EHBP1 Is Critically Involved in the Dendritic Arbor Formation and Is Coupled to Factors Promoting Actin Filament Formation.

The coordinated action of a plethora of factors is required for the organization and dynamics of membranous structures critically underlying the development and function of cells, organs, and organisms. The evolutionary acquisition of additional amino acid motifs allows for expansion and/or specification of protein functions. We identify a thus far unrecognized motif specific for chordata EHBP1 proteins and demonstrate that this motif is critically required for interaction with syndapin I, an F-BAR domain-containing, membrane-shaping protein predominantly expressed in neurons. Gain-of-function and loss-of-function studies in rat primary hippocampal neurons (of mixed sexes) unraveled that EHBP1 has an important role in neuromorphogenesis. Surprisingly, our analyses uncovered that this newly identified function of EHBP1 did not require the domain responsible for Rab GTPase binding but was strictly dependent on EHBP1's syndapin I binding interface and on the presence of syndapin I in the developing neurons. These findings were underscored by temporally and spatially remarkable overlapping dynamics of EHBP1 and syndapin I at nascent dendritic branch sites. In addition, rescue experiments demonstrated the necessity of two additional EHBP1 domains for dendritic arborization, the C2 and CH domains. Importantly, the additionally uncovered critical involvement of the actin nucleator Cobl in EHBP1 functions suggested that not only static association with F-actin via EHBP1's CH domain is important for dendritic arbor formation but also actin nucleation. Syndapin interactions organize ternary protein complexes composed of EHBP1, syndapin I, and Cobl, and our functional data show that only together these factors give rise to proper cell shape during neuronal development.

Phosphorylation in the intrinsically disordered region of F-BAR protein Imp2 regulates its contractile ring recruitment.

The F-BAR protein Imp2 is an important contributor to cytokinesis in the fission yeast Schizosaccharomyces pombe. Because cell cycle-regulated phosphorylation of the central intrinsically disordered region (IDR) of the Imp2 paralog Cdc15 controls Cdc15 oligomerization state, localization and ability to bind protein partners, we investigated whether Imp2 is similarly phosphoregulated. We found that Imp2 is endogenously phosphorylated on 28 sites within its IDR, with the bulk of phosphorylation being constitutive. In vitro, the casein kinase 1 (CK1) isoforms Hhp1 and Hhp2 can phosphorylate 17 sites, and Cdk1 (also known as Cdc2) can phosphorylate the remaining 11 sites. Mutations that prevent Cdk1 phosphorylation result in precocious Imp2 recruitment to the cell division site, and mutations designed to mimic these phosphorylation events delay Imp2 accumulation at the contractile ring (CR). Mutations that eliminate CK1 phosphorylation sites allow CR sliding, and phosphomimetic substitutions at these sites reduce Imp2 protein levels and slow CR constriction. Thus, like Cdc15, the Imp2 IDR is phosphorylated at many sites by multiple kinases. In contrast to Cdc15, for which phosphorylation plays a major cell cycle regulatory role, Imp2 phosphorylation is primarily constitutive, with milder effects on localization and function. This article has an associated First Person interview with the first author of the paper.

Design and Fabrication of a Flexible Gravimetric Sensor Based on a Thin-Film Bulk Acoustic Wave Resonator.

Sensing systems are becoming less and less invasive. In this context, flexible materials offer new opportunities that are impossible to achieve with bulky and rigid chips. Standard silicon sensors cannot be adapted to curved shapes and are susceptible to big deformations, thus discouraging their use in wearable applications. Another step forward toward minimising the impacts of the sensors can be to avoid the use of cables and connectors by exploiting wireless transmissions at ultra-high frequencies (UHFs). Thin-film bulk acoustic wave resonators (FBARs) represent the most promising choice among all of the piezoelectric microelectromechanical system (MEMS) resonators for the climbing of radio frequencies. Accordingly, the fabrication of FBARs on flexible and wearable substrates represents a strategic step toward obtaining a new generation of highly sensitive wireless sensors. In this work, we propose the design and fabrication of a flexible gravimetric sensor based on an FBAR on a polymeric substrate. The resonator presents one of the highest electromechanical coupling factors in the category of flexible AlN-based FBARs, equal to 6%. Moreover, thanks to the polymeric support layer, the presence of membranes can be avoided, which leads to a faster and cheaper fabrication process and higher robustness of the structure. The mass sensitivity of the device was evaluated, obtaining a promising value of 23.31 ppm/pg. We strongly believe that these results can pave the way to a new class of wearable MEMS sensors that exploit ultra-high-frequency (UHF) transmissions.

