Browsing by Author "Shankar, Karthik"
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Item All wired up: an exploration of the electrical properties of microtubules and tubulin(2020) Kalra, Aarat P.; Eakins, Boden B.; Patel, Sahil D.; Ciniero, Gloria; Rezania, Vahid; Shankar, Karthik; Tuszynski, Jack A.Microtubules are hollow, cylindrical polymers of the protein α, β tubulin, that interact mechanochemically with a variety of macromolecules. Due to their mechanically robust nature, microtubules have gained attention as tracks for precisely directed transport of nanomaterials within lab-on-a-chip devices. Primarily due to the unusually negative tail-like C-termini of tubulin, recent work demonstrates that these biopolymers are also involved in a broad spectrum of intracellular electrical signaling. Microtubules and their electrostatic properties are discussed in this Review, followed by an evaluation of how these biopolymers respond mechanically to electrical stimuli, through microtubule migration, electrorotation and C-termini conformation changes. Literature focusing on how microtubules act as nanowires capable of intracellular ionic transport, charge storage, and ionic signal amplification is reviewed, illustrating how these biopolymers attenuate ionic movement in response to electrical stimuli. The Review ends with a discussion on the important questions, challenges, and future opportunities for intracellular microtubule-based electrical signaling.Item Investigation of the electrical properties of microtubule ensembles under cell-like conditions(2020) Aarat, Kalra P.; Patel, Sahil D.; Bhuiyan, Asadullah; Preto, Jordane; Scheuer, Kyle G.; Mohammed, Usman; Lewis, John D.; Rezania, Vahid; Shankar, Karthik; Tuszynski, Jack A.Microtubules (MTs) are cylindrical polymers composed of the heterodimers of protein α, β- tubulin that play a variety of well-recognised intracellular roles, such as maintaining the shape and rigidity of the cell, aiding in positioning and stabilisation of the mitotic spindle for allowing chromosomal segregation, acting as ‘rails’ for macromolecular transport and forming cilia and flagella for cell movement. Since the tubulin dimer possesses a high negative electric charge of ~23e and a large intrinsic high dipole moment of approximately 1750 D [1,2], MTs have been implicated in electrically-mediated biological roles [3,4,5,6]. They have been modelled as nanowires capable of enhancing ionic transport [7,8], and simulated to receive and attenuate electrical oscillations [4,9,10,11]. In solution, MTs have been shown to align with applied electric fields [2,12,13,14,15,16]. Recently, MTs have also been modelled as the primary cellular targets for low-intensity (1–2 V), intermediate-frequency (100–300 kHz) electric fields termed TTFields (tumour-treating electric fields) that inhibit cancer cell proliferation, in particular glioma [17,18,19]. Indeed, MTs have been reported to decrease buffer solution resistance [12,13], leading to a conductance peak at frequencies close to the TTField regime [20]. While these studies show that MTs are highly sensitive to external electric fields, answers to the questions ‘How do MTs effect a solution’s capacitance?’ and ‘What is the capacitance of a single MT?’ are still elusive and crucial to the determination of the dielectric properties of living cells. The tubulin concentration in mammalian cells varies in the micromolar range (~10–25 μM) [21,22]. In vitro, polymerizing tubulin at such high concentrations can lead to the formation of entangled networks, confounding quantification of the individual MT response to electric fields. Electro-rotation, di-electrophoresis and impedance spectroscopy are thus performed using low concentrations of tubulin, in the nanomolar regime, to enable robust observation of individual MTs.Item Modeling microtubule counterion distributions and conductivity using the Poisson-Boltzmann equation(2021) Eakins, Boden B.; Patel, Sahil D.; Kalra, Aarat P.; Rezania, Vahid; Shankar, KarthikMicrotubules are highly negatively charged proteins which have been shown to behave as bio-nanowires capable of conducting ionic currents. The electrical characteristics of microtubules are highly complicated and have been the subject of previous work; however, the impact of the ionic concentration of the buffer solution on microtubule electrical properties has often been overlooked. In this work we use the non-linear Poisson Boltzmann equation, modified to account for a variable permittivity and a Stern Layer, to calculate counterion concentration profiles as a function of the ionic concentration of the buffer. We find that for low-concentration buffers ([KCl] from 10 μM to 10 mM) the counterion concentration is