A Highlights from MBoC Selection: Formation of membrane ridges and scallops by the F-BAR protein Nervous Wreck

Eukaryotic cells are defined by extensive intracellular compartmentalization, which requires dynamic membrane remodeling. FER/Cip4 homology-Bin/amphiphysin/Rvs (F-BAR) domain family proteins form crescent-shaped dimers, which can bend membranes into buds and tubules of defined geometry and lipid composition. However, these proteins exhibit an unexplained wide diversity of membrane-deforming activities in vitro and functions in vivo. We find that the F-BAR domain of the neuronal protein Nervous Wreck (Nwk) has a novel higher-order structure and membrane-deforming activity that distinguishes it from previously described F-BAR proteins. The Nwk F-BAR domain assembles into zigzags, creating ridges and periodic scallops on membranes in vitro. This activity depends on structural determinants at the tips of the F-BAR dimer and on electrostatic interactions of the membrane with the F-BAR concave surface. In cells, Nwk-induced scallops can be extended by cytoskeletal forces to produce protrusions at the plasma membrane. Our results define a new F-BAR membrane-deforming activity and illustrate a molecular mechanism by which positively curved F-BAR domains can produce a variety of membrane curvatures. These findings expand the repertoire of F-BAR domain mediated membrane deformation and suggest that unique modes of higher-order assembly can define how these proteins sculpt the membrane.     

EFC/F-BAR proteins and the N-WASP-WIP complex induce membrane curvature-dependent actin polymerization

Extended Fer-CIP4 homology (EFC)/FCH-BAR (F-BAR) domains generate and bind to tubular membrane structures of defined diameters that are involved in the formation and fission of endocytotic vesicles. Formin-binding protein 17 (FBP17) and Toca-1 contain EFC/F-BAR domains and bind to neural Wiskott-Aldrich syndrome protein (N-WASP), which links phosphatidylinositol (4,5)-bisphosphate (PIP(2)) and the Rho family GTPase Cdc42 to the Arp2/3 complex. The N-WASP-WASP-interacting protein (WIP) complex, a predominant form of N-WASP in cells, is known to be activated by Toca-1 and Cdc42. Here, we show that N-WASP-WIP complex-mediated actin polymerization is activated by phosphatidylserine-containing membranes depending on membrane curvature in the presence of Toca-1 or FBP17 and in the absence of Cdc42 and PIP(2). Cdc42 further promoted the activation of actin polymerization by N-WASP-WIP. Toca-1 or FBP17 recruited N-WASP-WIP to the membrane. Conserved acidic residues near the SH3 domain of Toca-1 and FBP17 positioned the N-WASP-WIP to be spatially close to the membrane for activation of actin polymerization. Therefore, curvature-dependent actin polymerization is stimulated by spatially appropriate interactions of EFC/F-BAR proteins and the N-WASP-WIP complex with the membrane.     

Ultrastructural freeze-fracture immunolabeling identifies plasma membrane-localized syndapin II as a crucial factor in shaping caveolae

Membrane topology control is thought to involve peripheral membrane proteins of the F-BAR domain family including syndapins. These proteins are predestined to shape membranes by partial insertion and by imposing their curved shape onto the lipid bilayer. Direct observation of such functions on cellular membranes, however, was precluded by the difficulty to combine high-resolution imaging with visualization of membrane topology. Here, we report the ultrastructural visualization of endogenous syndapin II at the plasma membrane of NIH 3T3 cells using a combination of freeze-fracturing, immunogold labeling and transmission electron microscopy. Surprisingly, syndapin II was detected at flat and curved membrane areas. Ultrastructural colocalization with caveolin 1 identified syndapin II-positive invaginations as caveolae. Consistent with the syndapin II F-BAR domain interacting with caveolin 1, F-BAR overexpression affected caveolin 1 localization. Syndapin II knockdown did not alter caveolin 1 expression or plasma membrane recruitment. Instead, syndapin II knockdown reduced the density of caveolae and strongly increased the number of caveolin 1 molecules at flat membrane areas. Comparative immunoelectron microscopy and tilt series revealed that syndapin II was asymmetrically localized at the neck of caveolae. Double-immunogold labeling showed that the caveolae-shaping molecule PTRF/cavin 1 behaved similarly and that syndapin II and PTRF/cavin 1 colocalized. Visualization of a transiently membrane-binding F-BAR protein in direct relation to membrane topology of mammalian cells thereby revealed that syndapin II binds to both flat and curved membranes in vivo and that it plays an important role in caveolar shaping, a role that it shares with PTRF/cavin 1.     

