Membrane Transduction Research Articles

MeCP2 is a naturally supercharged protein with cell membrane transduction capabilities.

The intrinsically disordered protein MeCP2 is a global transcriptional regulator encoded by the MECP2 gene. Although the structured domains of MeCP2 have been the subject of multiple studies, its unstructured regions have not been that extensively characterized. In this work, we show that MeCP2 possesses properties akin to those of supercharged proteins. By utilizing its unstructured portions, MeCP2 can successfully transduce across cell membranes and localize to heterochromatic foci in the nuclei, displaying uptake levels a third lower than a MeCP2 construct fused to the cell-penetrating peptide TAT. MeCP2 uptake can further be enhanced by the addition of compounds that promote endosomal escape following cellular trafficking by means of macropinocytosis. Using a combination of in silico prediction algorithms and live-cell imaging experiments, we mapped the sequence in MeCP2 responsible for its cellular incorporation, which bears a striking resemblance to TAT itself. Transduced MeCP2 was shown to interact with HDAC3. These findings provide valuable insight into the properties of MeCP2 and may be beneficial for devising future protein-based treatment strategies.

High frequency venting of MEMS ultrasonic transducers and sensors: materials solutions.

Abstract This paper introduces the concept of ultrasonic venting. Similar to acoustic vents, ultrasonic vents refer to the aperture in air-coupled MEMS ultrasonic transducers (either PMUT or CMUT) intended to allow the equalization of internal and external pressures, the transfer of heat and the pass of ultrasonic waves, while impeding the penetration of fluids or particles that can affect the transducer membrane. To that end, vents are covered with a porous membrane whose properties are tuned to meet the afore mentioned requirements. The main difficulty in ultrasonic venting, compared with acoustic venting, is that the required “transparency” to ultrasonic waves is much more difficult to achieve. This involves two main problems as both transmission loss and frequency distortion are much larger at ultrasonic frequencies than in the audio range. The objectives of this paper are: to measure the response of acoustic venting materials in the ultrasonic frequency range, to determine the usability of these materials in ultrasonic vents, and to extract useful information for the design of efficient ultrasonic venting materials. Transmission coefficient spectra of different acoustic venting materials is measured in the frequency range 0.2 –2.7 MHz. The origin of the ultrasonic losses and frequency distortion are analysed as well as the role of mode conversion, internal interferences, modes interference, etc. Results reveal that none of the acoustic venting materials analysed can be used in ultrasonic venting applications, but the obtained knowledge about the response of these materials in the ultrasonic frequency range permit to advance in the selection of successful candidate materials for this application.

Human tactile sensing and sensorimotor mechanism: from afferent tactile signals to efferent motor control

In tactile sensing, decoding the journey from afferent tactile signals to efferent motor commands is a significant challenge primarily due to the difficulty in capturing population-level afferent nerve signals during active touch. This study integrates a finite element hand model with a neural dynamic model by using microneurography data to predict neural responses based on contact biomechanics and membrane transduction dynamics. This research focuses specifically on tactile sensation and its direct translation into motor actions. Evaluations of muscle synergy during in -vivo experiments revealed transduction functions linking tactile signals and muscle activation. These functions suggest similar sensorimotor strategies for grasping influenced by object size and weight. The decoded transduction mechanism was validated by restoring human-like sensorimotor performance on a tendon-driven biomimetic hand. This research advances our understanding of translating tactile sensation into motor actions, offering valuable insights into prosthetic design, robotics, and the development of next-generation prosthetics with neuromorphic tactile feedback.

On the Role and Structural Outlook of Carboxy-Terminal Domain of Anabaena Sensory Rhodopsin

Anabaena Sensory Rhodopsin, ASR represents a small family of sensory rhodopsins among a vast family of microbial rhodopsins. In contrast to other microbial sensory rhodopsins, which are known to mediate photostimulated signal relay using cognate membrane transducer, ASR interacts to plausible soluble oligomeric transducer protein. Though the atomic resolution structure ASR is available, it only included only the transmembrane domain. Interestingly the available structure is remarkably different from other microbial sensory rhodopsins, with presence of water. Additionally, the sequence of missing cytoplasmic domain contains a series of positively charged Arginine residues in it. Authors have outlined the influence of this domain in addition to flexible structural outlook using Deep Mind’s Nova Fold-2. Authors propose that charged amino acids are vital in ASR-transducer interaction. Subsequent attempts to explore the molecular basis of ASR and cognate transducer binding would be vital to understand the unique photo-mediated signal cascade in Anabaena PCC 7120.

