Electrical Oscillations in Two-Dimensional Microtubular Structures

Microtubules (MTs) are unique components of the cytoskeleton formed by hollow cylindrical structures of αβ tubulin dimeric units. The structural wall of the MT is interspersed by nanopores formed by the lateral arrangement of its subunits. MTs are also highly charged polar polyelectrolytes, capable of amplifying electrical signals. The actual nature of these electrodynamic capabilities remains largely unknown. Herein we applied the patch clamp technique to two-dimensional MT sheets, to characterize their electrical properties. Voltage-clamped MT sheets generated cation-selective oscillatory electrical currents whose magnitude depended on both the holding potential, and ionic strength and composition. The oscillations progressed through various modes including single and double periodic regimes and more complex behaviours, being prominent a fundamental frequency at 29 Hz. In physiological K+ (140 mM), oscillations represented in average a 640% change in conductance that was also affected by the prevalent anion. Current injection induced voltage oscillations, thus showing excitability akin with action potentials. The electrical oscillations were entirely blocked by taxol, with pseudo Michaelis-Menten kinetics and a KD of ~1.29 μM. The findings suggest a functional role of the nanopores in the MT wall on the genesis of electrical oscillations that offer new insights into the nonlinear behaviour of the cytoskeleton.Fil: Cantero, Maria del Rocio. Universidad de Buenos Aires. Facultad de Odontología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pérez, Paula Luciana. Universidad de Buenos Aires. Facultad de Odontología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Smoler, Mariano. Universidad de Buenos Aires. Facultad de Odontología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Villa Etchegoyen, Cecilia. Universidad de Buenos Aires. Facultad de Odontología; ArgentinaFil: Cantiello, Horacio Fabio. Universidad de Buenos Aires. Facultad de Odontología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Ultrasonic spectroscopy of sessile droplets coupled to optomechanical sensors

We describe a system for interrogating the acoustic properties of sub-nanoliter liquid samples within an open microfluidics platform. Sessile droplets were deposited onto integrated optomechanical sensors, which possess ambient-medium-noise-limited sensitivity and can thus passively sense the thermally driven acoustic spectrum of the droplets. The droplet acoustic breathing modes manifest as resonant features in the thermomechanical noise spectrum of the sensor, in some cases hybridized with the sensor's own vibrational modes. Excellent agreement is found between experimental observations and theoretical predictions, over the entire ~ 0 - 40 MHz operating range of our sensors. With suitable control over droplet size and morphology, this technique has the potential for precision acoustic sensing of small-volume biological and chemical samples

Engineering mechanobiology: the bacterial exclusively-mechanosensitive ion channel MscL as a future tool for neuronal stimulation technology

