Surface topography of microtubule walls decorated with monomeric and dimeric kinesin constructs

The surface topography of opened-up microtubule walls (sheets) decorated wi th monomeric and dimeric kinesin motor domains was investigated by freeze-d rying and unidirectional metal shadowing. Electron microscopy of surface-sh adowed specimens produces images with a high signal/noise ratio, which enab le a direct observation of surface features below 2 nm detail. Here we inve stigate the inner and outer surface of microtubules and tubulin sheets with and without decoration by kinesin motor domains. Tubulin sheets are flatte ned walls of microtubules, keeping lateral protofilament contacts intact. S urface shadowing reveals the following features: (i) when the microtubule o utside is exposed the surface relief is dominated by the bound motor domain s. Monomeric motor constructs generate a strong 8 nm periodicity, correspon ding to the binding of one motor domain per beta -tubulin heterodimer. This surface periodicity largely disappears when dimeric kinesin motor domains are used for decoration, even though it is still visible in negatively stai ned or frozen hydrated specimens, This could be explained by disorder in th e binding of the second (loosely tethered) kinesin head, and/or disorder in the coiled-coil tail. (ii) Both surfaces of undecorated sheets or microtub ules, as well as the inner surface of decorated sheets, reveal a strong 4 n m repeat (due to the periodicity of tubulin monomers) and a weak 8 nm repea t (due to slight differences between alpha- and beta -tubulin). The differe nces between alpha- and beta -tubulin on the inner surface are stronger tha n expected from cryo-electron microscopy of unstained microtubules, indicat ing the existence of tubulin subdomain-specific surface properties that ref lect the surface corrugation and hence metal deposition during evaporation. The 16 nm periodicity visible in some negatively stained specimens (caused by the pairing of cooperatively bound kinesin dimers) is not detected by s urface shadowing.