The design strain sensitivity of the schenberg spherical resonant antenna for gravitational waves

The main purpose of this study is to review the Schenberg resonant antenna transfer function and to recalculate the antenna design strain sensitivity for gravitational waves. We consider the spherical antenna with six transducers in the semi dodecahedral configuration. When coupled to the antenna, the transducer-sphere system will work as a mass-spring system with three masses. The first one is the antenna effective mass for each quadrupole mode, the second one is the mass of the mechanical structure of the transducer first mechanical mode and the third one is the effective mass of the transducer membrane that makes one of the transducer microwave cavity walls. All the calculations are done for the degenerate (all the sphere quadrupole mode frequencies equal) and non-degenerate sphere cases. We have come to the conclusion that the “ultimate” sensitivity of an advanced version of Schenberg antenna (aSchenberg) is around the standard quantum limit (although the parametric transducers used could, in principle, surpass this limit). However, this sensitivity, in the frequency range where Schenberg operates, has already been achieved by the two aLIGOs in the O3 run, therefore, the only reasonable justification for remounting the Schenberg antenna and trying to place it in the sensitivity of the standard quantum limit would be to detect gravitational waves with another physical principle, different from the one used by laser interferometers. This other physical principle would be the absorption of the gravitational wave energy by a resonant mass like Schenberg.

A low-frequency acoustic sensing scheme based on cladding mode of large-angle tilted fiber Bragg grating

A novel low frequency acoustic sensor based on the cladding mode of large-angle tilted fiber Bragg grating (TFBG) is proposed and verified in this work. It mainly uses the characteristic that the coupling mode of the core and cladding mode in TFBG is easy to change when the TFBG experiences micro-bend, which will finally causes a dramatic drift in the spectrum. By combining a large-angle TFBG with the designed polyethylene terephthalate (PET) transducer diaphragm and cavity structure, an effective low-frequency acoustic sensing system is obtained in this work. Under the action of applied acoustic wave, the transducer membrane will have periodic vibrations, which will makes the fixed TFBG dynamically bend, directly leading to a wavelength shift of the cladding mode spectrum. The experimental results show that the sensing system can achieve high-sensitivity acoustic detection in a frequency range of 45–220 Hz, and a maximum acoustic pressure sensitivity of 115.88 mV/Pa at 54 Hz. Moreover, the minimum detection sound pressure can achieve 539.2 μPa/Hz<sup>1/2</sup>@54 Hz. Therefore, the sensor has the advantages of high sensitivity, good repeatability, simple structure, easy processing, etc. It has a great development prospect in the low-frequency acoustic detection related application fields.

Oligomerization state of the functional bacterial twin-arginine translocation (Tat) receptor complex

The twin-arginine translocation (Tat) system transports folded proteins across bacterial and plastid energy transducing membranes. Ion leaks are generally considered to be mitigated by the creation and destruction of the translocation conduit in a cargo-dependent manner, a mechanism that enables tight sealing around a wide range of cargo shapes and sizes. In contrast to the variable stoichiometry of the active translocon, the oligomerization state of the receptor complex is considered more consistently stable but has proved stubbornly difficult to establish. Here, using a single molecule photobleaching analysis of individual inverted membrane vesicles, we demonstrate that Tat receptor complexes are tetrameric in native membranes with respect to both TatB and TatC. This establishes a maximal diameter for a resting state closed pore. A large percentage of Tat-deficient vesicles explains the typically low transport efficiencies observed. This individual reaction chamber approach will facilitate examination of the effects of stochastically distributed molecules.

