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R H Wade

Publications and source records attributed to R H Wade.

At least 37 records · Page 2Linked to original sources
PubMed1998

Nucleotide-dependent conformations of the kinesin dimer interacting with microtubules.

BACKGROUND: Kinesins are crucial to eukaryotic cells. They are a superfamily of motor proteins that use ATP hydrolysis to move along microtubules. Many of these motors are heterotetramers with two heavy and two light chains. The heavy chain has a globular motor domain that interacts with microtubules and shows a similar sequence throughout the family. Compared with myosin and dynein, kinesin provides a 'simple' model for understanding molecular motors. RESULTS: Electron cryomicroscopy and three-dimensional reconstruction methods have been used to investigate microtubule-kinesin dimer complexes in different nucleotide states. Three-dimensional maps were obtained in the presence of 5'-adenylylimidodiphosphate (AMP-PNP), ADP-AIF4, ADP and apyrase. In all cases, kinesin has one attached and one free head per tubulin heterodimer. The attached heads appear very similar whereas the free heads show distinct conformations and orientations depending on their nucleotide states. CONCLUSIONS: The kinesin dimer is likely to undergo considerable conformational changes during its ATP hydrolysis cycle. In all nucleotide states, the kinesin dimer attaches to a microtubule using one motor domain with the other motor domain hanging free. Only the free domain changes conformation in the presence of different nucleotides, suggesting that it, or the region linking both motor domains to the coiled coil, is the determinant of directionality. These results give some structural clues as to how kinesin moves along microtubules and we describe possible models of kinesin movement based on currently available data.

Adenosine Triphosphate↗
PubMed1998

Organisation and structure of microtubules and microtubule-motor protein complexes.

We present a short overview of the current status of work on the organisation and structure of microtubules and of microtubule-motor protein complexes. At present there is great interest in obtaining structural information that can help us to understand the movement of the kinesin family of microtubule associated molecular motors. Using electron cryomicroscopy and image reconstruction methods three dimensional maps of microtubule-motor complexes have been obtained in the presence of different nucleotides. We address a number of principles involved in different aspects of this work.

Animals↗
PubMed1998

Pre-operative planning in orthopaedics: a study of surgeons' opinions.

Pre-operative planning is important in orthopaedic surgery in both trauma and elective orthopaedic surgery. We know of no study into surgeons attitudes to pre-operative planning. A postal questionnaire was used to ascertain attitudes to planning. 94% of consultants and 100% of trainees felt that planning was important but half, respectively, routinely planned fracture treatment. Only 24% of trainees and 44% consultants traced fracture configurations from radiographs. 88% of trainees and 91% of consultants felt that planning should be undertaken more often with 94% of consultants expressing a view that trainees should do more planning. Pre-operative planning is good practice and from our study we have shown both trainees and consultants feel it should be undertaken more often.

Attitude of Health Personnel↗
PubMed1997

Tomography without tilt: three-dimensional imaging of microtubule/motor complexes.

Usually structures such as microtubules are supposed to have surface lattices built from families of continuous helices, giving electron micrographs that can be analyzed by helical diffraction theory with a view to obtaining three-dimensional reconstructions. In the case of microtubules the helical surface lattice may be interrupted by discontinuities, called seams, in which case the usual helical reconstruction approach is no longer applicable. Even so, by virtue of their "superhelical" protofilaments, microtubules are still helical structures and we use this feature to treat a microtubule image as a set of projections equivalent to images obtained in a single axis tilt series. The main thrust of this article is to discuss how to obtain images and image parameters best suited to a tomographic approach to three-dimensional reconstruction. The method is tested by comparing helical and back-projection reconstructions of appropriate microtubules both with and without surface lattice decoration by kinesin family motor proteins. Tomographic reconstruction gives an independent demonstration that in vitro assembled microtubules have a B-type surface lattice. We show that 3-start, 15-protofilament microtubules have a seam, whereas 4-start microtubules have no seam and possess complete helical symmetry. Monomer motor domains attach to the outer ridge of the protofilaments and extend along the protofilament toward the plus end.

Animals↗
PubMed1997

Microtubule structure and dynamics.