largely independent of the buffer's ionic concentration, but for physiological-concentration buffers ([KCl] from 100 to 500 mM) the counterion concentration varies dramatically with changes in the buffer's ionic concentration. We then calculate the conductivity of microtubule-counterion complexes, which are found to be more conductive than the buffer when the buffer's ionic concentrations is less than ≈100 mM and less conductive otherwise. These results demonstrate the importance of accounting for the ionic concentration of the buffer when analyzing microtubule electrical properties both under laboratory and physiological conditions. We conclude by calculating the basic electrical parameters of microtubules over a range of ionic buffer concentrations applicable to nanodevice and medical applications.Item A nanometric probe of the local proton concentration in microtubule-based biophysical systems(2022) Kalra, Aarat P.; Eakins, Boden B.; Vagin, Sergei I.; Wang, Hui; Patel, Sahil D.; Winter, Philip; Aminpour, Maral; Lewis, John D.; Rezania, Vahid; Shankar, Karthik; Scholes, Gregory D.; Tuszynski, Jack A.; Rieger, Bernhard; Meldrum, AlkiviathesWe show a double-functional fluorescence sensing paradigm that can retrieve nanometric pH information on biological structures. We use this method to measure the extent of protonic condensation around microtubules, which are protein polymers that play many roles crucial to cell function. While microtubules are believed to have a profound impact on the local cytoplasmic pH, this has been hard to show experimentally due to the limitations of conventional sensing techniques. We show that subtle changes in the local electrochemical surroundings cause a double-functional sensor to transform its spectrum, thus allowing a direct measurement of the protonic concentration at the microtubule surface. Microtubules concentrate protons by as much as one unit on the pH scale, indicating a charge storage role within the cell via the localized ionic condensation. These results confirm the bioelectrical significance of microtubules and reveal a sensing concept that can deliver localized biochemical information on intracellular structures.Item Response to alternating electric fields of tubulin dimers and microtubule ensembles in electrolytic solutions(2017) Santelices, Iara B.; Friesen, Douglas E.; Bell, Clayton; Hough, Cameron M.; Xiao, Jack; Kalra, Aarat P.; Kar, Piyush; Freedman, Holly; Rezania, Vahid; Lewis, John D.; Shankar, Karthik; Tuszynski, Jack A.Microtubules (MTs), which are cylindrical protein filaments that play crucial roles in eukaryotic cell functions, have been implicated in electrical signalling as biological nanowires. We report on the small-signal AC (“alternating current”) conductance of electrolytic solutions containing MTs and tubulin dimers, using a microelectrode system. We find that MTs (212 nM tubulin) in a 20-fold diluted BRB80 electrolyte increase solution conductance by 23% at 100 kHz, and this effect is directly proportional to the concentration of MTs in solution. The frequency response of MT-containing electrolytes exhibits a concentration-independent peak in the conductance spectrum at 111 kHz (503 kHz FWHM that decreases linearly with MT concentration), which appears to be an intrinsic property of MT ensembles in aqueous environments. Conversely, tubulin dimers (42 nM) decrease solution conductance by 5% at 100 kHz under similar conditions. We attribute these effects primarily to changes in the mobility of ionic species due to counter-ion condensation effects, and changes in the solvent structure and solvation dynamics. These results provide insight into MTs’ ability to modulate the conductance of aqueous electrolytes, which in turn, has significant implications for biological information processing, especially in neurons, and for intracellular electrical communication in general.Item Revealing and attenuating the electrostatic properties of tubulin and its polymers(2021) Kalra, Aarat P.; Patel, Sahil D.; Eakins, Boden B.; Riddell, Saralyn; Kumar, Pawan; Winter, Philip; Preto, Jordane; Carlson, Kris W.; Lewis, John D.; Rezania, Vahid; Tuszynski, Jack A.; Shankar, KarthikTubulin is an electrostatically negative protein that forms cylindrical polymers termed microtubules, which are crucial for a variety of intracellular roles. Exploiting the electrostatic behavior of tubulin and microtubules within functional microfluidic and optoelectronic devices is limited due to the lack of understanding of tubulin behavior as a function of solvent composition. This work displays the tunability of tubulin surface charge using dimethyl sulfoxide (DMSO) for the first time. Increasing the DMSO volume fractions leads to the lowering of tubulin's