Apparent stiffness of vimentin intermediate filaments in living cells and its relation with other cytoskeletal polymers

The cytoskeleton is a complex network of interconnected biopolymers intimately involved in the generation and transmission of forces. Several mechanical properties of microtubules and actin filaments have been extensively explored in cells. In contrast, intermediate filaments (IFs) received comparatively less attention despite their central role in defining cell shape, motility and adhesion during physiological processes as well as in tumor progression. Here, we explored relevant biophysical properties of vimentin IFs in living cells combining confocal microscopy and a filament tracking routine that allows localizing filaments with ~20 nm precision. A Fourier-based analysis showed that IFs curvatures followed a thermal-like behavior characterized by an apparent persistence length (lp*) similar to that measured in aqueous solution. Additionally, we determined that certain perturbations of the cytoskeleton affect lp* and the lateral mobility of IFs as assessed in cells in which either the microtubule dynamic instability was reduced or actin filaments were partially depolymerized. Our results provide relevant clues on how vimentin IFs mechanically couple with microtubules and actin filaments in cells and support a role of this network in the response to mechanical stress.Fil: Smoler, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Coceano, Giovanna. Royal Institute of Technology; SueciaFil: Testa, Ilaria. Royal Institute of Technology; SueciaFil: Bruno, Luciana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Calculo. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Calculo; ArgentinaFil: Levi, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentin

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

Dynamics of T-Junction Solution Switching Aimed at Patch Clamp Experiments.

Solutions exchange systems are responsible for the timing of drug application on patch clamp experiments. There are two basic strategies for generating a solution exchange. When slow exchanges are bearable, it is easier to perform the exchange inside the tubing system upstream of the exit port. On the other hand, fast, reproducible, exchanges are usually performed downstream of the exit port. As both strategies are combinable, increasing the performance of upstream exchanges is desirable. We designed a simple method for manufacturing T-junctions (300 μm I.D.) and we measured the time profile of exchange of two saline solutions using a patch pipette with an open tip. Three factors were found to determine the timing of the solution switching: pressure, travelled distance and off-center distance. A linear relationship between the time delay and the travelled distance was found for each tested pressure, showing its dependence to the fluid velocity, which increased with pressure. The exchange time was found to increase quadratically with the delay, although a sizeable variability remains unexplained by this relationship. The delay and exchange times increased as the recording pipette moved away from the center of the stream. Those increases became dramatic as the pipette was moved close to the stream borders. Mass transport along the travelled distance between the slow fluid at the border and the fast fluid at the center seems to contribute to the time course of the solution exchange. This effect would be present in all tubing based devices. Present results might be of fundamental importance for the adequate design of serial compound exchangers which would be instrumental in the discovery of drugs that modulate the action of the physiological agonists of ion channels with the purpose of fine tuning their physiology

Time course of the solution exchange at two propelling pressures for a constant travelled distance of 20 mm at the optimal position.

<p>(A) Complete exchange. From top to bottom: pinch valve voltage command; measured response at 0.4 bar; idem at 0.1 bar. The current was measured while holding the pipette at -100 mV. Dashed line indicates t_on and t₉₀. (B) Detail of the forward exchange. Measured current (black) and fitted equation (red) at 0.4 bar; idem at 0.1 bar; fitted equation. Red circles indicate the t₁₀ and t₉₀. Delay time and exchange time (ET) are defined as t₁₀-t_on and t₉₀-t₁₀, respectively.</p

The effect of the off-center distance.

<p>The upper diagram is a schematic representation of a cross section of the stream. Three different recording positions are shown (red circle 1 is limit zone, red circle 2 is optimal position and red circle 3 is a suboptimal position). 1; 2; and 3 are the records of each position and dashed red line indicates the maximum response at all positions.</p

T-junction manufacture and operation.

<p>(A) Silicone tubing and punch. A 0.5 mm interval ruler is apparent on the bottom. (B) A hole is drilled perpendicular to the central hole. (C) After inserting a polyethylene tubing the T-junction is completed. (D) General device. <i>I</i>. N₂ tank. <i>II</i>. Filter <i>III</i>. Pressurized reservoir of solution. <i>IV</i>. 3-way solenoid pinch valves. <i>V</i>. A 24 V custom made valve driver. <i>VI</i>. Merger system of two T-junctions staked in XYZ translator. <i>VII</i>. Open tip recording pipette. <i>VIII</i>. Patch clamp amplifier. <i>IX</i>. A multifunction data acquisition connected to PC. (E) Schematic representation of experimental configuration. (F) Image of experimental configuration. The image was taken using a web camera coupled to the setup microscope. (G) Operation of the solution switching. A pulse of test solution (violet) was inserted into the control solution (pink).</p

Time course of the solution exchange slowed down away from the optimal position.

<p>Exchanges were measured at 0, 15 and 30 μm away from the optimal position for 0.1 or 0.4 bar and for distances of 1, 20, 51, or 70 mm. The small line above each scale bar indicates the scale bar of the previous distance.</p

Fraction of response that is described by an error function (α).

<p>Table represents the variation of α with both pressure and distance traveled for interface. Data of α was listed as mean ± SD (n = 4) and were statistically analyzed by two-way ANOVA. The interaction between two factors were significant (P < 0.0001) and F = 25.07. The comparisons were made using Bonferroni post-test against the 1 mm column values with a significance level of P < 0.05 (*) and P < 0.001 (***).</p><p>Fraction of response that is described by an error function (α).</p

Sagittal section of the time course of the solution exchange.

<p>The vertical yellow bar represents 350 μm; ticks indicate the position of each recorded trace used to interpolate the data. The yellow horizontal traces indicate the command voltage applied to the valve, (notice that the time increases from right to left. In this way the plot gives the right impression that the center goes faster than the borders). Red arrows indicate the ton. The diffusive nature of the profile is clearly increasing with distance and lowering pressure.</p

Time course of the forward exchange at different distances (horizontal panels) and pressures (line color) at the optimal position.

<p>Experimental data is indicated on black; fits to the equation (same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133187#pone.0133187.g002" target="_blank">Fig 2</a>) are indicated on color. (A) The delay between the time of valve activation (arrow, t_on) and the exchange increased with both distance and pressure. (B) Expanded time scales showed that the fit covered the data in all cases. The correlation coefficients for 1 mm were the following: 0.986, 0.980 and 0.974 for 0.1, 0.2 and 0.4 bar respectively. For 20 mm, they were: 0.984; 0.988 and 0.987 while for 51 mm: 0.985, 0.975 and 0.982. Finally for 70 mm were: 0.983, 0.982 and 0.980.</p