The study of microtubules always manages to surprise and fascinate us, and it has done so yet again over the past year as significant progress has been made in the areas of microtubule nucleation, growth and structural polarity. Microtubule nucleation has been the subject of publications that show the involvement of gamma-tubulin-containing complexes as nucleating templates in the microtubule-organizing centre. It is unclear how this nucleation is compatible with microtubule growth, which appears to take place by an unusual, and perhaps unique, process involving sheet-like extensions that continuously close into tubes as growth proceeds. The related, and longstanding, problem is that of the relationship between tubulin dimer structure and microtubule polarity. This problem appears to be solved. A number of approaches have converged to suggest that the tubulin dimer is organized with beta-tubulin pointing towards the microtubule fast-growing plus end and with alpha-tubulin towards the minus end. Specific decoration with kinesin monomers shows that all microtubules examined to date are basically organized as B-lattices.

Microtubules↗
PubMed1996

Three-dimensional structure of functional motor proteins on microtubules.

BACKGROUND: Kinesins are a superfamily of motor proteins that use ATP hydrolysis to fuel movement along microtubules and participate in many crucial phases of the eukaryotic cell cycle. Usually these motors are heterotetramers of two heavy and two light chains, and have globular motor domains on the two heavy chains. Most kinesins move towards the microtubule 'plus end', but some, such as ncd (nonclaret disjunctional protein), move in the opposite direction. Heavy chain dimers produced by overexpression are viable motors. RESULTS: In order to establish whether the opposite directionality of kinesin and ncd dimers is related to notable conformational differences, we have used electron cryo-microscopy and three-dimensional reconstruction methods to investigate the structure of kinesin and ncd dimers attached to microtubules in the presence of AMP-PNP (5'-adenylylimidodiphosphate), a nonhydrolyzable ATP analogue. Three-dimensional maps of the motor-microtubule complexes show the motors to have one unattached, and one attached head per tubulin dimer. The polarity of the reconstructions was determined for each individual microtubule. Attachment occurs on the crest of a protofilament at the end of the tubulin dimer that points towards the plus end of the microtubule. The attached head extends over the next tubulin molecule along the protofilament. The unattached heads of kinesin and ncd have distinctly different conformations. CONCLUSIONS: The attached heads of kinesin and ncd appear to be similar and to interact with the same region of the plus end-oriented tubulin subunits. The free heads, however, are quite different, which suggests that directionality could be determined by differences in the dimer conformations. Work is in progress to obtain three-dimensional maps in the presence of different nucleotides with the aim of understanding how these motors move along microtubules.

Animals↗
PubMed1996

Determination of microtubule polarity by cryo-electron microscopy.

BACKGROUND: Microtubules are tubular polymers of tubulin dimers, which are arranged head-to-tail in protofilaments that run lengthwise along the microtubules, giving them an overall structural polarity. Many of the functions of microtubules depend on this polarity, including directed intracellular transport and chromosome segregation during mitosis. The determination of microtubule polarity for lengthwise views of microtubules observed by electron microscopy has not previously been possible. Here, we present methods for directly determining the polarity of individual microtubules imaged by cryo-electron microscopy. RESULTS: When observed in vitreous ice by cryo-electron microscopy, microtubules with skewed protofilaments show arrowhead moiré patterns. We have used centrosome nucleated microtubules to relate the directionality of the moiré patterns to microtubule polarity. We show that the arrowheads point towards the plus end of microtubules with protofilaments having a right-handed skew, and towards the minus end of microtubules with protofilaments having a left-handed skew. We describe two methods for determining the handedness of the protofilament skew. The first method uses two or more tilted views. The second method involves analysis of the diffraction patterns of the microtubule images. CONCLUSIONS: It is now possible to determine directly the polarity of in vitro assembled microtubules from cryo-electron micrographs. This will be helpful in a number of types of studies, including studies of the three-dimensional structure of microtubules interacting with motor proteins, as knowledge of the polarity of the microtubule is essential to understand motor directionality.

Cold Temperature↗
PubMed1995

How does taxol stabilize microtubules?

BACKGROUND: The antimitotic agent taxol is an important new drug for the treatment of certain cancers. It blocks the cell cycle in its G1 or M phases by stabilizing the microtubule cytoskeleton against depolymerization. RESULTS: We have used electron cryomicroscopy and image analysis to investigate the structure of microtubules assembled in vitro, and found that their fine structure was sensitive to the presence of taxol. The conformation of the microtubule lattice depended on whether the drug was added before or after assembly. The structure of preassembled microtubules changed only slightly when taxol was added; a larger change was observed when microtubules were assembled in the presence of the drug. In both cases, taxol-containing microtubules were stable over many days at, or below, room temperature. CONCLUSIONS: As in another recent investigation using guanylyl-(alpha,beta)-methylene-diphosphonate (a non-hydrolyzable GTP analogue), microtubule stabilization with taxol is accompanied by a conformational change in the microtubule surface lattice and, implicitly, in the tubulin dimer. We speculate that a general mechanism may underlie the stabilization of microtubules by different agents.