Design and Dynamics of Oil Filled Flexural Ultrasonic Transducers for Elevated Pressures

The flexural ultrasonic transducer has traditionally been limited to proximity measurement applications, such as car-parking systems and industrial metrology. Principally, their classical form is unsuitable for environments above atmospheric 1 bar pressure, due to an internal air cavity which creates a pressure imbalance across the transducer’s vibrating membrane. This imbalance leads to physical deformation and degradation of the transducer’s structure, restricting the membrane’s capacity to vibrate at resonance to transmit and receive ultrasound. There is a requirement for ultrasonic sensors which can withstand environments of elevated pressure, for example in ultrasonic gas metering. Recent research demonstrated the dynamic performance of flexural ultrasonic transducers with vented structures, allowing the pressure to balance across the transducer membrane. However, a hermetically sealed transducer is a more practical and robust solution, where the internal components of the transducer, such as the piezoelectric ceramic disc, will be protected from harmful environmental fluids. In this research, the design and fabrication of a new form of flexural ultrasonic transducer for environments of elevated pressure is demonstrated, where the internal air cavity is filled with an incompressible fluid in the form of a non-volatile oil. Dynamic performance is measured through acoustic microphone measurements, electrical impedance analysis, and pulse-echo ultrasound measurement. Together with finite element analysis, stable ultrasound measurement is achieved above 200 bar in air, opening the possibility for reliable ultrasound measurement in hostile environments of elevated pressure.

Insights into Site‐1 Protease Recognition of a Sigma Regulator Involved in Cell Surface Signaling During Pseudomonas capeferrumPseudobactin BN7/8 Import

Gram‐negative CSS pathways are composed of three key components, an outer membrane transducer that senses extracellular stimuli, an inner membrane sigma regulator responsible for transducing a signal from the periplasm to the cytoplasm, and an extracytoplasmic function (ECF) sigma factor which activates transcription of target genes (1). In our model system, the Pseudomonas capeferrumpseudobactin BN7/8 (Pup) import system, these proteins are PupB, PupR, and PupI, respectively. Our recent insights into this CSS system indicate the C‐terminal cell‐surface signaling domain (CCSSD) of PupR and the N‐terminal signaling domain (NTSD) of PupB form a stable complex in the periplasm (2). This complex stabilizes the PupR CCSSD prior to signal transduction (2). Subsequently, CSS signal transduction is activated by regulated intramembrane proteolysis (RIP) of the sigma regulator by an initial site‐1 protease followed by a site‐2 protease (3, 4). Recent data has implicated the C‐terminal processing protease, Prc (or Tsp), as the site‐1 protease in PseudomonasCSS (4). Therefore, our objective is to investigate the recognition and degradation of the PupR CCSSD by Prc in the presence and absence of the PupB NTSD utilizing both active protease and catalytically inactive mutants, Prc‐His6 S485A and Prc‐His6 K510A. We hypothesize that the site‐1 protease, Prc, will recognize and cleave the PupR CCSSD unless shielded by PupB NTSD in non‐signaling conditions. Here we present the 2.0 Å structure of Prc‐His6 S485A solved by X‐ray crystallography and additional structural information of Prc‐His6 K510A by size exclusion chromatography small angle X‐ray scattering. Furthermore, we characterize the interaction of Prc‐His6 K510A with MBP‐PupR CCSSD in the presence and absence of PupB NTSD by affinity pull‐down assays and isothermal titration calorimetry, verifying the tighter affinity of the PupR CCSSD for the PupB and supporting its potential shielding role. Additionally, we show proteolytic degradation of MBP‐PupR CCSSD by active Prc‐His6 and analyze the proteolytic fragments by SDS‐PAGE and mass spectrometry. Together these data provide new structural insights into how CSS is activated by RIP following extracellular stimulus. Thus, we support a new CCS activation model wherein the sigma regulator is shielded from Prc by interacting with the transducer NTSD until an event alters this interaction allowing RIP.1. N. Noinaj, et al., TonB‐Dependent Transporters: Regulation, Structure, and Function. Annual Review of Microbiology 64, 43‐60 (2010).2. J. L. Jensen, et al., Structural basis of cell surface signaling by a conserved sigma regulator in Gram‐negative bacteria. Journal of Biological Chemistry (2020).3. R. C. Draper, et al., Differential proteolysis of sigma regulators controls cell‐surface signalling in Pseudomonas aeruginosa. Mol Microbiol 82, 1444‐1453 (2011).4. K. C. Bastiaansen, et al., The Prc and RseP proteases control bacterial cell‐surface signalling activity. Environ Microbiol 16, 2433‐2443 (2014).