Regulation of caveolae through cholesterol-depletion-dependent tubulation mediated by PACSIN2.

The membrane-shaping ability of PACSIN2 (also known as syndapin II), which is mediated by its F-BAR domain, has been shown to be essential for caveolar morphogenesis, presumably through the shaping of the caveolar neck. Caveolar membranes contain abundant cholesterol. However, the role of cholesterol in PACSIN2-mediated membrane deformation remains unclear. Here, we show that the binding of PACSIN2 to the membrane can be negatively regulated by cholesterol. We prepared reconstituted membranes based on the lipid composition of caveolae. The reconstituted membrane with cholesterol had a weaker affinity for the F-BAR domain of PACSIN2 than a membrane without cholesterol. Consistent with this, upon depletion of cholesterol from the plasma membrane, PACSIN2 localized at tubules that had caveolin-1 at their tips, suggesting that cholesterol inhibits membrane tubulation mediated by PACSIN2. The tubules induced by PACSIN2 could be representative of an intermediate of caveolae endocytosis. Consistent with this, the removal of caveolae from the plasma membrane upon cholesterol depletion was diminished in the PACSIN2-deficient cells. These data suggest that PACSIN2-mediated caveolae internalization is dependent on the amount of cholesterol, providing a mechanism for cholesterol-dependent regulation of caveolae.This article has an associated First Person interview with the first author of the paper.

Syndapin constricts microvillar necks to form a united rhabdomere in Drosophila photoreceptors.

Drosophila photoreceptors develop from polarized epithelial cells that have apical and basolateral membranes. During morphogenesis, the apical membranes subdivide into a united bundle of photosensory microvilli (rhabdomeres) and a surrounding supporting membrane (stalk). By EMS-induced mutagenesis screening, we found that the F-Bin/Amphiphysin/Rvs (F-BAR) protein syndapin is essential for apical membrane segregation. The analysis of the super-resolution microscopy, STORM and the electron microscopy suggest that syndapin localizes to the neck of the microvilli at the base of the rhabdomere. Syndapin and moesin are required to constrict the neck of the microvilli to organize the membrane architecture at the base of the rhabdomere, to exclude the stalk membrane. Simultaneous loss of syndapin along with the microvilli adhesion molecule chaoptin significantly enhanced the disruption of stalk-rhabdomere segregation. However, loss of the factors involving endocytosis do not interfere. These results indicated syndapin is most likely functioning through its membrane curvature properties, and not through endocytic processes for stalk-rhabdomere segregation. Elucidation of the mechanism of this unconventional domain formation will provide novel insights into the field of cell biology.

Membrane curvature underlies actin reorganization in response to nanoscale surface topography.

Surface topography profoundly influences cell adhesion, differentiation, and stem cell fate control. Numerous studies using a variety of materials demonstrate that nanoscale topographies change the intracellular organization of actin cytoskeleton and therefore a broad range of cellular dynamics in live cells. However, the underlying molecular mechanism is not well understood, leaving why actin cytoskeleton responds to topographical features unexplained and therefore preventing researchers from predicting optimal topographic features for desired cell behavior. Here we demonstrate that topography-induced membrane curvature plays a crucial role in modulating intracellular actin organization. By inducing precisely controlled membrane curvatures using engineered vertical nanostructures as topographies, we find that actin fibers form at the sites of nanostructures in a curvature-dependent manner with an upper limit for the diameter of curvature at ∼400 nm. Nanotopography-induced actin fibers are branched actin nucleated by the Arp2/3 complex and are mediated by a curvature-sensing protein FBP17. Our study reveals that the formation of nanotopography-induced actin fibers drastically reduces the amount of stress fibers and mature focal adhesions to result in the reorganization of actin cytoskeleton in the entire cell. These findings establish the membrane curvature as a key linkage between surface topography and topography-induced cell signaling and behavior.

High-Order Harmonic Film Bulk Acoustic Resonator Based on a Polymer Reflector.

A film bulk acoustic resonator (FBAR), based on a polymer air cavity, is presented. The polymer reflective layer on the polymer air cavity can serve both as the reflective layer and the function layer for inducing the high-order mode resonance. With the aluminum nitride as the piezoelectric layer, the resonance frequency of the FBAR can reach 6.360 GHz, based on the finite element method. The product of the corresponding frequency and the quality factor, f × Q is more than 2 × 1013. This design model provides a good solution for the high-frequency filters and high-sensitivity sensor designs.

Growth arrest specific protein 7 inhibits tau fibrillogenesis.