The development of novel approaches to stimulate neuronal circuits is crucial to understand the physiology of neuronal networks, and to provide new strategies to treat neurological disorders. Nowadays, chemical, electrical or optical approaches are the main exploited strategies to interrogate and dissect neuronal circuit functions. However, although all these methods have contributed to achieve important insights into neuroscience research field, they all present relevant limitations for their use in in-vivo studies or clinical applications. For example, while chemical stimulation does not require invasive surgical procedures, it is difficult to control the pharmacokinetics and the spatial selectivity of the stimulus; electrical stimulation provides high temporal bandwidth, but it has low spatial resolution and it requires implantation of electrodes; optical stimulation provides subcellular resolution but the low depth penetration in dense tissue still requires the invasive insertion of stimulating probes. Due to all these drawbacks, there is still a strong need to develop new stimulation strategies to remotely activate neuronal circuits as deep as possible. The development of remote stimulation techniques would allow the combination of functional and behavioral studies, and the design of novel and minimally invasive prosthetic approaches. Alternative approaches to circumvent surgical implantation of probes include transcranial electrical, thermal, magnetic, and ultrasound stimulation. Among v these methods, the use of magnetic and ultrasound (US) fields represents the most promising vector to remotely convey information to the brain tissue. Both magnetic and low-intensity US fields provide an efficient mean for delicate and reversible alteration of cells and tissues through the generation of local mechanical perturbations. In this regard, advances in the mechanobiology research field have led to the discovery, design and engineering of cellular transduction pathways to perform stimulation of cellular activity. Furthermore, the use of US pressure fields is attracting considerable interest due to its potential for the development of miniaturized, portable and implantation-free US stimulation devices. The purpose of my PhD research activity was the establishment of a novel neuronal stimulation paradigm adding a cellular selectivity to the US stimulation technology through the selective mechano-sensitization of neuronal cells, in analogy to the well-established optogenetic approach. In order to achieve the above mentioned goal, we propose the cellular overexpression of mechanosensitive (MS) ion channels, which could then be gated upon the application of an US generated pressure field. Therefore, we selected the bacterial large conductance mechanosensitive ion channel (MscL), an exclusively-MS ion channel, as ideal tool to develop a mechanogenetic approach. Indeed, the MscL with its extensive characterization represents a malleable nano-valve that could be further engineered with respect to channel sensitivity, conductance and gating mechanism, in order to obtain the desired biophysical properties to achieve reliable and efficient remote mechanical stimulation of neuronal activity. In the first part of the work, we report the development of an engineered MscL construct, called eMscL, to induce the heterologous expression of the bacterial protein in rodent primary neuronal cultures. Furthermore, we report the structural and functional characterization of neuronal cells expressing the eMscL channel, at both single-cell and network levels, in order to show that the functional expression of the engineered MscL channel induces an effective vi neuronal sensitization to mechanical stimulation, which does not affect the physiological development of the neuronal itself. In the second part of the work, we report the design and development of a water tank-free ultrasound delivery system integrated to a custom inverted fluorescence microscope, which allows the simultaneous US stimulation and monitoring of neuronal network activity at single resolution. Overall, this work represents the first development of a genetically mechanosensitized neuronal in-vitro model. Moreover, the developed US delivery system provides the platform to perform high-throughput and reliable investigation, testing and calibration of the stimulation protocols. In this respect, we propose, and envisage in the near future, the exploitation of the engineered MscL ion channel as a mature tool for novel neuro-technological applications

A doublecortin containing microtubule-associated protein is implicated in mechanotransduction in Drosophila sensory cilia

Mechanoreceptors are sensory cells that transduce mechanical stimuli into electrical signals and mediate the perception of sound, touch and acceleration. Ciliated mechanoreceptors possess an elaborate microtubule cytoskeleton that facilitates the coupling of external forces to the transduction apparatus. In a screen for genes preferentially expressed in Drosophila campaniform mechanoreceptors, we identified DCX-EMAP, a unique member of the EMAP family (echinoderm–microtubule-associated proteins) that contains two doublecortin domains. DCX-EMAP localizes to the tubular body in campaniform receptors and to the ciliary dilation in chordotonal mechanoreceptors in Johnston's organ, the fly's auditory organ. Adult flies carrying a piggyBac insertion in the DCX-EMAP gene are uncoordinated and deaf and display loss of mechanosensory transduction and amplification. Electron microscopy of mutant sensilla reveals loss of electron-dense materials within the microtubule cytoskeleton in the tubular body and ciliary dilation. Our results establish a catalogue of candidate genes for Drosophila mechanosensation and show that one candidate, DCX-EMAP, is likely to be required for mechanosensory transduction and amplification