Reverse Electron Transfer by Respiratory Complex I Catalyzed in a Modular Proteoliposome System.

Respiratory complex I is an essential metabolic enzyme that uses the energy from NADH oxidation and ubiquinone reduction to translocate protons across an energy transducing membrane and generate the proton motive force for ATP synthesis. Under specific conditions, complex I can also catalyze the reverse reaction, Δp-linked oxidation of ubiquinol to reduce NAD+ (or O2), known as reverse electron transfer (RET). Oxidative damage by reactive oxygen species generated during RET underpins ischemia reperfusion injury, but as RET relies on several converging metabolic pathways, little is known about its mechanism or regulation. Here, we demonstrate Δp-linked RET through complex I in a synthetic proteoliposome system for the first time, enabling complete kinetic characterization of RET catalysis. We further establish the capability of our system by showing how RET in the mammalian enzyme is regulated by the active-deactive transition and by evaluating RET by complex I from several species in which direct assessment has not been otherwise possible. We thus provide new insights into the reversibility of complex I catalysis, an important but little understood mechanistic and physiological feature.

Endocytosis Involved d-Oligopeptide of Tryptophan and Arginine Displays Ordered Nanostructures and Cancer Cell Stereoselective Toxicity by Autophagy.

Owing to their self-aggregation propensity and selective interaction with the anionic membranes, the peptides rich in tryptophan (Trp) and arginine (Arg) are considered for the development of novel anticancer therapeutics. However, the structural insights from the perspective of backbone chirality and spatial orientation of side chains into the selective toxicity of peptides are limited. Here, we investigated the selectivity and cellular uptake of HHC36, a Trp/Arg-rich nonapeptide, and its d-enantiomer (allDHHC36) and a retroinverso analogue in the lung A549 and breast MDA-MB-231 cancer cells. We realized that the d-peptides can specifically induce autophagy at nontoxic concentrations only in the A549 cells supported from the LC 3-II immunostaining expression in the vicinity of the nucleus and the ultrastructural analysis revealing the autophagosome formation. The autophagic flux was also remarkable in the cells exposed to d-peptides at a far lower concentration in synergism with doxorubicin (DOX). In marked contrast, nonselective cell death was observed only if a high amount of HHC36 was applied. HHC36 tended to irregular collagen-like fibrils relative to allDHHC36 that distinctly formed higher-order coiled nanostructures. Interestingly, the short d-peptide fragments were generated in a harsh oxidative condition. Compared with the direct membrane transduction of HHC36, the entry of d-peptides into the lung cancer cells was controlled by endocytosis through the contribution of heparan sulfate proteoglycans (HSPGs) and cholesterol (CHO). However, both l- and d-peptides feasibly crossed the membrane and localized inside the S-phase-arrested cell nucleus. This suggested the likelihood of peptide intercalation with DNA that might differently appear in selective and/or nonselective deaths. These results unraveled the d-handedness-selective toxicity of a self-assembling Trp/Arg-rich sequence that is dependent on the cell type from the aspects of the density of anionic charges and CHO in the outer leaflet of the plasma membrane, as well as the intracellular redox imbalance that may drive the formation of toxic peptide nanostructure fragments.

Oligomerization state of the functional bacterial twin arginine translocation (Tat) receptor complex

The twin-arginine translocation (Tat) system transports folded proteins important for growth and survival across bacterial and plastid energy transducing membranes. Ion leaks are generally considered to be mitigated by the creation and destruction of the translocation conduit in a cargo-dependent manner, a mechanism that enables tight sealing around a wide range of cargo shapes and sizes. Since the Tat system is conserved among bacteria and absent in mammalian cells, it is an attractive target for antibacterial therapies.

