphatases and RhoGTPases. Most possess an SH3 domain that facilitates the recruitment and activation of WASP/N-WASP. Thus, F-BAR domain proteins affect remodeling of both membrane and the actin cytoskeleton. The purpose of this review is to highlight the role of F-BAR proteins in coupling WASP/N-WASP to cytoskeletal remodeling. A role for F-BAR/WASP interaction in human diseases affecting nervous, blood, and neoplastic tissues is discussed.     

Coordination of Membrane and Actin Cytoskeleton Dynamics during Filopodia Protrusion

Leading edge protrusion of migrating cells involves tightly coordinated changes in the plasma membrane and actin cytoskeleton. It remains unclear whether polymerizing actin filaments push and deform the membrane, or membrane deformation occurs independently and is subsequently stabilized by actin filaments. To address this question, we employed an ability of the membrane-binding I-BAR domain of IRSp53 to uncouple the membrane and actin dynamics and to induce filopodia in expressing cells. Using time-lapse imaging and electron microscopy of IRSp53-I-BAR-expressing B16F1 melanoma cells, we demonstrate that cells are not able to protrude or maintain durable long extensions without actin filaments in their interior, but I-BAR-dependent membrane deformation can create a small and transient space at filopodial tips that is subsequently filled with actin filaments. Moreover, the expressed I-BAR domain forms a submembranous coat that may structurally support these transient actin-free protrusions until they are further stabilized by the actin cytoskeleton. Actin filaments in the I-BAR-induced filopodia, in contrast to normal filopodia, do not have a uniform length, are less abundant, poorly bundled, and display erratic dynamics. Such unconventional structural organization and dynamics of actin in I-BAR-induced filopodia suggests that a typical bundle of parallel actin filaments is not necessary for generation and mechanical support of the highly asymmetric filopodial geometry. Together, our data suggest that actin filaments may not directly drive the protrusion, but only stabilize the space generated by the membrane deformation; yet, such stabilization is necessary for efficient protrusion.     

Regulation of Membrane-Shape Transitions Induced by I-BAR Domains

I-BAR proteins are well-known actin-cytoskeleton adaptors and have been observed to be involved in the formation of plasma membrane protrusions (filopodia). I-BAR proteins contain an all-helical, crescent-shaped IRSp53-MIM domain (IMD) dimer that is believed to be able to couple with a membrane shape. This coupling could involve the sensing and even the generation of negative plasma membrane curvature. Indeed, the in vitro studies have shown that IMDs can induce inward tubulation of liposomes. While N-BAR domains, which generate positive membrane curvature, have received a considerable amount of attention from both theory and experiments, the mechanisms of curvature coupling through IMDs are comparatively less studied and understood. Here we used a membrane-shape stability assay developed recently in our lab to quantitatively characterize IMD-induced membrane-shape transitions. We determined a membrane-shape stability diagram for IMDs that reveals how membrane tension and protein density can comodulate the generation of IMD-induced membrane protrusions. From comparison to analytical theory, we determine three key parameters that characterize the curvature coupling of IMD. We find that the curvature generation capacity of IMDs is significantly stronger compared to that of endophilin, an N-BAR protein known to be involved in plasma membrane shape transitions. Contrary to N-BAR domains, where amphipathic helix insertion is known to promote its membrane curvature generation, for IMDs we find that amphipathic helices inhibit membrane shape transitions, consistent with the inverse curvature that IMDs generate. Importantly, in both of these types of BAR domains, electrostatic interactions affect membrane-binding capacity, but do not appear to affect the curvature generation capacity of the protein. These two types of BAR domain proteins show qualitatively similar membrane shape stability diagrams, suggesting an underlying ubiquitous mechanism by which peripheral proteins regulate membrane curvature.     

Coordination between the actin cytoskeleton and membrane deformation by a novel membrane tubulation domain of PCH proteins is involved in endocytosis

The conserved FER-CIP4 homology (FCH) domain is found in the pombe Cdc15 homology (PCH) protein family members, including formin-binding protein 17 (FBP17). However, the amino acid sequence homology extends beyond the FCH domain. We have termed this region the extended FC (EFC) domain. We found that FBP17 coordinated membrane deformation with actin cytoskeleton reorganization during endocytosis. The EFC domains of FBP17, CIP4, and other PCH protein family members show weak homology to the Bin-amphiphysin-Rvs (BAR) domain. The EFC domains bound strongly to phosphatidylserine and phosphatidylinositol 4,5-bisphosphate and deformed the plasma membrane and liposomes into narrow tubules. Most PCH proteins possess an SH3 domain that is known to bind to dynamin and that recruited and activated neural Wiskott-Aldrich syndrome protein (N-WASP) at the plasma membrane. FBP17 and/or CIP4 contributed to the formation of the protein complex, including N-WASP and dynamin-2, in the early stage of endocytosis. Furthermore, knockdown of endogenous FBP17 and CIP4 impaired endocytosis. Our data indicate that PCH protein family members couple membrane deformation to actin cytoskeleton reorganization in various cellular processes.     

