Browsing by Author "Winter, Philip"
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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 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.