Drosophila F-BAR protein Syndapin contributes to coupling the plasma membrane and contractile ring in cytokinesis

Cytokinesis is a highly ordered cellular process driven by interactions between central spindle microtubules and the actomyosin contractile ring linked to the dynamic remodelling of the plasma membrane. The mechanisms responsible for reorganizing the plasma membrane at the cell equator and its coupling to the contractile ring in cytokinesis are poorly understood. We report here that Syndapin, a protein containing an F-BAR domain required for membrane curvature, contributes to the remodelling of the plasma membrane around the contractile ring for cytokinesis. Syndapin colocalizes with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) at the cleavage furrow, where it directly interacts with a contractile ring component, Anillin. Accordingly, Anillin is mislocalized during cytokinesis in Syndapin mutants. Elevated or diminished expression of Syndapin leads to cytokinesis defects with abnormal cortical dynamics. The minimal segment of Syndapin, which is able to localize to the cleavage furrow and induce cytokinesis defects, is the F-BAR domain and its immediate C-terminal sequences. Phosphorylation of this region prevents this functional interaction, resulting in reduced ability of Syndapin to bind to and deform membranes. Thus, the dephosphorylated form of Syndapin mediates both remodelling of the plasma membrane and its proper coupling to the cytokinetic machinery.     

A Hinge in the Distal End of the PACSIN 2 F-BAR Domain May Contribute to Membrane-Curvature Sensing

The protein kinase C and casein kinase 2 substrates in neurons (PACSINs) represent a subfamily of membrane-binding proteins characterized by an amino-terminal Bin-Amphiphysin-Rvs (F-BAR) domain. PACSINs link membrane trafficking with actin dynamics and regulate the localization of distinct cargo molecules. The F-BAR domain forms a dimer essential for lipid binding. We have obtained crystals of authentic murine PACSIN 2 that contain an ordered F-BAR domain, indicating that additional domains are flexibly connected to F-BAR. The structure shares similarity to other BAR domains and exhibits special features unique to PACSINs. These include the uneven distribution of charged residues on the concave molecular surface and a so-called wedge loop that is driven into the membrane upon binding of PACSIN. The murine PACSIN 2 F-BAR domain requires dimerization for sensing of curved membranes, and the present structure also provides a mechanism for higher-order oligomer formation. Importantly, comparison of murine with human and Drosophila PACSIN 2 F-BAR domains reveals stark differences in the orientation of distal helical segments leading to a wider crescent shape of murine PACSIN 2. We define hinge residues for these movements that may help PACSINs sense and concomitantly reinforce membrane curvature.     

Mapping of the basic amino-acid residues responsible for tubulation and cellular protrusion by the EFC/F-BAR domain of pacsin2/Syndapin II

The extended Fes-CIP4 homology (EFC)/FCH-BAR (F-BAR) domain tubulates membranes. Overexpression of the pacsin2 EFC/F-BAR domain resulted in tubular localization inside cells and deformed liposomes into tubules in vitro. We found that overexpression of the pacsin2 EFC/F-BAR domain induced cellular microspikes, with the pacsin2 EFC/F-BAR domain concentrated at the neck. The hydrophobic loops and the basic amino-acid residues on the concave surface of the pacsin2 EFC/F-BAR domain are essential for both the microspike formation and tubulation. Since the curvature of the neck of the microspike and that of the tubulation share similar geometry, the pacsin2 EFC/F-BAR domain is considered to facilitate both microspike formation and tubulation.