Here we show that Gas7 inhibits phosphorylated tau fibrillogenesis by binding to phosphorylated tau at its non-WW domain, presumably F-BAR domain. We revealed that Gas7 binds to the third repeat domain of tau, the core element of tau oligomerization and the C-terminal domain of tau and alters the conformation not to form fibrils. These results suggest that Gas7 may serve to protect against Alzheimer's disease and other tauopathies by preventing tau fibrillogenesis.

The Design of a Frame-Like ZnO FBAR Sensor for Achieving Uniform Mass Sensitivity Distributions.

In this paper, an infinite circular ZnO thin film bulk acoustic resonator (FBAR) with a frame-like electrode operating at the thickness-extensional (TE) mode is studied. Two-dimensional scalar differential equations established for the problem in the Cartesian coordinate system are successfully solved by transforming them into normal Bessel equations and modified Bessel equations in the cylindrical coordinate system. Resonant frequencies and vibration distributions are obtained for this frame-like FBAR sensor. A nearly uniform mass sensitivity distribution in the active area is achieved by designing proper electrode size and mass ratio of the driving electrode to the ZnO film. Numerical results show that compared with the reported ring electrode FBAR sensor, the novel frame-like electrode FBAR can achieve a maximum optimization ratio (up to 97.90%) on the uniformity of the mass sensitivity distribution in the active area under the same structural parameters, which is also higher than the optimization ratio 77.63% obtained by the reported double-ring electrode design. Moreover, the mechanism to achieve a very uniform mass sensitivity distribution in the active area by the frame-like electrode is explained in detail according to dispersion curves. Namely, when the resonant frequency of the FBAR sensor is close to the cut-off frequency of the active region in the dispersion curve, the mass sensitivity distribution is nearly uniform. These conclusions provide a theoretical guidance for the design and optimization of ZnO FBAR mass sensors with high performance.

Coordinated autoinhibition of F-BAR domain membrane binding and WASp activation by Nervous Wreck.

Membrane remodeling by Fes/Cip4 homology-Bin/Amphiphysin/Rvs167 (F-BAR) proteins is regulated by autoinhibitory interactions between their SRC homology 3 (SH3) and F-BAR domains. The structural basis of autoregulation, and whether it affects interactions of SH3 domains with other cellular ligands, remain unclear. Here we used single-particle electron microscopy to determine the structure of the F-BAR protein Nervous Wreck (Nwk) in both soluble and membrane-bound states. On membrane binding, Nwk SH3 domains do not completely dissociate from the F-BAR dimer, but instead shift from its concave surface to positions on either side of the dimer. Unexpectedly, along with controlling membrane binding, these autoregulatory interactions inhibit the ability of Nwk-SH3a to activate Wiskott-Aldrich syndrome protein (WASp)/actin related protein (Arp) 2/3-dependent actin filament assembly. In Drosophila neurons, Nwk autoregulation restricts SH3a domain-dependent synaptopod formation, synaptic growth, and actin organization. Our results define structural rearrangements in Nwk that control F-BAR-membrane interactions as well as SH3 domain activities, and suggest that these two functions are tightly coordinated in vitro and in vivo.

An Overview of High Frequency Acoustic Sensors-QCMs, SAWs and FBARs-Chemical and Biochemical Applications.

Acoustic devices have found wide applications in chemical and biosensing fields owing to their high sensitivity, ruggedness, miniaturized design and integration ability with on-field electronic systems. One of the potential advantages of using these devices are their label-free detection mechanism since mass is the fundamental property of any target analyte which is monitored by these devices. Herein, we provide a concise overview of high frequency acoustic transducers such as quartz crystal microbalance (QCM), surface acoustic wave (SAW) and film bulk acoustic resonators (FBARs) to compare their working principles, resonance frequencies, selection of piezoelectric materials for their fabrication, temperature-frequency dependency and operation in the liquid phase. The selected sensor applications of these high frequency acoustic transducers are discussed primarily focusing on the two main sensing domains, i.e., biosensing for working in liquids and gas/vapor phase sensing. Furthermore, the sensor performance of high frequency acoustic transducers in selected cases is compared with well-established analytical tools such as liquid chromatography mass spectrometry (LC-MS), gas chromatographic (GC) analysis and enzyme-linked immunosorbent assay (ELISA) methods. Finally, a general comparison of these acoustic devices is conducted to discuss their strengths, limitations, and commercial adaptability thus, to select the most suitable transducer for a particular chemical/biochemical sensing domain.

Structural History of Human SRGAP2 Proteins.