Antineoplastic Agents, Phytogenic↗
PubMed1995

Quaternary structure of casein kinase 2. Characterization of multiple oligomeric states and relation with its catalytic activity.

The structure-activity relationship of casein kinase 2 (CK2) was examined with regard to its previously reported property to self-aggregate in vitro. Sedimentation velocity and electron microscopy studies showed that the purified kinase exhibited four major, different oligomeric forms in aqueous solution. This self-polymerization was a reproducible and fully reversible process, highly dependent upon the ionic strength of the medium, suggesting that electrostatic interactions are mostly involved. At high salt concentrations (e.g. 0.5 M NaCl), CK2 appears as spherical moieties with a 18.7 +/- 1.6 nm average diameter, roughly corresponding to the alpha 2 beta 2 protomer, as deduced by measurements of the Stokes radius and by light scattering studies. At lower ionic strength (e.g. 0.2 M NaCl), the protomers associate to form ring-like structures with a diameter (averaging 36.6 +/- 2.1 nm) and Stokes radius indicating that they are most likely made of four circularly associated alpha 2 beta 2 protomers. At 0.1 M NaCl, two additional polymeric structures were visualized: thin filaments (16.4 +/- 1.4 nm average), as long as 1 to 5 microns, and thick and shorter filaments (28.5 +/- 1.6 nm average). Examination of the molecular organization of CK2 under different catalytic conditions revealed that the ring-like structure is the favored conformation adopted by the enzyme in the presence of saturating concentrations of substrates and cofactors. During catalysis, well-known cofactors like MgCl2 or spermine are the main factors governing the stabilization of the active ring-like structure. On the other hand, inhibitory high salt concentrations promote the dissociation of the active ring-like structure into protomers. Such observations suggest a strong correlation between the ring-like conformation of the enzyme and optimal specific activity. Thus, CK2 may be considered as an associating-dissociating enzyme, and this remarkable property supports the hypothesis of a cooperative and allosteric regulation of the kinase in response to appropriate regulatory ligands possibly taking place in intact cells.

Amino Acid Sequence↗
PubMed1995

Toward understanding the structure and interactions of microtubules and motor proteins.

To obtain an overall three-dimensional picture of the interaction between microtubules and the motor proteins of the kinesin family it will be necessary to take account of both atomic resolution structures obtained by X-ray crystallography and medium resolution reconstructions obtained by electron cryomicroscopy. We examine the problems associated with obtaining the required structural information from electron micrographs of vitreous ice-embedded microtubules decorated with motor domains. We find that the minus-end directed motor, ncd, decorates microtubules with an 80 A periodicity as for kinesin. Our theoretical analysis and experiments with ncd illustrate the difficulty in determining unambiguously the surface lattice organization by diffraction analysis of micrographs. 3D reconstructions of decorated microtubules are required to accurately locate the motor domains. Helical diffraction theory is not usually applicable because microtubules are cylindrical structures that rarely have complete helical symmetry. We propose using a back-projection method based on the long pitch helices formed by individual protofilaments. Model reconstructions show that this approach is feasible.

Computer Simulation↗
PubMed1995

Structural changes accompanying GTP hydrolysis in microtubules: information from a slowly hydrolyzable analogue guanylyl-(alpha,beta)-methylene-diphosphonate.

We have used cryoelectron microscopy to try to understand the structural basis for the role of GTP hydrolysis in destabilizing the microtubule lattice. We have measured a structural difference introduced into microtubules by replacing GTP with guanylyl-(alpha,beta)-methylene-diphosphonate (GMPCPP). In a stable GMPCPP microtubule lattice, the moiré patterns change and the tubulin subunits increase in size by 1.5 A. This information provides a clue to the role of hydrolysis in inducing the structural change at the end of a microtubule during the transition from a growing to a shrinking phase.

Animals↗
PubMed1994

Cryoelectron microscopy of macromolecular complexes.

Although there are many macromolecular complexes which play extremely important roles in biology, and despite continued progress in X-ray crystallographic and NMR methods, it is still very difficult to obtain atomic level structural information about such large assemblies. It is now clear that a powerful approach is to combine structural information obtained at different levels. Cryoelectron microscopy of frozen-hydrated samples together with computer based 3-D reconstruction can give structural information at the quaternary level. This can then be combined with atomic level structures of individual components, obtained by X-ray crystallography or NMR to build-up a detailed picture of the overall architecture of the complexes and of the interactions between the components. In our laboratory we are particularly interested in developing the complementarity between the different structural approaches. The aim of this short review is to briefly present our ongoing work using cryoelectron microscopy of vitreous ice-embedded samples as a quantitative tool to investigate the assembly and organization of two important biological structures, namely, microtubules and viruses, in particular the bluetongue virus.