Structured summary

MINT-7710892: EFCS pacsin2 (uniprotkb:Q9UNF0) and EFCS pacsin2 (uniprotkb:Q9UNF0) bind (MI:0407) by X-ray crystallography (MI:0114)     

Phosphoinositides affect both the cellular distribution and activity of the F-BAR-containing RhoGAP Rgd1p in yeast

Cell polarity is a key element of development in most eukaryotes. The Rho GTPase-activating protein Rgd1p positively regulates the GTPase activity of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively, in the budding yeast Saccharomyces cerevisiae. Rgd1p contains an F-BAR domain at its N-terminal end in addition to its RhoGAP domain at its C-terminal end. We demonstrate here that phospholipids discriminate between the GTPase activities of Rho3p and Rho4p through Rgd1p and specifically stimulate the RhoGAP activity on Rho4p. The central region of the protein contiguous to the F-BAR domain is required for this stimulation. The F-BAR region binds to phosphoinositides in vitro and also plays a key role in the localization of Rgd1p to the bud tip and neck during the cell cycle. Studies of heat-sensitive mutants lacking phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-biphosphate suggested that Rgd1p initially binds to Golgi membranes via phosphatidylinositol 4-phosphate and is then transported to the plasma membrane, where it binds phosphatidylinositol 4,5-biphosphate. We demonstrate here the dual effects of phosphoinositides on a RhoGTPase-activating protein. Phosphoinositides both regulate the recruitment and trafficking of Rgd1p to membranes via the F-BAR domain and specifically stimulate GTPase-activating protein activity, consistent with functional interplay between lipids, RhoGAP, and its related GTPases in yeast growth.     

Structural basis of membrane invagination by F-BAR domains

BAR superfamily domains shape membranes through poorly understood mechanisms. We solved structures of F-BAR modules bound to flat and curved bilayers using electron (cryo)microscopy. We show that membrane tubules form when F-BARs polymerize into helical coats that are held together by lateral and tip-to-tip interactions. On gel-state membranes or after mutation of residues along the lateral interaction surface, F-BARs adsorb onto bilayers via surfaces other than their concave face. We conclude that membrane binding is separable from membrane bending, and that imposition of the module's concave surface forces fluid-phase bilayers to bend locally. Furthermore, exposure of the domain's lateral interaction surface through a change in orientation serves as the crucial trigger for assembly of the helical coat and propagation of bilayer bending. The geometric constraints and sequential assembly of the helical lattice explain how F-BAR and classical BAR domains segregate into distinct microdomains, and provide insight into the spatial regulation of membrane invagination.     

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.     

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.     

Setting the F-BAR: Functions and regulation of the F-BAR protein family

F-BAR domain proteins serve as transient linkers between the cell cortex and the cytoskeleton in multiple biological contexts. Recent studies have detailed roles for this protein family in endocytosis, cytokinesis, neurotransmission, motility, and cellular morphogenesis. Here, we review emerging functional information regarding the recently recognized F-BAR domain family and the regulatory mechanisms whereby F-BAR proteins are deployed in diverse processes.     

Versatile Membrane Deformation Potential of Activated Pacsin

Endocytosis is a fundamental process in signaling and membrane trafficking. The formation of vesicles at the plasma membrane is mediated by the G protein dynamin that catalyzes the final fission step, the actin cytoskeleton, and proteins that sense or induce membrane curvature. One such protein, the F-BAR domain-containing protein pacsin, contributes to this process and has been shown to induce a spectrum of membrane morphologies, including tubules and tube constrictions in vitro. Full-length pacsin isoform 1 (pacsin-1) has reduced activity compared to its isolated F-BAR domain, implicating an inhibitory role for its C-terminal Src homology 3 (SH3) domain. Here we show that the autoinhibitory, intramolecular interactions in pacsin-1 can be released upon binding to the entire proline-rich domain (PRD) of dynamin-1, resulting in potent membrane deformation activity that is distinct from the isolated F-BAR domain. Most strikingly, we observe the generation of small, homogenous vesicles with the activated protein complex under certain experimental conditions. In addition, liposomes prepared with different methods yield distinct membrane deformation morphologies of BAR domain proteins and apparent activation barriers to pacsin-1''s activity. Theoretical free energy calculations suggest bimodality of the protein-membrane system as a possible source for the different outcomes, which could account for the coexistence of energetically equivalent membrane structures induced by BAR domain-containing proteins in vitro. Taken together, our results suggest a versatile role for pacsin-1 in sculpting cellular membranes that is likely dependent both on protein structure and membrane properties.     