TRPA1 CHANNELS IN COCHLEAR SUPPORTING CELLS REGULATE HEARING SENSITIVITY AFTER NOISE EXPOSURE

TRPA1 channels are sensors for noxious stimuli in a subset of nociceptive neurons. TRPA1 channels are also expressed in cells of the mammalian inner ear, but their function in this tissue remains unknown given that Trpa1–/– mice exhibit normal hearing, balance and sensory mechanotransduction. Here we show that non-sensory (supporting) cells of the hearing organ in the cochlea detect tissue damage via the activation of TRPA1 channels and subsequently modulate cochlear amplification through active cellshape changes. We found that cochlear supporting cells of wild type but not Trpa1–/– mice generate inward currents and robust long-lasting Ca2+ responses after stimulation with TRPA1 agonists. These Ca2+ responses often propagated between different types of supporting cells and were accompanied by prominent tissue displacements. The most prominent shape changes were observed in pillar cells which here we show possess Ca2+-dependent contractile machinery. Increased oxidative stress following acoustic overstimulation leads to the generation of lipid peroxidation byproducts such as 4-hydroxynonenal (4-HNE) that could directly activate TRPA1. Therefore, we exposed mice to mild noise and found a longer-lasting inhibition of cochlear amplification in wild type than in Trpa1–/– mice. Our results suggest that TRPA1-dependent changes in pillar cell shape can alter the tissue geometry and affect cochlear amplification. We believe this novel mechanism of cochlear regulation may protect or fine-tune the organ of Corti after noise exposure or other cochlear injuries

Tubulin response to intense nanosecond-scale electric field in molecular dynamics simulation

Intense pulsed electric fields are known to act at the cell membrane level and are already being exploited in biomedical and biotechnological applications. However, it is not clear if electric pulses within biomedically-attainable parameters could directly influence intra-cellular components such as cytoskeletal proteins. If so, a molecular mechanism of action could be uncovered for therapeutic applications of such electric fields. To help clarify this question, we first identified that a tubulin heterodimer is a natural biological target for intense electric fields due to its exceptional electric properties and crucial roles played in cell division. Using molecular dynamics simulations, we then demonstrated that an intense - yet experimentally attainable - electric field of nanosecond duration can affect the bβ-tubulin’s C-terminus conformations and also influence local electrostatic properties at the GTPase as well as the binding sites of major tubulin drugs site. Our results suggest that intense nanosecond electric pulses could be used for physical modulation of microtubule dynamics. Since a nanosecond pulsed electric field can penetrate the tissues and cellular membranes due to its broadband spectrum, our results are also potentially significant for the development of new therapeutic protocols

The Role of Pkd1 in Mouse Inner Ear Hair Cells

The polycystic kidney disease-1 (Pkd1) gene encodes a large transmembrane protein (polycystin-1 or PC-1) that is reported to function as a fluid flow-sensor in the kidney. As a member of the transient receptor potential (TRP) family, PC-1 has also been hypothesized to play a role in the elusive mechanoelectrical transduction (MET) channel in inner ear hair cells based on PC-1 role of fluid flow sensing and calcium uptake into renal epithelial cells. However, two independent mouse lines with PC-1 mutations exhibit normal MET channel function despite hearing loss and ultra-structural abnormalities of stereocilia that remain properly polarized at adult ages. These findings indicate that PC-1 plays an essential role in stereocilia structure and maintenance, but not directly in MET channel function and planar cell polarity. We also demonstrate that PC-1 is co-localized with F-actin in hair cell stereocilia as well as with the actin based microvilli in a renal epithelia cell line. These results not only provide a unique hair cell stereocilia phenotype, but also ultimately may lead to a further understanding of the mechanisms behind polycystic kidney disease