In the development of the human brain, human-specific genes are considered to play key roles, conferring its unique advantages and vulnerabilities. At the time of Homo lineage divergence from Australopithecus, SRGAP2C gradually emerged through a process of serial duplications and mutagenesis from ancestral SRGAP2A (3.4-2.4 Ma). Remarkably, ectopic expression of SRGAP2C endows cultured mouse brain cells, with human-like characteristics, specifically, increased dendritic spine length and density. To understand the molecular mechanisms underlying this change in neuronal morphology, we determined the structure of SRGAP2A and studied the interplay between SRGAP2A and SRGAP2C. We found that: 1) SRGAP2A homo-dimerizes through a large interface that includes an F-BAR domain, a newly identified F-BAR extension (Fx), and RhoGAP-SH3 domains. 2) SRGAP2A has an unusual inverse geometry, enabling associations with lamellipodia and dendritic spine heads in vivo, and scaffolding of membrane protrusions in cell culture. 3) As a result of the initial partial duplication event (∼3.4 Ma), SRGAP2C carries a defective Fx-domain that severely compromises its solubility and membrane-scaffolding ability. Consistently, SRGAP2A:SRAGP2C hetero-dimers form, but are insoluble, inhibiting SRGAP2A activity. 4) Inactivation of SRGAP2A is sensitive to the level of hetero-dimerization with SRGAP2C. 5) The primal form of SRGAP2C (P-SRGAP2C, existing between ∼3.4 and 2.4 Ma) is less effective in hetero-dimerizing with SRGAP2A than the modern SRGAP2C, which carries several substitutions (from ∼2.4 Ma). Thus, the genetic mutagenesis phase contributed to modulation of SRGAP2A's inhibition of neuronal expansion, by introducing and improving the formation of inactive SRGAP2A:SRGAP2C hetero-dimers, indicating a stepwise involvement of SRGAP2C in human evolutionary history.

A curvature-dependent membrane binding by tyrosine kinase Fer involves an intrinsically disordered region.

Tyrosine kinases are important enzymes that mediate signal transduction at the plasma membrane. While the significance of membrane localization of tyrosine kinases has been well evaluated, the role of membrane curvature in their regulation is unknown. Here, we demonstrate that an intrinsically disordered region in the tyrosine kinase Fer acts as a membrane curvature sensor that preferentially binds to highly curved membranes in vitro. This region forms an amphipathic α-helix upon interaction with curved membranes, aligning hydrophobic residues on one side of the helical structure. Further, the tyrosine kinase activity of Fer is significantly enhanced by the membrane in a manner dependent on curvature. We propose a model for the regulation of Fer based on an intramolecular interaction and the curvature-dependent membrane binding mediated by its intrinsically disordered region.

Flexible Film Bulk Acoustic Wave Filters toward Radiofrequency Wireless Communication.

This paper presents a flexible radiofrequency filter with a central frequency of 2.4 GHz based on film bulk acoustic wave resonators (FBARs). The flexible filter consists of five air-gap type FBARs, each comprised of an aluminum nitride piezoelectric thin film sandwiched between two thin-film electrodes. By transfer printing the inorganic film structure from a silicon wafer to an ultrathin polyimide substrate, high electrical performance and mechanical flexibility are achieved. The filter has a peak insertion loss of -1.14 dB, a 3 dB bandwidth of 107 MHz, and a temperature coefficient of frequency of -27 ppm °C-1 . The passband and roll-off characteristics of the flexible filter are comparable with silicon-based commercial products. No electrical performance degradation and mechanical failure occur under bending tests with a bending radius of 2.5 mm or after 100 bending cycles. The flexible FBAR filters are believed to be promising candidates for future flexible wireless communication systems.

Deciphering the BAR code of membrane modulators.

The BAR domain is the eponymous domain of the "BAR-domain protein superfamily", a large and diverse set of mostly multi-domain proteins that play eminent roles at the membrane cytoskeleton interface. BAR domain homodimers are the functional units that peripherally associate with lipid membranes and are involved in membrane sculpting activities. Differences in their intrinsic curvatures and lipid-binding properties account for a large variety in membrane modulating properties. Membrane activities of BAR domains are further modified and regulated by intramolecular or inter-subunit domains, by intermolecular protein interactions, and by posttranslational modifications. Rather than providing detailed cell biological information on single members of this superfamily, this review focuses on biochemical, biophysical, and structural aspects and on recent findings that paradigmatically promote our understanding of processes driven and modulated by BAR domains.

A role for F-BAR protein Rga7p during cytokinesis in S. pombe.