Missing-in-metastasis and IRSp53 deform PI(4,5)P2-rich membranes by an inverse BAR domain-like mechanism

The actin cytoskeleton plays a fundamental role in various motile and morphogenetic processes involving membrane dynamics. We show that actin-binding proteins MIM (missing-in-metastasis) and IRSp53 directly bind PI(4,5)P(2)-rich membranes and deform them into tubular structures. This activity resides in the N-terminal IRSp53/MIM domain (IMD) of these proteins, which is structurally related to membrane-tubulating BAR (Bin/amphiphysin/Rvs) domains. We found that because of a difference in the geometry of the PI(4,5)P(2)-binding site, IMDs induce a membrane curvature opposite that of BAR domains and deform membranes by binding to the interior of the tubule. This explains why IMD proteins induce plasma membrane protrusions rather than invaginations. We also provide evidence that the membrane-deforming activity of IMDs, instead of the previously proposed F-actin-bundling or GTPase-binding activities, is critical for the induction of the filopodia/microspikes in cultured mammalian cells. Together, these data reveal that interplay between actin dynamics and a novel membrane-deformation activity promotes cell motility and morphogenesis.     

Dissecting BAR Domain Function in the Yeast Amphiphysins Rvs161 and Rvs167 during Endocytosis

BAR domains are protein modules that bind to membranes and promote membrane curvature. One type of BAR domain, the N-BAR domain, contains an additional N-terminal amphipathic helix, which contributes to membrane-binding and bending activities. The only known N-BAR-domain proteins in the budding yeast Saccharomyces cerevisiae, Rvs161 and Rvs167, are required for endocytosis. We have explored the mechanism of N-BAR-domain function in the endocytosis process using a combined biochemical and genetic approach. We show that the purified Rvs161–Rvs167 complex binds to liposomes in a curvature-independent manner and promotes tubule formation in vitro. Consistent with the known role of BAR domain polymerization in membrane bending, we found that Rvs167 BAR domains interact with each other at cortical actin patches in vivo. To characterize N-BAR-domain function in endocytosis, we constructed yeast strains harboring changes in conserved residues in the Rvs161 and Rvs167 N-BAR domains. In vivo analysis of the rvs endocytosis mutants suggests that Rvs proteins are initially recruited to sites of endocytosis through their membrane-binding ability. We show that inappropriate regulation of complex sphingolipid and phosphoinositide levels in the membrane can impinge on Rvs function, highlighting the relationship between membrane components and N-BAR-domain proteins in vivo.     

Bending over backwards: BAR proteins and the actin cytoskeleton in mammalian receptor-mediated endocytosis

The role of the actin cytoskeleton during receptor-mediated endocytosis (RME) has been well characterized in yeast for many years. Only more recently has the interplay between the actin cytoskeleton and RME been extensively explored in mammalian cells. These studies have revealed the central roles of BAR proteins in RME, and have demonstrated significant roles of BAR proteins in linking the actin cytoskeleton to this cellular process. The actin cytoskeleton generates and transmits mechanical force to promote the extension of receptor-bound endocytic vesicles into the cell. Many adaptor proteins link and regulate the actin cytoskeleton at the sites of endocytosis. This review will cover key effectors, adaptors and signalling molecules that help to facilitate the invagination of the cell membrane during receptor-mediated endocytosis, including recent insights gained on the roles of BAR proteins. The final part of this review will explore associations of alterations to genes encoding BAR proteins with cancer.     

On the role of membrane anisotropy and BAR proteins in the stability of tubular membrane structures

Recent studies have demonstrated that actin filaments are not crucial for the short-term stability of tubular membrane protrusions originating from the cell surface. It has also been demonstrated that prominin nanodomains and curvature inducing I-BAR proteins could account for the stability of the membrane protrusion. Here we constructed an axisymmetric model of a membrane protrusion that excludes actin filaments in order to investigate the contributions of prominin nanodomains (rafts) and I-BAR proteins to the membrane protrusion stability. It was demonstrated that prominin nanodomains and I-BAR proteins can stabilize the membrane protrusion only over a specific range of spontaneous curvature. On the other hand, high spontaneous curvature and/or high density of I-BAR proteins could lead to system instability and to non-uniform contraction in the radial direction of the membrane protrusion. In agreement with previous studies, it was also shown that the isotropic bending energy of lipids is not sufficient to explain the stability of the observed tubular membrane protrusion without actin filaments.     

