Evaluation of biosensor surfaces for the detection of microtubule perturbation

Abstract

Dual polarization interferometry (DPI) and resonant mirror (RM) methods were used to characterize the growth of microtubules (MTs) on biosensor surfaces. The structure and dynamics of MTs play an important role in cell division and are a target for many anti-cancer drugs. Evidence from DPI demonstrated the growth of MTs on streptavidin-biotinylated-tubulin surfaces from the increase in mass and thickness, with a simultaneous decrease in density. The initial increase in thickness of 0.236 nm/min suggested the elongation of protofilaments before they join laterally to form the MT, where the rate of growth increased to 0.436 nm/min. Continuous mass increases were also observed when tubulin was added to a similar underlying RM surface. Tubulin binding to these surfaces was also temperature dependent, increasing the absolute response with MT stabilizers, while inhibiting binding with destabilizers when temperature was changed from 15 to 37 degrees C. Finally, the initial rates of tubulin assembly (mean+/-SD, n=3) with MT-stabilizer agents were significantly higher at 1.50+/-0.27 and 1.04+/-0.13 arcseconds/s, respectively, compared to 0.37+/-0.11 arcseconds/s for tubulin containing GTP only. In the presence of the MT destabilizers, colchicine and dolastatin 10, the slopes of initial rates were lower than in their absence at 0.05+/-0.01 and 0.27+/-0.08 arcseconds/s, respectively. This provides evidence for the ability of surface-based optical sensors to distinguish between MT stabilizers and destabilizers, while also paving the path to develop other methods to screen for MT-perturbing agents using the same underlying surface engineering.

Figure 1

Resolved mass, thickness, and density of tubulin assembly as assessed with DPI. (A) The increase in mass when streptavidin, biotinylated-tubulin, and tubulin were added indicated specific binding to each of the underlying layers (B) The increase in thickness with decreasing density after the addition of tubulin to biotinylated tubulin suggested the formation of MTs on the surface.

Figure 2

(A) Schematic representation of the formation of a uniform streptavidin layer on a biotin chip followed by the growth of tubulin protofilaments on biotinylated tubulin. (B) As protofilaments continue to grow in length, they join laterally to form MTs.

Figure 3

Binding profile of tubulin to a biotinylated tubulin surface in 1 M MSG buffer (pH 6.6) with GTP at 37 °C using the RM biosensor. The response continuously increases until a buffer wash is performed and the process repeated.

Figure 4

Temperature dependence of tubulin binding to biotinylated tubulin using the RM biosensor. “Sample Channel” represents the observed response of the instrument upon the addition of tubulin to a biotinylated tubulin surface at 15 °C followed by an increase in temperature to 37 °C, where the dotted line indicates the start of temperature change. “Reference Channel” represents the change in response due to the change in temperature. “Difference” represents the subtraction of “Reference Channel” from the “Sample Channel” to give the absolute change in response due to the change in temperature.

Figure 5

The absolute change in response starting from the time of temperature change from 15 to 37 °C. Paclitaxel addition gave a similar response to tubulin with GTP, but remained more stable from MT disassembly afterwards. Colchicine caused a decrease in the response, after an initial increase in response, but completely inhibited binding when it was pre-incubated with tubulin.

Figure 6

Comparison of the slopes of initial rates of MT assembly with various MT-perturbing agents. Paclitaxel and epothilone B, both MT stabilizers, exhibited greater slopes of initial rates in comparison with the DMSO control. MT destabilizers, colchicine and dolastatin, gave lower values than the control. Values are reported as a mean with n=3 and the error bars representing the standard deviation. The pair-wise P values in comparison with the tubulin control were 0.035, 0.005, 0.033, and 0.838 for paclitaxel, epothilone B, colchicine, and dolastatin 10, respectively.