Evaluation of biosensor surfaces for the detection of microtubule perturbation.
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.
Daghestani
Hikmat N
HN
Department of Structural Biology, University of Pittsburgh, BST3 10017, 3501 Fifth Ave, Pittsburgh, PA 15213, USA.
Fernig
David G
DG
Day
Billy W
BW
D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002417:Cattle; D007368:Interferometry; D008027:Light; D008870:Microtubules / Q000378:metabolism*; D011485:Protein Binding; D013499:Surface Properties; D013696:Temperature; D014404:Tubulin / Q000378:metabolism*
D014404:Tubulin