negative surface charge, eventually causing it to become positive in solutions >80% DMSO. As determined by electrophoretic mobility measurements, this change in surface charge is directionally reversible, i.e., permitting control between −1.5 and + 0.2 cm2 (V s)−1. When usually negative microtubules are exposed to these conditions, the positively charged tubulin forms tubulin sheets and aggregates, as revealed by an electrophoretic transport assay. Fluorescence-based experiments also indicate that tubulin sheets and aggregates colocalize with negatively charged g-C3N4 sheets while microtubules do not, further verifying the presence of a positive surface charge. This study illustrates that tubulin and its polymers, in addition to being mechanically robust, are also electrically tunable.Item Revealing and attenuating the electrostatic properties of tubulin and microtubules(2020) Kalra, Aarat P.; Patel, Sahil D.; Winter, Philip; Wang, Hui; Carlson, Kris W.; Rezania, Vahid; Lewis, John; Meldrum, Al; Shankar, Karthik; Tuszynski, Jack A.Cancer treatment modalities such as chemotherapy and radiation therapy involve several side effects including an increased risk of getting cancer a second time. TTField (Tumour-treating field) therapy is a novel cancer-treatment modality that has attained U.S FDA approval for treatment of Glioblastoma Multiforme. TTFields are low intensity (1-2 V/cm), intermediate frequency (100-300 kHz) electric fields that have been shown to drastically lower tumor growth. While the only side-effects of TTField exposure are mild skin rashes, the exact mechanism of how TTFields act is not well understood. Models of TTFields posit that they target α, β- tubulin, a highly negatively charged (−50e), high dipole moment (∼1750 D) protein. Tubulin forms hollow cylindrical polymers termed microtubules, which form bundles that are crucial for mitosis and macromolecular transport. When exposed to TTFields, tubulin is expected to re-align and spatially relocate in response, inhibiting microtubule growth and interfering with mitosis. Using photolithography for microelectrode fabrication, we exposed unpolymerized tubulin containing solutions to AC electric fields and measured solution conductance. Interestingly, while we found that the presence of microtubules increased solution ionic conductance with a peak at TTField-like frequencies, the presence of unpolymerized tubulin reduced ionic conductance. Next, we used a parallel-plate electrode setup to compare the capacitance of solutions containing unpolymerized tubulin to those containing microtubules at identical physiological tubulin concentrations and ionic strengths. We found that while the presence of microtubules increased solution capacitance appreciably, the presence of unpolymerized tubulin did not. We are presently quantifying the chemical nature of the counterionic cloud around tubulin using a pH-sensitive fluorophore and Dynamic Light Scattering. Our results, in addition to displaying the significance of the tubulin polymerization state on the solution’ dielectric properties, also indicate that TTFields may target ion-tubulin interactions to inhibit tumour growth.Item Tubulin and microtubules as molecular targets for ttfield therapy(2019) Kalra, Aarat P.; Patel, Sahil D.; Bhuiyan, Asadullah; Rezania, Vahid; Lewis, John; Shankar, Karthik; Tuszynski, Jack A.TTField (Tumor-treating field) therapy utilizes low intensity intermediate frequency AC electric fields to reduce the spread of cancer. While it has attained FDA approval for the treatment of recurrent glioblastoma multiforme, the exact molecular targets of TTField therapy are not well understood. Microtubules are pipe-like polymers of the highly charged (–31 e) and strongly dipolar (dipole moment 1666 D) protein, α, β- tubulin. Studies on the electrical properties of microtubules have recently gained interest, with them being modelled as molecular targets of TTFields. Here, we experimentally show that while tubulin polymerized into microtubules leads to an increase in solution capacitance, unpolymerized tubulin has no appreciable effect. To the best of our knowledge, we present the first experimental quantification of the capacitance of a 20 μm-long microtubule. Using these results, we calculate the resonant frequency of a microtubule meshwork in a cell-like environment to be in the TTField regime. Our results utilize high ionic strength solutions and cell-like concentrations of tubulin to show the potential of microtubules as the targets of TTField action and as intracellular charge-storage devices. We conclude with a hypothesis of an electrically-tunable cell, where the dielectric properties of the cytoskeleton alter local and global charge storage and transport.