Modelling on the nanomechanics of cytoskeletal filaments

Cytoskeleton is a structure that enables cells to maintain their shape and internal organization. The proper functions of cytoskeleton depend crucially on the mechanical responses and properties of its component filaments (e.g., microtubules and actin filaments). Thus, an in-depth understanding of cytoskeletal filaments mechanics is essential in revealing how cells fulfil their biological functions via cytoskeleton, stimulating the innovative idea in designing biomimetic structure or materials and facilitating to develop novel techniques in disease diagnosis and treatment. This thesis thus focuses on studying the inherent and environmental factors that determine the nanomechanics of cytoskeletal components, i.e., the monomeric feature of cytoskeletal filaments at microscale, the relation between the helical structure and the mechanical properties, and the interaction between the protein filaments and the surrounding environment, such as cytosol, filamentous proteins, electrical fields, etc. The thesis starts with a comprehensive review of the existing cytoskeletal filaments models. It is followed by the molecular structural mechanics models developed for microtubules and actin filaments. Subsequently, the models with monomeric feature were employed to identify the origin of the inter-protofilament sliding and its role in bending and vibration of microtubules. After that, helix structure effects on the mechanics of cytoskeletal filaments were explored. A three-dimensional transverse vibration was reported for microtubules with chiral structures, where the bending axis of the cross-section rotates in an anticlockwise direction and the adjacent half-waves oscillate in different planes. The tension-induced bending was also studied for actin filaments as a result of the helicity. Then, the subcellular environment effect on the filament mechanics was explored. Attempts were also made to reveal the physics of the experimentally observed localized buckling of microtubules and the crucial role of the cross-linker in regulating microtubule stiffness. Also the role of actin-binding proteins in determining the stiffness of actin bundle was examined during the formation of filopodia protrusion. Finally, the studies were carried out for the microtubule vibration excited by the alternating external electric field. Strong correlation was achieved between the tubulin interaction and the frequency shift. Meanwhile, the unique feature of nanoscale microtubule-cytosol interface was studied in detail. Large reduction of the viscous damping of cytosol was achieved in the presence of the nanoscale solid-liquid interface. At the end of the thesis, the contributions of the dissertation research were summarised and remarks were given on future research directions

Artichoke, a novel LRR protein, localizes to the supporting extracelular matrix of ciliated sensory organs in Drosophila and is necessary for its assembly and physiology

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid. Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 20-12-201

Ultrastructural characterization of microtubules at high resolution in the mammalian peripheral nervous system

Mechanotransduction is the ability of living organisms to sense and respond to mechanical forces by converting them into a biological response. In mammals, mechanotransduction is mediated by specialized sensory neurons which are capable of detecting a wide range of mechanical stimuli, relying on the presence of mechanotransducer channels on sensory nerve endings. Surprisingly, little is known about the properties of mechanotransducers in mammals and thus the mechanisms that convert mechanical forces into electrical signals at the peripheral endings of sensory neurons and, especially how the cytoskeleton influences it. In previous work, we have found that mice lacking the -tubulin acetyltransferase Atat1 display a significant decrease in mechanosensitivity across all major fiber types innervating the skin, strongly affecting light touch and pain, with no impact on other sensory modalities1. We also assessed that such a phenotype does not arise from wide-ranging effects on the development, morphology and structure of peripheral sensory neurons but may be caused by the lack of a sub-membrane ring of acetylated -tubulin that somehow sets the mechanical rigidity of the cells, rendering them more resistant to mechanical deformation1. How -tubulin acetylation is capable of setting cellular rigidity remains poorly understood. Here, an ultrastructural analysis on sensory nerve endings was performed to examine whether the lack of -tubulin acetylation affect microtubules (MTs) organization and structure along the sensory neuron axis, from the soma of DRG neurons to their peripheral endings. Superresolution microscopy analysis on DRG neurons shows that the lack of -tubulin acetylation does not affect the overall MTs organization. Moreover, combining high-resolution transmission electron microscopy with image analysis, I investigated MT morphology and distribution in the saphenous nerve from Atat1control and Atat1cKO mice. Our results demonstrated that no major differences were observed between MTs from the Atat1cKO compared to the Atat1control when minor axis, eccentricity, solidity and perimeter length were compared. These results were also confirmed by Cryo-EM observations, suggesting that lack of acetylation does not affect MTs ultrastructure in mammals in the absence of mechanical stress. Finally, the von Frey assay shows that mice lacking Atat1 not only display a profound loss of light touch and pain sensitivity but, in addition, they develop allodynia only beginning at day 21 post SNI (spared nerve injury), suggesting that microtubule acetylation play an important role in hypersensitivity to mechanical stimuli associated with chronic pain