The SH3 domains of two PCH family members cooperate in assembly of the Schizosaccharomyces pombe contractile ring

Schizosaccharomyces pombe cdc15 homology (PCH) family members participate in many cellular processes by bridging the plasma membrane and cytoskeleton. Their F-BAR domains bind and curve membranes, whereas other domains, typically SH3 domains, are expected to provide cytoskeletal links. We tested this prevailing model of functional division in the founding member of the family, Cdc15, which is essential for cytokinesis in S. pombe, and in the related PCH protein, Imp2. We find that the distinct functions of Imp2 and Cdc15 are SH3 domain independent. However, the Cdc15 and Imp2 SH3 domains share an essential role in recruiting proteins to the contractile ring, including Pxl1 and Fic1. Together, Pxl1 and Fic1, a previously uncharacterized C2 domain protein, add structural integrity to the contractile ring and prevent it from fragmenting during division. Our data indicate that the F-BAR proteins Cdc15 and Imp2 contribute to a single biological process with both distinct and overlapping functions.     

Bar domain proteins: a role in tubulation, scission and actin assembly in clathrin-mediated endocytosis

Endocytosis is an important way for cells to take up liquids and particles from their environment. It requires membrane bending to be coupled with membrane fission, and the actin cytoskeleton has an active role in membrane remodelling. Here, we review recent research into the function of Bin-Amphiphysin-Rvs (BAR) domain proteins, which can sense membrane curvature and recruit actin to membranes. BAR proteins interact with the endocytic and cytoskeletal machinery, including the GTPase dynamin (which mediates vesicle fission), N-WASP (an Arp2/3 complex regulator) and synaptojanin (a phosphoinositide phosphatase). We describe three classes of BAR domains, BAR, N-BAR and F-BAR, providing examples of each discussing and how they function in linking membranes to the actin cytoskeleton in endocytosis.     

ProSAP1 and membrane nanodomain-associated syndapin I promote postsynapse formation and function

Insights into mechanisms coordinating membrane remodeling, local actin nucleation, and postsynaptic scaffolding during postsynapse formation are important for understanding vertebrate brain function. Gene knockout and RNAi in individual neurons reveal that the F-BAR protein syndapin I is a crucial postsynaptic coordinator in formation of excitatory synapses. Syndapin I deficiency caused significant reductions of synapse and dendritic spine densities. These syndapin I functions reflected direct, SH3 domain–mediated associations and functional interactions with ProSAP1/Shank2. They furthermore required F-BAR domain-mediated membrane binding. Ultra-high-resolution imaging of specifically membrane-associated, endogenous syndapin I at membranes of freeze-fractured neurons revealed that membrane-bound syndapin I preferentially occurred in spines and formed clusters at distinct postsynaptic membrane subareas. Postsynaptic syndapin I deficiency led to reduced frequencies of miniature excitatory postsynaptic currents, i.e., to defects in synaptic transmission phenocopying ProSAP1/Shank2 knockout, and impairments in proper synaptic ProSAP1/Shank2 distribution. Syndapin I–enriched membrane nanodomains thus seem to be important spatial cues and organizing platforms, shaping dendritic membrane areas into synaptic compartments.     

PACSIN 1 forms tetramers via its N-terminal F-BAR domain

The ability of protein kinase C and casein kinase 2 substrate in neurons (PACSIN)/syndapin proteins to self-polymerize is crucial for the simultaneous interactions with more than one Src homology 3 domain-binding partner or with lipid membranes. The assembly of this network has profound effects on the neural Wiskott-Aldrich syndrome protein-mediated attachment of the actin polymerization machinery to vesicle membranes as well as on the movement of the corresponding vesicles. Also, the sensing of vesicle membranes and/or the induction of membrane curvature are more easily facilitated in the presence of larger PACSIN complexes. The N-terminal Fes-CIP homology and Bin-Amphiphysin-Rvs (F-BAR) domains of several PACSIN-related proteins have been shown to mediate self-interactions, whereas studies using deletion mutants derived from closely related proteins led to the view that oligomerization depends on the formation of a trimeric complex via a coiled-coil region present in these molecules. To address whether the model of trimeric complex formation is applicable to PACSIN 1, the protein was recombinantly expressed and tested in four different assays for homologous interactions. The results showed that PACSIN 1 forms tetramers of about 240 kDa, with the self-interaction having a K(D) of 6.4 x 10(-8) M. Ultrastructural analysis of these oligomers after negative staining showed that laterally arranged PACSIN molecules bind to each other via a large globular domain and form a barrel-like structure. Together, these results demonstrate that the N-terminal F-BAR domain of PACSIN 1 forms the contact site for a tetrameric structure, which is able to simultaneously interact with multiple Src homology 3 binding partners.     