Vibro-tactile display for the visually impaired using miniature electrostatic transducers

A vibro-tactile electronic display is being developed to aid the visually impaired community by increasing access to graphical and textual information. A high-density vibro-tactile arrayed display would improve the user experience while moving away from external displays on the market. We propose to use millimeter scale electrostatic transducers operating at frequencies well below resonance as candidate array pixel elements. Motion detectable by a fingertip, mechanical impedance of a fingertip, and frequency at which fingertips can detect contact were considered. Motion of 5μm with a fingertip touching the device and 10 μm without a finger touching the device is desired with the device at a mechanical impedance of 480N/m, which is twice the mechanical impedance of the fingertip (240N/m measured by Diller and Kyung). Transducer membrane thickness, force on the fingertip, and gap width are being optimized through prototyping and finite element analysis. A prototyped row of pixels was manufactured using 0.051 mm thick metalized Mylar film with an air gap of 0.02mm and a diameter of 1 to 2 mm per pixel, driven by AC electrostatics at 400 V peak to peak. Motion analysis is being conducted using a laser vibrometer. Preliminary results demonstrate motion of 10 μm peak to peak at low frequencies, with further analysis and optimization ongoing.

Membrane-Based Scanning Force Microscopy

We report the development of a scanning force microscope based on an ultra-sensitive silicon nitride membrane transducer. Our development is made possible by inverting the standard microscope geometry - in our instrument, the substrate is vibrating and the scanning tip is at rest. We present first topography images of samples placed on the membrane surface. Our measurements demonstrate that the membrane retains an excellent force sensitivity when loaded with samples and in the presence of a scanning tip. We discuss the prospects and limitations of our instrument as a quantum-limited force sensor and imaging tool.

Labeling of polyprenylated macromolecules using mild Ene reactions

Abstract Protein posttranslational modifications play pivotal roles in a wide range of functions such as gene expression regulation, differentiation, cell migration, various human diseases and so on. Polyprenylated group are a lipid modification only found in the Ras super family protein. It was demonstrated that farnesyl, geranylgeranyl groups are covalently attached into sulfur atom on cysteine of Caxx motif, playing essential roles in anchoring on plasma membrane and signal transduction etc. We first reported a straightforward chemical approach to labelling of polyprenylated macromolecules. Our method mainly relied on a novel fluorination/Ene reaction, exhibiting application potential in the study of proteome-wide mapping of protein prenylation target.

Regulation of bacterial ATP synthase activity: A gear-shifting or a pawl-ratchet mechanism?

The F1 Fo -ATP synthase, a widely distributed nanomotor responsible of ATP synthesis, rotates its central rotor reversibly: In the clockwise direction when viewed from the Fo (with the observer facing the positive side of the energy transducing membrane and looking down into the negative side of the membrane), it functions as ATP synthase, while in counterclockwise sense, it operates as a proton-pumping ATP hydrolase. Regulation exerted by naturally occurring inhibitory proteins of the enzyme appears to function by avoiding ATP hydrolysis while preserving ATP synthesis. The work of Liu et al. describes an unbiased, elegant analytical pipeline that provides important insights into the inhibitory role of the ε-subunit of the bacterial F1 Fo -ATP synthase invivo. We discuss if a gear-shifting versus a pawl-ratchet mechanism may explain the regulatory role of the ε-subunit.

Development of a Wireless Telemetry Sensor Device to Measure Load and Deformation in Orthopaedic Applications.

Due to sensor size and supporting circuitry, in-vivo load and deformation measurements are currently restricted to applications within larger orthopaedic implants. The objective of this study is to repurpose a commercially available low-power, miniature, wireless, telemetric, tire-pressure sensor (FXTH87) to measure load and deformation for future use in orthopaedic and biomedical applications. The capacitive transducer membrane was modified, and compressive deformation was applied to the transducer to determine the sensor signal value and the internal resistive force. The sensor package was embedded within a deformable enclosure to illustrate potential applications of the sensor for monitoring load. To reach the maximum output signal value, sensors required compressive deformation of 350 ± 24 µm. The output signal value of the sensor was an effective predictor of the applied load on a calibrated plastic strain member, over a range of 35 N. The FXTH87 sensor can effectively sense and transmit load-induced deformations. The sensor does not have a limit on loads it can measure, as long as deformation resulting from the applied load does not exceed 350 µm. The proposed device presents a sensitive and precise means to monitor deformation and load within small-scale, deformable enclosures.