F-BAR proteins are known to participate in cytokinesis, but their mechanisms are not well understood. Here we investigated Rga7p, an Schizosaccharomyces pombe F-BAR protein with a RhoGAP domain. Localization of Rga7p to the cytokinetic cleavage furrow depends on its F-BAR domain, actin filaments, the formins Cdc12p and For3p, and the presence of a contractile ring. Rga7p is not required for the constriction of the contractile ring but does participate in the transport of a ß-glucan synthetase (Bgs4p) from the late Golgi compartments to plasma membrane that is adjacent to the contractile ring. Cells without Rga7p moved Bgs4p normally from the poles to the Golgi complex near to the cell center, but Bgs4p then moved slowly from the late Golgi compartments to the cleavage site. The late arrival and lower than normal numbers of Bgs4p result in septal defects late in cytokinesis, and in the lysis of separating cells, similar to that in cells with mutations in the cwg1(+) gene (which encodes Bgs4p).

Optimization of a VOC Sensor with a Bilayered Diaphragm Using FBAR as Strain Sensing Elements.

Film bulk acoustic resonators (FBARs) are widely applied in mass bio-sensing and pressure sensors, owing to their extreme sensitivity and integration ability, and ability to miniaturize circuits. A volatile organic compound (VOC) sensor with a polymer-coated diaphragm, using FBARs as a strain sensing element is proposed and optimized. This vapor sensor is based on organic vapor-induced changes of mechanical deformation of the micro-diaphragm. The four FBARs are located at the edge of the bi-layer diaphragm comprising silicon nitride and silicon oxide for strain extraction. In this work, the strain distribution of the FBAR area under vapor loads is obtained using the finite element analysis (FEA) and the response frequency changes of the FBARs under vapor loads are obtained based on both the first-principle methods to deduce the elastic coefficient variation of aluminum nitride film in FBARs under the bending stresses and the Mason equivalent circuit model of the sensor using ADS software. Finally, optimizations are performed on both the bilayered diaphragm structure and sensing film. The diaphragm with a 0.7 µm silicon nitride layer and a 0.5 µm silicon oxide layer are considered to be the optimized design. The optimal coverage area of the sensing film for the diaphragm is around 0.8.

Stepwise and cooperative assembly of a cytokinetic core complex in Saccharomyces cerevisiae.

Actomyosin ring (AMR) contraction and the synthesis of an extracellular septum are interdependent pathways that mediate cytokinesis in the yeast Saccharomyces cerevisiae and other eukaryotes. How these interdependent pathways are physically connected is central for understanding cytokinesis. The yeast IQGAP (Iqg1p) belongs to the conserved AMR. The F-BAR-domain-containing protein Hof1p is a member of a complex that stimulates cell wall synthesis. We report here on the stepwise formation of a physical connection between both proteins. The C-terminal IQ-repeats of Iqg1p first bind to the essential myosin light chain before both proteins assemble with Hof1p into the Mlc1p-Iqg1p-Hof1p (MIH) bridge. Mutations in Iqg1p that disrupt the MIH complex alter Hof1p targeting to the AMR and impair AMR contraction. Epistasis analyses of two IQG1 alleles that are incompatible with formation of the MIH complex support the existence and functional significance of a large cytokinetic core complex. We propose that the MIH complex acts as hinge between the AMR and the proteins involved in cell wall synthesis and membrane attachment.

F-BAR domain protein Rga7 collaborates with Cdc15 and Imp2 to ensure proper cytokinesis in fission yeast.

F-BAR domain proteins act as linkers between the cell cortex and cytoskeleton, and are involved in membrane binding and bending. Rga7 is one of the seven F-BAR proteins present in the fission yeast Schizosaccharomyces pombe. In addition to the F-BAR domain in the N-terminal region, Rga7 possesses a Rho GTPase-activating protein (GAP) domain at its C-terminus. We show here that Rga7 is necessary to prevent fragmentation of the contracting ring and incorrect septum synthesis. Accordingly, cultures of cells lacking Rga7 contain a higher percentage of dividing cells and more frequent asymmetric or aberrant septa, which ultimately might cause cell death. The Rga7 F-BAR domain is necessary for the protein localization to the division site and to the cell tips, and also for the Rga7 roles in cytokinesis. In contrast, Rga7 GAP catalytic activity seems to be dispensable. Moreover, we demonstrate that Rga7 cooperates with the two F-BAR proteins Cdc15 and Imp2 to ensure proper cytokinesis. We have also detected association of Rga7 with Imp2, and its binding partners Fic1 and Pxl1. Taken together, our findings suggest that Rga7 forms part of a protein complex that coordinates the late stages of cytokinesis.

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.