Direct interaction of actin filaments with F‐BAR protein pacsin2

Two mechanisms have emerged as major regulators of membrane shape: BAR domain‐containing proteins, which induce invaginations and protrusions, and nuclear promoting factors, which cause generation of branched actin filaments that exert mechanical forces on membranes. While a large body of information exists on interactions of BAR proteins with membranes and regulatory proteins of the cytoskeleton, little is known about connections between these two processes. Here, we show that the F‐BAR domain protein pacsin2 is able to associate with actin filaments using the same concave surface employed to bind to membranes, while some other tested N‐BAR and F‐BAR proteins (endophilin, CIP4 and FCHO2) do not associate with actin. This finding reveals a new level of complexity in membrane remodeling processes.     

Membrane protrusion powers clathrin-independent endocytosis of interleukin-2 receptor

Endocytosis controls many functions including nutrient uptake, cell division, migration and signal transduction. A clathrin- and caveolin-independent endocytosis pathway is used by important physiological cargos, including interleukin-2 receptors (IL-2R). However, this process lacks morphological and dynamic data. Our electron microscopy (EM) and tomography studies reveal that IL-2R-pits and vesicles are initiated at the base of protrusions. We identify the WAVE complex as a specific endocytic actor. The WAVE complex interacts with IL-2R, via a WAVE-interacting receptor sequence (WIRS) present in the receptor polypeptide, and allows for receptor clustering close to membrane protrusions. In addition, using total internal reflection fluorescent microscopy (TIRF) and automated analysis we demonstrate that two timely distinct bursts of actin polymerization are required during IL-2R uptake, promoted first by the WAVE complex and then by N-WASP. Finally, our data reveal that dynamin acts as a transition controller for the recruitment of Arp2/3 activators required for IL-2R endocytosis. Altogether, our work identifies the spatio-temporal specific role of factors initiating clathrin-independent endocytosis by a unique mechanism that does not depend on the deformation of a flat membrane, but rather on that of membrane protrusions.     

VE-cadherin-induced Cdc42 signaling regulates formation of membrane protrusions in endothelial cells

The cytoplasmic domain of cadherins and the associated catenins link the cytoskeleton with signal transduction pathways. To study the signaling function of non-junctional VE-cadherin, which can form during the loss VE-cadherin homotypic adhesion, wild type VE-cadherin or VE-cadherin cytoplasmic domain (DeltaEXD) was expressed in sub-confluent endothelial cells. We observed that Cdc42 was activated in transfected cells and that these cells also developed Cdc42-dependent >70-microm-long plasma membrane protrusions. The formation of these structures required actin polymerization, and they developed specifically in endothelial cells as compared with epithelial cells. Expression of the VE-cadherin cytoplasmic domain lacking the beta-catenin binding site also induced Cdc42 activation; thus, its activation cannot be ascribed to beta-catenin binding. However, these cells were not able to form the protrusions. These results suggest that the cytoplasmic domain of non-junctional VE-cadherin can serve as a scaffold involved in Cdc42 activation at the endothelial plasma membrane. beta-Catenin and the associated alpha-catenin may serve as support sites for actin polymerization, leading to formation of long plasma membrane protrusions. Thus, non-junctional VE-cadherin actively participates in inside-out signaling at the plasma membrane, leading to the development of endothelial membrane protrusions.     

BAR proteins in cancer and blood disorders

Remodeling of the membrane and cytoskeleton is involved in a wide range of normal and pathologic cellular function. These are complex, highly-coordinated biochemical and biophysical processes involving dozens of proteins. Serving as a scaffold for a variety of proteins and possessing a domain that interacts with plasma membranes, the BAR family of proteins contribute to a range of cellular functions characterized by membrane and cytoskeletal remodeling. There are several subgroups of BAR proteins: BAR, N-BAR, I-BAR, and F-BAR. They differ in their ability to induce angles of membrane curvature and in their recruitment of effector proteins. Evidence is accumulating that BAR proteins contribute to cancer cell invasion, T cell trafficking, phagocytosis, and platelet production. In this review, we discuss the physiological function of BAR proteins and discuss how they contribute to blood and cancer disorders.