Jeune-Smith Yolaine, Agarwal Ashutosh, Hess Henry
Department of Materials Science and Engineering, University of Florida, USA.
J Vis Exp. 2010 Nov 3(45):2006. doi: 10.3791/2006.
Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport of cargo. While kinesins tail domain binds to a variety of cargoes, kinesins head domains utilize the chemical energy stored in ATP molecules to step along the microtubule lattice. The long, stiff microtubules serve as tracks for long-distance intracellular transport. These motors and filaments can also be employed in microfabricated synthetic environments as components of molecular shuttles. In a frequently used design, kinesin motors are anchored to the track surface through their tails, and functionalized microtubules serve as cargo carrying elements, which are propelled by these motors. These shuttles can be loaded with cargo by utilizing the strong and selective binding between biotin and streptavidin. The key components (biotinylated tubulin, streptavidin, and biotinylated cargo) are commercially available. Building on the classic inverted motility assay, the construction of molecular shuttles is detailed here. Kinesin motor proteins are adsorbed to a surface precoated with casein; microtubules are polymerized from biotinylated tubulin, adhered to the kinesin and subsequently coated with rhodamine-labeled streptavidin. The ATP concentration is maintained at subsaturating concentration to achieve a microtubule gliding velocity optimal for loading cargo. Finally, biotinylated fluorescein-labeled nanospheres are added as cargo. Nanospheres attach to microtubules as a result of collisions between gliding microtubules and nanospheres adhering to the surface. The protocol can be readily modified to load a variety of cargoes such as biotinylated DNA, quantum dots or a wide variety of antigens via biotinylated antibodies.
细胞已经进化出复杂的分子机制，如驱动蛋白运动蛋白和微管丝，以支持细胞内货物的主动运输。虽然驱动蛋白的尾部结构域与多种货物结合，但驱动蛋白的头部结构域利用ATP分子中储存的化学能量沿着微管晶格移动。长而硬的微管作为细胞内长距离运输的轨道。这些马达和细丝也可以作为分子穿梭器的组件应用于微制造的合成环境中。在一种常用的设计中，驱动蛋白马达通过其尾部锚定在轨道表面，功能化的微管作为货物运载元件，由这些马达推动。这些穿梭器可以利用生物素和链霉亲和素之间强而选择性的结合来装载货物。关键组件（生物素化微管蛋白、链霉亲和素和生物素化货物）可商购。基于经典的反向运动分析，这里详细介绍分子穿梭器的构建。驱动蛋白运动蛋白吸附到预先用酪蛋白包被的表面；微管由生物素化微管蛋白聚合而成，附着在驱动蛋白上，随后用罗丹明标记的链霉亲和素包被。ATP浓度保持在亚饱和浓度，以实现最适合装载货物的微管滑动速度。最后，添加生物素化的荧光素标记纳米球作为货物。由于滑动的微管与附着在表面的纳米球之间的碰撞，纳米球附着在微管上。该方案可以很容易地修改，以通过生物素化抗体装载各种货物，如生物素化DNA、量子点或各种抗原。
