F-BAR Protein Research Articles

F-BAR proteins CIP4 and FBP17 function in cortical neuron radial migration and process outgrowth.

During embryonic development, radial migration of newly born cortical neurons is a complex process that underlies the proper formation of the neocortex, the outermost layers of neurons in the brain. Disruptions in radial migration results in profound effects on cognitive function and can lead to devastating developmental disabilities. To better understand this critical process in brain development we examined two members of the F-BAR family of membrane bending proteins, CIP4 and FBP17, which are present in the developing brain. We demonstrate that intracellular concentrations of these proteins must be tightly regulated. Increasing or decreasing levels of either protein has profound effects on neuronal morphology and proper radial migration, suggesting they are key players in cortical development.

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.

Azoles activate type I and type II programmed cell death pathways in crop pathogenic fungi

Triazoles are widely used to control pathogenic fungi. They inhibit the ergosterol biosynthetic pathway, but the precise mechanisms leading to fungicidal activities in many fungal pathogens are poorly understood. Here, we elucidate the mode of action of epoxiconazole and metconazole in the wheat pathogen Zymoseptoria tritici and the rice blast fungus Magnaporthe oryzae. We show that both azoles have fungicidal activity and reduce fluidity, but not integrity, of the plasma membrane. This impairs localisation of Cdc15-like F-BAR proteins, resulting in defective actin ring assembly and incomplete septation. However, mutant studies and pharmacological experiments in vitro and in planta show that azole lethality is due to a combination of reactive oxygen species-induced apoptosis and macroautophagy. Simultaneous inhibition of both programmed cell death pathways abolishes azole-induced cell death. Other classes of ergosterol biosynthesis inhibitors also induce apoptosis and macroautophagy, suggesting that activation of these two cell death pathways is a hallmark of ergosterol synthesis-targeting fungicides. This knowledge will inform future crop protection strategies.

Phosphorylation of the F-BAR protein Hof1 drives septin ring splitting in budding yeast

A double septin ring accompanies cytokinesis in yeasts and mammalian cells. In budding yeast, reorganisation of the septin collar at the bud neck into a dynamic double ring is essential for actomyosin ring constriction and cytokinesis. Septin reorganisation requires the Mitotic Exit Network (MEN), a kinase cascade essential for cytokinesis. However, the effectors of MEN in this process are unknown. Here we identify the F-BAR protein Hof1 as a critical target of MEN in septin remodelling. Phospho-mimicking HOF1 mutant alleles overcome the inability of MEN mutants to undergo septin reorganisation by decreasing Hof1 binding to septins and facilitating its translocation to the actomyosin ring. Hof1-mediated septin rearrangement requires its F-BAR domain, suggesting that it may involve a local membrane remodelling that leads to septin reorganisation. In vitro Hof1 can induce the formation of intertwined septin bundles, while a phosphomimetic Hof1 protein has impaired septin-bundling activity. Altogether, our data indicate that Hof1 modulates septin architecture in distinct ways depending on its phosphorylation status.

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

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

PACSIN2 Orchestrates Actin Reorganization during Megakaryocyte Maturation

Thrombocytopenia, defined by a platelet count below 150,000/µL, poses significant mortality risks, primarily due to bleeding. Blood platelets are produced by bone marrow megakaryocytes (MKs) in unique cellular processes that require polyploidization and extensive intracellular membrane rearrangements leading to the formation of the demarcation membrane system (DMS), the surface-connected membrane reservoir for future platelets. The membrane-deforming F-BAR protein PACSIN2 is one of the most abundant and conserved BAR proteins in platelets, and exomechip analysis and genome-wide association studies have associated PACSIN2 single nucleotide variants with platelet count and mean platelet volume in humans. Our previous work has shown that PACSIN2 localizes with the initiating DMS in MKs in a process regulated by the cytoskeletal and scaffolding protein filamin A (FlnA). Recently, we have found that Pacsin2 -/- mice display mild thrombocytopenia with slightly enlarged platelets and severe thrombus formation defects due to increased activation of platelet integrin β1. Here we investigated the role of PACSIN2 in MK maturation and platelet production. Cultured fetal liver cell-derived Pacsin2 -/- MKs formed proplatelets normally compared to controls. Pacsin2 -/- bone marrow sections displayed MK count comparable to control mice, and freshly isolated bone marrow Pacsin2 -/- MKs had normal ploidy, indicating normal MK maturation. The observed effects of PACSIN2 deficiency led to a defective formation of the DMS within MKs. In PACSIN2 deficient MKs, the DMS exhibited a less distinct structure, making it challenging to delineate clear platelet territories. Interestingly, the lack of PACSIN2 also impacted the spatial distribution of GPIbα and integrin β1 membrane receptors. These receptors, typically dispersed within the cytoplasm in normal MKs, were concentrated at the cell periphery in Pacsin2 -/-MKs. A similar relocation was observed for polymerized actin. This finding underscores the pivotal role of PACSIN2 in actin reorganization within MKs, which in turn influences the rearrangement of GPIbα and integrin β1. The data imply that receptor and actin cytoskeleton rearrangements are critical to DMS formation and subsequent platelet production in vivo.

