Surface Topography of Microtubule Walls Decorated with Monomeric and Dimeric Kinesin Constructs

Abstract

The surface topography of opened-up microtubule walls (sheets) decorated with monomeric and dimeric kinesin motor domains was investigated by freezedrying and unidirectional metal shadowing. Electron microscopy of surface-shadowed specimens produces images with a high signal/noise ratio, which enable a direct observation of surface features below 2 nm detail. Here we investigate the inner and outer surface of microtubules and tubulin sheets with and without decoration by kinesin motor domains. Tubulin sheets are flattened walls of microtubules, keeping lateral protofilament contacts intact. Surface shadowing reveals the following features: (i) when the microtubule outside is exposed the surface relief is dominated by the bound motor domains. Monomeric motor constructs generate a strong 8 nm periodicity, corresponding to the binding of one motor domain per ␣-␤-tubulin heterodimer. This surface periodicity largely disappears when dimeric kinesin motor domains are used for decoration, even though it is still visible in negatively stained or frozen hydrated specimens. This could be explained by disorder in the binding of the second (loosely tethered) kinesin head, and/or disorder in the coiled-coil tail. (ii) Both surfaces of undecorated sheets or microtubules, as well as the inner surface of decorated sheets, reveal a strong 4 nm repeat (due to the periodicity of tubulin monomers) and a weak 8 nm repeat (due to slight differences between ␣-and ␤-tubulin). The differences between ␣-and ␤-tubulin on the inner surface are stronger than expected from cryo-electron microscopy of unstained microtubules, indicating the existence of tubulin subdomain-specific surface properties that reflect 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 surface shadowing.

Surface-decoration of Microtubules by Human Tau

“…The kinesin decoration pattern and the surface features of co-decorated sheets are essentially identical with that of tubulin sheets decorated exclusively with motor domains; in both cases a strong axial 8 nm repeat is observed, corresponding to a single motor complexed to one ab-tubulin dimer. 20 Similar to the assemblies found with K19, large extended sheets appeared, indicating an increased lateral stability between protofilaments due to the presence of tau. These features are observed both with surface-shadowed and iceembedded samples ( Figure 7B) and they are slightly different from MT -kinesin complexes, which often reveal long extended single protofilaments, or very small bundles of protofilaments, e.g.…”

Processive movement of single kinesins on crowded microtubules visualized using quantum dots

“…Intermediate binding ratios were also reported under certain conditions (Hoenger et al, 2000;Skiniotis et al, 2003), indicating that probably the details of the binding kinetics are important for the exact binding stoichiometry (Vilfan et al, 2001). Therefore, we estimate that in our experiment with AMP-PNP at the maximum kinesin density probably some of the kinesin dimers are attached via only one head to the microtubule, while some others are attached with both heads corresponding to a maximum binding ratio being at most 1:1 and at least 1:2.…”

“…The presence of a fluorescence label on the crowding agent had the advantage of allowing us to measure the degree of crowdedness on the microtubule by TIRF microscopy under exactly the same steady-state conditions under which the runs of quantum dot-labelled kinesins were observed. An important first result was the observation that the maximum density of kinesins under steady-state conditions, that is, when kinesins move along the microtubule, was considerably lower as compared to a static situation in the presence of AMP-PNP ( Figure 2B), where densities of either one kinesin dimer per tubulin dimer or per two tubulin dimers have been reported in cryo-electron microscopy experiments (Arnal and Wade, 1998;Hirose et al, 1999;Hoenger et al, 2000;Skiniotis et al, 2003). Therefore, our binding curves indicate that the maximum steady-state density of kinesin-GFPs walking on a microtubule in the presence of ATP is at most one kinesin dimer per two tubulin dimers and at least one kinesin dimer per four tubulin dimers.…”

A Structural Analysis of the Interaction between ncd Tail and Tubulin Protofilaments

“…Sheets of that magnitude cannot be observed in preparations of plain MTs due to their intrinsic instability. 19,20 Some areas are ordered enough to reveal diffraction spots, corresponding to a lateral spacing of parallel protofilaments of 5.4(^0.14) nm (boxed area) like in regular tubulin sheets. 17,18 The 3D reconstruction of microtubules decorated with NT6…”