Microtubules, reconstruction

That microtubules exhibit an intrinsic polarity (i.e., with protofilaments running parallel and tubulin protomers regularly arranged in a head-to-tail manner) is obvious from experiments other than those involving image reconstruction. This intrinsic polarity was observed by Rosenbaum and Child (1976) and Witman (1975), who demonstrated biased tubulin addition to microtubules in vitro. This biased addition is a consequence of the nature of tubule-tubulin interactions at the two ends of the microtubule ... 

Fig. 5. 3D EM shows how kinesin and tau bind to microtubules. (A) Reconstruction of a microtubule decorated with kinesin heads (ochre). One kinesin head binds per afi-tubulin heterodimer (grey) and, due to its asymmetric form, can be used to distinguish between the subunits. (B) Inside view of a microtubule that was coassembled with gold-labeled tau and decorated with kinesin heads. The kinesin heads can be seen on the outside through the holes between the protofilaments. The labeled repeat motif of tau binds to the inside face of microtubule. The averaged nanogold density (yellow), which is attached to a repeat motif of tau through a linker, can only be seen near the Taxol binding site of -tubulin, but not on the a subunit (Kar et al, 2003a). The ribbon diagram of the refined zinc-sheet structure is also shown for reference (see Figure 3).

A detailed view of the kinesin-microtubule complex has been obtained by combining high-resolution structures of the individual components from X-ray crystallography (kinesin) and electron diffraction (tubulin Lowe et al., 2001 Nogales et al, 1998) with low-resolution models of kinesin-decorated microtubules obtained by cryoelectron microscopy and image reconstruction (Hirose et al, 1999 Hoenger et al., 2000 Kikkawa et al, 2001 Kozielski et al., 1998 Rice et al., 1999 Skiniotis et al., 2003 Wendt... 

Hoenger, A., Sack, S., Thormahlen, M., Marx, A., Muller, J., Gross, H., and Mandelkow, E. (1998). Image reconstructions of microtubules decorated with monomeric and dimeric kinesins Comparison with x-ray structure and implications for motility. /. CeU Biol. 141,419-430. 

The first structural location of the taxane binding site [42] placed it in the interprotofilament space, thus supporting the biochemical results. However, this changed when the first high resolution 3D structure of the paclitaxel-tubulin complex was solved by electron-crystallography of a two-dimensional zinc-induced tubulin polymer [5]. The fitting of this structure into a three-dimensional reconstruction of microtubules from cryoelectron microscopy allowed a pseudo atomic resolution model of microtubules [43] in which the paclitaxel binding site was placed inside the lumen of the microtubules hidden from the outer solvent. 

Fig. 3 Lateral interactions between protofilaments are shown in this ribbon diagram representing two dimers that have been docked into the three-dimensional reconstruction of a microtubule. The view is from the inside of the MT, with the p monomer at the top. The nucleotides (GDP and GTP) and Taxotere (TAX) are shown as spheres. The primary interactions across the interprotofilament interface involve the M loops from the dimer on the right with helix H3 on the left. (Reprinted with permission from [22]. Copyright 2000 Annual Reviews)...

A EXPERIMENTAL FIGURE 5-49 Deconvolution fluorescence microscopy yields high-resolution optical sections that can be reconstructed into one three-dimensional image. A macrophage cell was stained with fluorochrome-labeled reagents specific for DNA (blue), microtubules (green), and actin microfllaments (red). The series of fluorescent Images obtained at consecutive focal planes (optical... 

Cryo-electron microscopic 3D-reconstruction of an intact microtubule (Resolution ca. 0.8 nm). 

Fig. 21. Model of a late division nucleus of Gyrodinium reconstructed from serial section electron micrographs. The nucleus has divided Into two lobes perforated by bundles of microtubules (represented here by wires) along the axis of division. (From Kubai and RIs. 1969. J. Cell Biol., 40 508-528.)...

In this chapter, we discuss the theory behind SMLM, labeling strategies for the fluorescent probes, describe a workflow and a detailed protocol for fixation and immunostaining of neuronal microtubules, and provide some tips for successful super-resolution imaging, data analysis, and image reconstruction. 

Fig. 3 Representative example of the microtubule network in a proximal branch of a DIV4 hippocampal primary neuron stained with a primary antibody against a-tubulin and visualized via a secondary antibody labeled with Alexa Fluor 647. (a) Widefield overview, (b) Super-resolved SMLM image. Enlarged view of the area in the white teshown in lower panel, (c) Intensity profile of the cross section marked by the white line in (b). Dashed line corresponds to the widefield image, continuous line indicates the profile of the super-resolved image. Note that individual microtubules can only be distinguished in the reconstructed SMLM image...

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