Membrane shapers from two distinct superfamilies cooperate in the development of neuronal morphology.

Membrane-shaping proteins are driving forces behind establishment of proper cell morphology and function. Yet, their reported structural and in vitro properties are noticeably inconsistent with many physiological membrane topology requirements. We demonstrate that dendritic arborization of neurons is powered by physically coordinated shaping mechanisms elicited by members of two distinct classes of membrane shapers: the F-BAR protein syndapin I and the N-Ank superfamily protein ankycorbin. Strikingly, membrane-tubulating activities by syndapin I, which would be detrimental during dendritic branching, were suppressed by ankycorbin. Ankycorbin's integration into syndapin I-decorated membrane surfaces instead promoted curvatures and topologies reflecting those observed physiologically. In line with the functional importance of this mechanism, ankycorbin- and syndapin I-mediated functions in dendritic arborization mutually depend on each other and on a surprisingly specific interface mediating complex formation of the two membrane shapers. These striking results uncovered cooperative and interdependent functions of members of two fundamentally different membrane shaper superfamilies as a previously unknown, pivotal principle in neuronal shape development.

The fission yeast cytokinetic ring component Fic1 promotes septum formation.

In Schizosaccharomyces pombe, septum formation is coordinated with cytokinetic ring constriction but the mechanisms linking these events are unclear. In this study, we explored the role of the cytokinetic ring component Fic1, first identified by its interaction with the F-BAR protein Cdc15, in septum formation. We found that the fic1 phospho-ablating mutant, fic1-2A, is a gain-of-function allele that suppresses myo2-E1, the temperature-sensitive allele of the essential type-II myosin, myo2. This suppression is achieved by the promotion of septum formation and required Fic1's interaction with the F-BAR proteins Cdc15 and Imp2. Additionally, we found that Fic1 interacts with Cyk3 and that this interaction was likewise required for Fic1's role in septum formation. Fic1, Cdc15, Imp2, and Cyk3 are the orthologs of the Saccharomyces cerevisiae ingression progression complex, which stimulates the chitin synthase Chs2 to promote primary septum formation. However, our findings indicate that Fic1 promotes septum formation and cell abscission independently of the S. pombe Chs2 ortholog. Thus, while similar complexes exist in the two yeasts that each promote septation, they appear to have different downstream effectors.

GxcM-Fbp17/RacC-WASP signaling regulates polarized cortex assembly in migrating cells via Arp2/3.

The actin-rich cortex plays a fundamental role in many cellular processes. Its architecture and molecular composition vary across cell types and physiological states. The full complement of actin assembly factors driving cortex formation and how their activities are spatiotemporally regulated remain to be fully elucidated. Using Dictyostelium as a model for polarized and rapidly migrating cells, we show that GxcM, a RhoGEF localized specifically in the rear of migrating cells, functions together with F-BAR protein Fbp17, a small GTPase RacC, and the actin nucleation-promoting factor WASP to coordinately promote Arp2/3 complex-mediated cortical actin assembly. Overactivation of this signaling cascade leads to excessive actin polymerization in the rear cortex, whereas its disruption causes defects in cortical integrity and function. Therefore, apart from its well-defined role in the formation of the protrusions at the cell front, the Arp2/3 complex-based actin carries out a previously unappreciated function in building the rear cortical subcompartment in rapidly migrating cells.

Multiple polarity kinases inhibit phase separation of F-BAR protein Cdc15 and antagonize cytokinetic ring assembly in fission yeast.

