"All other approaches to achieving reliable guiding of microtubule transporters along predefined paths are costly and labour-intensive because they involve modifications of the surface topography [e.g. creating channels]," Stefan Diez of the Max Planck Institute of Molecular Cell Biology and Genetics told nanotechweb.org. "Our method demonstrates the possibility of getting patterned motors on planar surfaces without the need for topographical changes."
To carry out the technique, Diez and colleagues first attached the motor proteins to microtubules, which have a highly oriented regular structure. Microtubules are elongated protein structures found in cells. They take part in a number of cell processes, including vesicle transport by acting as a track for motor proteins, which move along the microtubule carrying cargo. The structures generally have a diameter of around 25 nm and are a few microns to a few millimetres long.
The researchers tried both a "biotemplated stamping" and a "biotemplated binding" approach for creating patterns of kinesin-1 and nonclaret disjunctional (Ncd) motor protein. In the stamping method, they attached kinesin-1 to template microtubules in the absence of adenosine triphosphate (ATP). Then they adsorbed the microtubules onto a surface so that the kinesin molecules bound to the surface with their motor domains pointing away from it. Finally, adding ATP caused the microtubule to "walk" off the track of kinesins, leaving them behind.
In the binding approach, the team added a template microtubule to the surface. They bound kinesin-1 or Ncd to the template using linker molecules or the second microtubule binding site in their tail domain, so that the proteins had their motor domains pointing away from the surface.
Once in place, the tracks of motor proteins were able to transport biotinylated microtubules with cargo such as streptavidin-coated quantum dots attached.
A key advantage of the technique is that the heads (motor domains) of the motor proteins point away from the surface – the researchers say this can't be achieved by direct protein patterning techniques such as microcontact printing or dip-pen nanolithography. Also, the absence of channels removes constraints on the size of cargo that the microtubules are able to transport.
These biotemplated nanopatterning techniques could have applications in the simple set-up of microtubule guiding and transport systems, in vitro investigations into the action of motor proteins and replicating complex subcellular machineries in synthetic environments. "The highly oriented deposition of proteins on surfaces will allow novel sensing and detection applications," said Diez.
Now the team plans to explore other ways of structuring planar surfaces with nanometre-wide protein patterns, for microtubule guiding and other applications. "These range all the way from cell-adhesion studies to molecular sorting systems," said Diez.
The researchers reported their work in Nano Letters.