We have been studying the adhesion characteristics of microtubules on chemically modified solid substrates with the goal of producing biologically active, patterned microtubules for the examination of two- dimensionally constrained molecular motor traffic in vitro. Microtubules are cylindrical microstructures 25 nm in diameter which self-assemble from tubulin protein in the presence of the nucleotide GTP. In the cell, microtubules act structurally as part of the cytoskeleton and also as substrates for active transport of organelles. This transport is driven by molecular motor proteins, such as kinesin and dynein, which "walk" along the microtubule in the presence of ATP carrying cargo along with them. We have investigated several different organosilane modified solid surfaces as substrates for microtubule and kinesin adhesion in vitro. Microtubules were found to adhere strongly to amino-silane modified surfaces but would not binding to clean glass surfaces. As a result we were able to create selectively adhered patterns of microtubules by immobilizing microtubules on patterns of amino-silane on glass. The microtubules could be aligned on the patterns by doing the immobilization in a fluid-flow field. Kinesin bead movement on the patterned microtubule surfaces was demonstrated, however, bead movement was not unidirectional so the flow-aligned microtubules did not have a constant polarity. To solve this problem, we are now investigating methods for creating patterns of kinesin as a means for creating active surfaces for moving and placing microtubules with controlled polarity. Several organosilane surfaces will be discussed which have shown the ability to selectively adhere kinesin. Finally, since microtubules are quite labile we have been investigating methods for improving their ruggedness and lifetime. We show that chemical cross-linking of microtubules results in rugged microtubules, with lifetimes of days rather than hours, without damaging their ability to act as substrates for ATP driven kinesin movement. This work will be discussed interms of possible applications in micro-machines and microtubule associated protein purification.
Supported by the Office of Naval Research.
David C. Turner, (202) 404-6021, FAX (202) 767-9594