The F-BAR protein Cdc15 is essential for cytokinesis in Schizosaccharomyces pombe and plays a key role in attaching the cytokinetic ring (CR) to the plasma membrane (PM). Cdc15's abilities to bind to the membrane and oligomerize via its F-BAR domain are inhibited by phosphorylation of its intrinsically disordered region (IDR). Multiple cell polarity kinases regulate Cdc15 IDR phosphostate, and of these the DYRK kinase Pom1 phosphorylation sites on Cdc15 have been shown in vivo to prevent CR formation at cell tips. Here, we compared the ability of Pom1 to control Cdc15 phosphostate and cortical localization to that of other Cdc15 kinases: Kin1, Pck1, and Shk1. We identified distinct but overlapping cohorts of Cdc15 phosphorylation sites targeted by each kinase, and the number of sites correlated with each kinases' abilities to influence Cdc15 PM localization. Coarse-grained simulations predicted that cumulative IDR phosphorylation moves the IDRs of a dimer apart and toward the F-BAR tips. Further, simulations indicated that the overall negative charge of phosphorylation masks positively charged amino acids necessary for F-BAR oligomerization and membrane interaction. Finally, simulations suggested that dephosphorylated Cdc15 undergoes phase separation driven by IDR interactions. Indeed, dephosphorylated but not phosphorylated Cdc15 undergoes liquid-liquid phase separation to form droplets in vitro that recruit Cdc15 binding partners. In cells, Cdc15 phosphomutants also formed PM-bound condensates that recruit other CR components. Together, we propose that a threshold of Cdc15 phosphorylation by assorted kinases prevents Cdc15 condensation on the PM and antagonizes CR assembly.

Polarity kinases that phosphorylate F-BAR protein Cdc15 have unique localization patterns during cytokinesis and contributions to preventing tip septation in Schizosaccharomyces pombe.

The Schizosaccharomyces pombe F-BAR protein, Cdc15, facilitates the linkage between the cytokinetic ring and the plasma membrane. Cdc15 is phosphorylated on many sites by four polarity kinases and this antagonizes membrane interaction. Dephosphorylation of Cdc15 during mitosis induces its phase separation, allowing oligomerization, membrane association, and protein partner binding. Here, using live cell imaging we examined whether spatial separation of Cdc15 from its four identified kinases potentially explains their diverse effects on tip septation and the mitotic Cdc15 phosphorylation state. We identified a correlation between kinase localization and their ability to antagonize Cdc15 cytokinetic ring and membrane localization.

A NanoCurvS platform for quantitative and multiplex analysis of curvature-sensing proteins.

The cell membrane is characterized by a rich variety of topographical features such as local protrusions or invaginations. Curvature-sensing proteins, including the Bin/Amphiphysin/Rvs (BAR) or epsin N-terminal homology (ENTH) family proteins, sense the bending sharpness and the positive/negative sign of these topographical features to induce subsequent intracellular signaling. A number of assays have been developed to study curvature-sensing properties of proteins in vitro, but it is still challenging to probe low curvature regime with the diameter of curvature from hundreds of nanometers to micrometers. It is particularly difficult to generate negative membrane curvatures with well-defined curvature values in the low curvature regime. In this work, we develop a nanostructure-based curvature sensing (NanoCurvS) platform that enables quantitative and multiplex analysis of curvature-sensitive proteins in the low curvature regime, in both negative and positive directions. We use NanoCurvS to quantitatively measure the sensing range of a negative curvature-sensing protein IRSp53 (an I-BAR protein) and a positive curvature-sensing protein FBP17 (an F-BAR protein). We find that, in cell lysates, the I-BAR domain of IRSp53 is able to sense shallow negative curvatures with the diameter-of-curvature up to 1500 nm, a range much wider than previously expected. NanoCurvS is also used to probe the autoinhibition effect of IRSp53 and the phosphorylation effect of FBP17. Therefore, the NanoCurvS platform provides a robust, multiplex, and easy-to-use tool for quantitative analysis of both positive and negative curvature-sensing proteins.

FCHSD2 is required for stereocilia maintenance in mouse cochlear hair cells.

Stereocilia are F-actin-based protrusions on the apical surface of inner-ear hair cells and are indispensable for hearing and balance perception. The stereocilia of each hair cell are organized into rows of increasing heights, forming a staircase-like pattern. The development and maintenance of stereocilia are tightly regulated, and deficits in these processes lead to stereocilia disorganization and hearing loss. Previously, we showed that the F-BAR protein FCHSD2 is localized along the stereocilia of cochlear hair cells and cooperates with CDC42 to regulate F-actin polymerization and cell protrusion formation in cultured COS-7 cells. In the present work, Fchsd2 knockout mice were established to investigate the role of FCHSD2 in hearing. Our data show that stereocilia maintenance is severely affected in cochlear hair cells of Fchsd2 knockout mice, which leads to progressive hearing loss. Moreover, Fchsd2 knockout mice show increased acoustic vulnerability. Noise exposure causes robust stereocilia degeneration as well as enhanced hearing threshold elevation in Fchsd2 knockout mice. Lastly, Fchsd2/Cdc42 double knockout mice show more severe stereocilia deficits and hearing loss, suggesting that FCHSD2 and CDC42 cooperatively regulate stereocilia maintenance.

Investigating the Ultrastructural Effects of F-BAR Proteins on Neuritogenesis by CLEM and Cryo-ET

Investigating the Ultrastructural Effects of F-BAR Proteins on Neuritogenesis by CLEM and Cryo-ET

Pacsin2 is required for endocytosis in the zebrafish pronephric tubule.

