Surface Topography of Microtubule Walls Decorated with Monomeric and Dimeric Kinesin Constructs
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Andreas Hoenger
Abstract
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 α-β-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 α- and β-tubulin). The differences between α- and β-tubulin on the inner surface are stronger than expected from cryo-electron microscopy of unstained microtubules, indicating the existence of tubulin sub-domain-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.
Copyright (c) 2000 by Walter de Gruyter GmbH & Co. KG
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- Alexander J. Varshavsky Felix Hoppe-Seyler Lecturer 2000
- The Ubiquitin System and the N-End Rule Pathway
- Paper of the Year 1999: Award to Igor Stagljar
- A Clockwork Organ
- The Transgeneticists Toolbox: Novel Methods for the Targeted Modification of Eukaryotic Genomes
- Interdependence of Filamentous Actin and Microtubules for Asymmetric Cell Division
- Genetic Analysis of Mammalian Cyclin-Dependent Kinases and Their Inhibitors
- Phosphorylcholine Substituents in Nematodes: Structures, Occurrence and Biological Implications
- Selenium in Biology: Facts and Medical Perspectives
- The Role of Se, Mo and Fe in the Structure and Function of Carbon Monoxide Dehydrogenase
- Molecular Basis for Interactions of the DnaK Chaperone with Substrates
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- Pathway Analysis and Metabolic Engineering in Corynebacterium glutamicum
- Metabolic Networks: a Signal-Oriented Approach to Cellular Models
- Representing and Analysing Molecular and Cellular Function Using the Computer
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- The Effect of Heat Shock on 20S/26S Proteasomes
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