Rigidity of wedge loop in PACSIN 3 protein is a key factor in dictating diameters of tubules

BAR (Bin/amphiphysin/Rvs) domain-containing proteins participate in cellular membrane remodeling. The F-BAR proteins normally generate low curvature tubules. However, in the PACSIN subfamily, the F-BAR domain from PACSIN 1 and 2 can induce both high and low curvature tubules. We found that unlike PACSIN 1 and 2, PACSIN 3 could only induce low curvature tubules. To elucidate the key factors that dictate the tubule curvature, crystal structures of all three PACSIN F-BAR domains were determined. A novel type of lateral interaction mediated by a wedge loop is observed between the F-BAR neighboring dimers. Comparisons of the structures of PACSIN 3 with PACSIN 1 and 2 indicate that the wedge loop of PACSIN 3 is more rigid, which influences the lateral interactions between assembled dimers. We further identified the residues that affect the rigidity of the loop by mutagenesis and determined the structures of two PACSIN 3 wedge loop mutants. Our results suggest that the rigidity-mediated conformations of the wedge loop correlate well with the various crystal packing modes and membrane tubulations. Thus, the rigidity of the wedge loop is a key factor in dictating tubule diameters.     

Proper synaptic vesicle formation and neuronal network activity critically rely on syndapin I

Synaptic transmission relies on effective and accurate compensatory endocytosis. F-BAR proteins may serve as membrane curvature sensors and/or inducers and thereby support membrane remodelling processes; yet, their in vivo functions urgently await disclosure. We demonstrate that the F-BAR protein syndapin I is crucial for proper brain function. Syndapin I knockout (KO) mice suffer from seizures, a phenotype consistent with excessive hippocampal network activity. Loss of syndapin I causes defects in presynaptic membrane trafficking processes, which are especially evident under high-capacity retrieval conditions, accumulation of endocytic intermediates, loss of synaptic vesicle (SV) size control, impaired activity-dependent SV retrieval and defective synaptic activity. Detailed molecular analyses demonstrate that syndapin I plays an important role in the recruitment of all dynamin isoforms, central players in vesicle fission reactions, to the membrane. Consistently, syndapin I KO mice share phenotypes with dynamin I KO mice, whereas their seizure phenotype is very reminiscent of fitful mice expressing a mutant dynamin. Thus, syndapin I acts as pivotal membrane anchoring factor for dynamins during regeneration of SVs.     

Fission yeast paxillin contains two Cdc15 binding motifs for robust recruitment to the cytokinetic ring

The F-BAR protein Cdc15 mediates attachment of the cytokinetic ring (CR) to the plasma membrane and is essential for cytokinesis in Schizosaccharomyces pombe. While its N-terminal F-BAR domain is responsible for oligomerization and membrane binding, its C-terminal SH3 domain binds other partners at a distance from the membrane. We previously demonstrated that the essential cytokinetic formin Cdc12, through an N-terminal motif, directly binds the cytosolic face of the F-BAR domain. Here, we show that paxillin-like Pxl1, which is important for CR stability, contains a motif highly related to that in formin Cdc12, and also binds the Cdc15 F-BAR domain directly. Interestingly, Pxl1 has a second site for binding the Cdc15 SH3 domain. To understand the importance of these two Pxl1-Cdc15 interactions, we mapped and disrupted both. Disrupting the Pxl1-Cdc15 F-BAR domain interaction reduced Pxl1 levels in the CR, whereas disrupting Pxl1’s interaction with the Cdc15 SH3 domain, did not. Unexpectedly, abolishing Pxl1-Cdc15 interaction greatly reduced but did not eliminate CR Pxl1 and did not significantly affect cytokinesis. These data point to another mechanism of Pxl1 CR recruitment and show that very little CR Pxl1 is sufficient for its cytokinetic function.