Design of a new pH‐activatable cell‐penetrating peptide for drug delivery into tumor cells

Cell-penetrating peptides (CPPs) have been considered as potential drug delivery vectors due to their remarkable membrane translocation capacity. However, lack of specificity and extreme systemic toxicity hamper their successful application for drug delivery. Here, we designed a new pH-activatable CPP, LHHLLHHLHHLLHH-NH2 (LH), by substitution of all lysines and two leucines of LKKLLKLLKKLLKL-NH2 (LK) with histidines. As expected, histidine-rich LH could be activated and penetrate into cells at pH 6.0, whereas its membrane transduction activity could be shielded at pH 7.4. In contrast, LK showed no obviously different cellular uptake at both pH conditions. Importantly, LH was significantly less cytotoxicity compared with LK at both pH values, suggesting a better safety for further application. In addition, after conjugation of camptothecin (CPT) with LH, this conjugate displayed remarkably pH-dependent antitumor activity than free CPT and LK-CPT. This study provides a new tumor pH-responsive CPP with low toxicity for selective anticancer drug delivery.

Membrane-Permeant, Environment-Sensitive Dyes Generate Biosensors within Living Cells.

Dyes with environment-sensitive fluorescence have proven useful to study the spatiotemporal dynamics of protein activity in living cells. When attached to proteins, their fluorescence can reflect protein conformational changes, post-translational modifications, or protein interactions. However, the utility of such dye-protein conjugates has been limited because it is difficult to load them into cells. They usually must be introduced using techniques that perturb cell physiology, limit throughput, or generate fluorescent vesicles (e.g., electroporation, microinjection, or membrane transduction peptides). Here we circumvent these problems by modifying a proven, environment-sensitive biosensor fluorophore so that it can pass through cell membranes without staining intracellular compartments and can be attached to proteins within living cells using unnatural amino acid (UAA) mutagenesis. Reactive groups were incorporated for attachment to UAAs or small molecules (mero166, azide; mero167, alkyne; mero76, carboxylic acid). These dyes are bright and fluoresce at long wavelengths (reaching ε = 100 000 M-1 cm-1, ϕ = 0.24, with excitation 565 nm and emission 594 nm). The utility of mero166 was demonstrated by in-cell labeling of a UAA to generate a biosensor for the small GTPase Cdc42. In addition, conjugation of mero166 to a small molecule produced a membrane-permeable probe that reported the localization of the DNA methyltransferase G9a in cells. This approach provides a strategy to access biosensors for many targets and to more practically harness the varied environmental sensitivities of synthetic dyes.

Development of a nonintrusive pressure sensor for polymer tubes based on dielectric elastomer membranes

In this work, a nonintrusive pressure sensor for contrast fluid injection in medical applications is developed, based on a dielectric elastomer (DE) membrane transducer. A DE is essentially a highly deformable capacitor, consisting of a thin elastomer membrane coated with compliant electrodes. A novel solution is proposed to perform a nonintrusive pressure measurement, in which a DE membrane is wrapped around the injection tube (patient line). While pressure is applied to the tube it expands, leading to an extension of the sensor and therefore to a significant increase in DE capacitance.First, a test rig is designed in order to measure the expansion of the polymer tube during pressure application. Multiple experiments are then performed to evaluate the tube deformation at different pressure levels. Subsequently, analytical models are developed for both fabric-reinforced injection tube and DE sensor, in order to predict the resulting capacitive change of the DE as well as to approximate the mechanical properties of the tube. Based on model predictions and tube characterization results, membrane DEs with different sensor geometries are developed, manufactured, and characterized. The sensor performance is finally assessed by means of several experiments, showing remarkable performance for pressures up to 62 bar.