Animals↗
PubMed1993

The molecular structure of corticotropin-induced secreted protein, a novel member of the thrombospondin family.

CISP (corticotropin-induced secreted protein) is a secreted protein recently purified in our laboratory from the conditioned medium of ACTH-treated bovine adrenocortical cells. Partial amino acid sequencing of CISP revealed homology with thrombospondins (TSPs), a family of adhesive proteins and in particular with TSP2. We report here the characterization of the molecular structure of CISP. Analysis of CISP by polyacrylamide gel electrophoresis in the absence or presence of SDS indicated an apparent molecular mass approximately equal to 600 kDa for the unreduced protein and an apparent molecular mass of 195 kDa after reduction by 2-mercaptoethanol. The sedimentation coefficient of CISP determined by ultracentrifugation on sucrose gradients was shifted from 9.7 S in the absence to 5.7 S in the presence of 2-mercaptoethanol. These data are consistent with a trimeric organization of the CISP molecule in which 195-kDa monomers would be linked together by disulfide bonds. The trimeric structure of CISP could be observed by rotary shadowing/electron microscopy, where CISP appeared to be composed of three equally electron-dense nodules and of a fourth nodule formed by the close association of three smaller fragments. The overall size of the molecule was 60 nm. We also observed that CISP is sulfated and glycosylated. Using glycosylation inhibitors, we could determine that CISP is synthesized as a 175-kDa core protein, is then matured into a 190-kDa high-mannose form and secreted as a 195-kDa mature protein. Inhibition of sulfation by chlorate did not prevent CISP secretion, whereas inhibition of glycosylation by tunicamycin blocked it. Taken together, these data indicate that the TSP2-related CISP molecule presents both structural and functional properties very similar to those of TSP1. CISP differs greatly, however, from TSP1 by the inducibility of its synthesis by cAMP.

Adrenal Cortex↗
PubMed1992

Lattice defects in microtubules: protofilament numbers vary within individual microtubules.

We have used cryo-electron microscopy of vitrified specimens to study microtubules assembled both from three cycle purified tubulin (3x-tubulin) and in cell free extracts of Xenopus eggs. In vitro assembled 3x-tubulin samples have a majority of microtubules with 14 protofilaments whereas in cell extracts most microtubules have 13 protofilaments. Microtubule polymorphism was observed in both cases. The number of protofilaments can change abruptly along individual microtubules usually by single increments but double increments also occur. For 3x-tubulin, increasing the magnesium concentration decreases the proportion of 14 protofilament microtubules and decreases the average separation between transitions in these microtubules. Protofilament discontinuities may correspond to dislocation-like defects in the microtubule surface lattice.

Animals↗
PubMed1992

Low pH deforms the influenza virus envelope.

Influenza virus membrane fusion is induced by low pH, which triggers an irreversible conformational change in the viral haemagglutinin (HA). The result of this change is the extrusion of the HA fusion peptide, after which it may act in the fusion of virus and endosomal membranes. Here we describe electron microscopic observations on low pH-treated virus after negative staining or cryo-electron microscopy of virus in the frozen hydrated state. The results indicate a destabilization of the virus membrane at low pH that can be reversed by returning the pH to neutral.

Hydrogen-Ion Concentration↗
PubMed1991

New data on the microtubule surface lattice.

The in vitro polymerisation of tubulin is a remarkable example of protein self-assembly in that several closely related microtubule structures coexist on the polymerisation plateau. Unfixed and unstained in vitro assembled microtubules were observed in vitreous ice by cryo-electron microscopy. New results are reported that considerably extend previous observations [47]. In ice, microtubule images have a distinctive contrast related to the number and skew of the protofilaments. The microtubules observed have from twelve to seventeen protofilaments. Comparison with thin sections of pelleted material allows a direct identification of images from microtubules with thirteen, fourteen and fifteen protofilaments. A surface lattice accommodation mechanism, previously proposed to explain how variable numbers of protofilaments can be incorporated into the basic thirteen protofilament structure, is described in detail. Our new experimental results are shown to be in overall agreement with the theoretical predictions. Only thirteen protofilament microtubules have unskewed protofilaments, this was confirmed by observations on axoneme fragments. The results imply that the microtubule surface lattice is based on a mixed packing which combines features of the standard A and B lattices.

Animals↗