Outer Surface Of Microtubule Research Articles

Detailed Structure of Single, Unaveraged Microtubules Revealed by Electron Microscopy Tomography

Tubulin carboxyterminal tail peptides are short (10-20 amino acids) acidic, unstructured peptides that vary significantly in sequence between tubulin isotypes and are exposed on the outer surface of microtubules. These peptides have, so far, eluded direct visualization. Electron microscopy tomography is used here to analyze stabilized microtubules embedded in a tungsten-based negative stain previously found to resist sustained electron beam irradiation. Sample stability is compatible with high electron doses, permitting collection of 142 images at various tilt angles with high signal-to-noise ratio. The resulting tomogram is reconstructed without the need of averaging multiple samples. After further optimizing collection of data and signal elaboration, images reveal the three dimensional structure of subunits as well as previously unresolved small stalks on the outer surface of the microtubule. Inspection of virtual sections demonstrates that the stalks are ∼ 2.5 nm long and ∼ 1 nm wide (at half length). Stalks protrude intermittently every ∼ 4 nm along the microtubule from the outer face of each monomer. These protrusions are well resolved from background in virtual sections perpendicular to the long axis of microtubule. These protrusions are likely the carboxyterminal peptides of tubulin. This hypothesis is supported by comparison with results obtained in the same way from microtubules whose C-terminals have been cleaved enzymatically. In summary, it is possible to resolve single peptides by amplitude contrast in a conventional electron microscope without the need of averaging data from multiple microtubules.

A New Mechanism of Kinesin Molility: Conversion of Random Motion to Directional Motion with Ratchet Structure

A dynamic model for the motility of kinesin along microtubule (MT), including stochastic-force generation and step formation, is proposed. In our model, force is generated by the impulse from the collision between the randomly moving long-chain stalk and the ratchet-shaped outer surface of microtubule. The force produces greater tension on the trailing head than on the leading head, and stochastically triggers the trailing head to flip over the leading head. Kinesin progresses along the protofilament in a non-conventional hand-over-hand fashion. We demonstrate the applicability of the dynamic model in a real vibrating simulation system including (a) ratchet structures similar to the outer surface of MT, (b) a bead chain connected to two heads, similarly to the stalk of the real kinesin motor, and (c) the attractive interaction between the heads of the simulated kinesin and microtubule. Most of the dynamical and statistical features of the motility of kinesin such as the step formation, the distribution of the dwell time, and the functional dependence of the velocity on the loading force (V-F relation) are reproduced in our simulating system. It is also found that the dependence of the velocity on the amplitude of vibration changes with the length of the stalk domain. Based on this finding, we propose an experiment to measure the dependence of the velocity of the motion of kinesin along MT as a function of the ATP concentration for the mutants of kinesin with various lengths of the stalk domain. Because the stalk domain has no effect on the motility of kinesin in the hand-over-hand model, such an experiment can discriminate between the hand-over-hand model and the dynamic model.

Identification of sites of phosphorylation by G-protein-coupled receptor kinase 2 in beta-tubulin.

G-protein-coupled receptor kinase 2 (GRK2) is known to specifically phosphorylate the agonist-bound forms of G-protein-coupled receptors (GPCRs). This strict specificity is due at least partly to activation of GRK2 by agonist-bound GPCRs, in which basic residues in intracellular regions adjacent to transmembrane segments are thought to be involved. Tubulin was found to be phosphorylated by GRK2, but it remains unknown if tubulin can also serve as both a substrate and an activator for GRK2. Purified tubulin, phosphorylated by GRK2, was subjected to biochemical analysis, and the phosphorylation sites in beta-tubulin were determined to be Thr409 and Ser420. In addition, the Ser444 in beta III-tubulin was also indicated to be phosphorylated by GRK2. The phosphorylation sites in tubulin for GRK2 reside in the C-terminal domain of beta-tubulin, which is on the outer surface of microtubules. Pretreatment of tubulin with protein phosphatase type-2A (PP2A) resulted in a twofold increase in the phosphorylation of tubulin by GRK2. These results suggest that tubulin is phosphorylated in situ probably by GRK2 and that the phosphorylation may affect the interaction of microtubules with microtubule-associated proteins. A GST fusion protein of a C-terminal region of beta I-tubulin (393-445 residues), containing 19 acidic residues but only one basic residue, was found to be a good substrate for GRK2, like full-length beta-tubulin. These results, together with the finding that GRK2 may phosphorylate synuclein and phosducin in their acidic domains, indicate that some proteins with very acidic regions but without basic activation domains could serve as substrates for GRK2.

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

The surface topography of opened-up microtubule walls (sheets) decorated with monomeric and dimeric kinesin motor domains was investigated by freeze-drying 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 alpha-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 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 alpha- and beta-tubulin). The differences between alpha- and beta-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.

Polysaccharides connected with microtubules in the lipotubuloids of Ornithogalum umbellatum L.

The presence of polysaccharides connected with microtubules in the lipotubuloids of Ornithogalum umbellatum L. was detected by the silver hexamine method and Ruthenium Red staining. Polysaccharides are localized on the outer surface of microtubules. Staining of polysaccharides by Ruthenium Red causes an increase in the microtubule walls thickness by 2.5 nm and in their diameter by 4.5 nm.