ABSTRACTEndocytosis mediates the cellular uptake of numerous molecules from the extracellular space and is a fundamentally important process. In the renal proximal tubule, the scavenger receptor megalin and its co-receptor cubilin mediate endocytosis of low molecular weight proteins from the renal filtrate. However, the extent to which megalin endocytosis relies on different components of the trafficking machinery remains relatively poorly defined in vivo. In this study, we identify a functional requirement for the F-BAR protein pacsin2 in endocytosis in the renal proximal tubule of zebrafish larvae. Pacsin2 is expressed throughout development and in all zebrafish tissues, similar to the mammalian orthologue. Within renal tubular epithelial cells, pacsin2 is enriched at the apical pole where it is localised to endocytic structures. Loss of pacsin2 results in reduced endocytosis within the proximal tubule, which is accompanied by a reduction in the abundance of megalin and endocytic organelles. Our results indicate that pacsin2 is required for efficient endocytosis in the proximal tubule, where it likely cooperates with other trafficking machinery to maintain endocytic uptake and recycling of megalin.

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

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

Mechanical loading of intraluminal pressure mediates wound angiogenesis by regulating the TOCA family of F-BAR proteins

Angiogenesis is regulated in coordinated fashion by chemical and mechanical cues acting on endothelial cells (ECs). However, the mechanobiological mechanisms of angiogenesis remain unknown. Herein, we demonstrate a crucial role of blood flow-driven intraluminal pressure (IP) in regulating wound angiogenesis. During wound angiogenesis, blood flow-driven IP loading inhibits elongation of injured blood vessels located at sites upstream from blood flow, while downstream injured vessels actively elongate. In downstream injured vessels, F-BAR proteins, TOCA1 and CIP4, localize at leading edge of ECs to promote N-WASP-dependent Arp2/3 complex-mediated actin polymerization and front-rear polarization for vessel elongation. In contrast, IP loading expands upstream injured vessels and stretches ECs, preventing leading edge localization of TOCA1 and CIP4 to inhibit directed EC migration and vessel elongation. These data indicate that the TOCA family of F-BAR proteins are key actin regulatory proteins required for directed EC migration and sense mechanical cell stretching to regulate wound angiogenesis.

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.

Computational investigation for endocytosis of CoVID-19 virus SARS-CoV-2 in cell membrane

CoVID-19 virus SARS-CoV-2 follows the endocytosis process to enter inside a cell to infect it. It is important to study the endocytosis of SARS-CoV-2 in cell membrane to prevent the pandemic of CoVID-19. In this paper we develop a finite element based computational model for endocytosis of SARS-CoV-2 in cell membrane and determine curvature generation on it during the process. The virus SARS-CoV-2 is modeled as a rigid spherical particle and cell membrane as an anisotropic elastic material, while its fluidic nature due to lipid exchange with infinite reservoir is preserved using suitable conditions. With the help of a contact pair created between the virus particle and cell membrane, endocytosis process is computationally studied and the curvature of membrane is evaluated as the time progresses during the endocytosis process. At the tip of the virus particle and half-radius distance from it, the membrane follows the curvature of virus very quickly. However, it takes more time for the membrane point located at a distance equal to the radius of the virus particle. This is compensated by the cytoplasmic peripheral proteins binding onto the inside surface of the cell membrane. The role of cytoplasmic peripheral BAR proteins is investigated by using a linear curvature-coupling model with protein concentrations. It is observed that F-BAR protein is more sensitive to the curvature of virus particle in comparison to the other BAR proteins. The sensitiveness deteriorates as the curvature is increased.

FBP17-mediated finger-like membrane protrusions in cell competition between normal and RasV12-transformed cells

SummaryAt the initial stage of carcinogenesis, cell competition often occurs between newly emerging transformed cells and the neighboring normal cells, leading to the elimination of transformed cells from the epithelial layer. For instance, when RasV12-transformed cells are surrounded by normal cells, RasV12 cells are apically extruded from the epithelium. However, the underlying mechanisms of this tumor-suppressive process still remain enigmatic. We first show by electron microscopic analysis that characteristic finger-like membrane protrusions are projected from both normal and RasV12 cells at their interface. In addition, FBP17, a member of the F-BAR proteins, accumulates in RasV12 cells, as well as surrounding normal cells, which plays a positive role in the formation of finger-like protrusions and apical elimination of RasV12 cells. Furthermore, cdc42 acts upstream of these processes. These results suggest that the cdc42/FBP17 pathway is a crucial trigger of cell competition, inducing “protrusion to protrusion response” between normal and RasV12-transformed cells.