Richard H. Wade

New data on the microtubule surface lattice

Summary— The in vitro polymerisation of tubulin is a remarkable example of protein self‐assembly in thet 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 photofilaments. 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.

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.

S-Trityl-L-cysteine Is a Reversible, Tight Binding Inhibitor of the Human Kinesin Eg5 That Specifically Blocks Mitotic Progression

Human Eg5, responsible for the formation of the bipolar mitotic spindle, has been identified recently as one of the targets of S-trityl-l-cysteine, a potent tumor growth inhibitor in the NCI 60 tumor cell line screen. Here we show that in cell-based assays S-trityl-l-cysteine does not prevent cell cycle progression at the S or G2 phases but inhibits both separation of the duplicated centrosomes and bipolar spindle formation, thereby blocking cells specifically in the M phase of the cell cycle with monoastral spindles. Following removal of S-trityl-l-cysteine, mitotically arrested cells exit mitosis normally. In vitro, S-trityl-l-cysteine targets the catalytic domain of Eg5 and inhibits Eg5 basal and microtubule-activated ATPase activity as well as mant-ADP release. S-Trityl-l-cysteine is a tight binding inhibitor (estimation of Ki,app <150 nm at 300 mm NaCl and 600 nm at 25 mm KCl). S-Trityl-l-cysteine binds more tightly than monastrol because it has both an ∼8-fold faster association rate and ∼4-fold slower release rate (6.1 μM–1 s–1 and 3.6 s–1 for S-trityl-l-cysteine versus 0.78 μM–1 s–1 and 15 s–1 for monastrol). S-Trityl-l-cysteine inhibits Eg5-driven microtubule sliding velocity in a reversible fashion with an IC50 of 500 nm. The S and d-enantiomers of S-tritylcysteine are nearly equally potent, indicating that there is no significant stereospecificity. Among nine different human kinesins tested, S-trityl-l-cysteine is specific for Eg5. The results presented here together with the proven effect on human tumor cell line growth make S-trityl-l-cysteine a very attractive starting point for the development of more potent mitotic inhibitors.

Characterization of microtubule protofilament numbers: How does the surface lattice accommodate?☆

Frozen-hydrated specimens of microtubules assembled in vitro were observed by cryoelectron microscopy. Specimens were of both pure tubulin, and of microtubule protein isolated by three cycles of assembly and disassembly. It is shown that the characteristic image contrast of individual microtubules allows the microtubule protofilament number to be determined unambiguously. Microtubules with 13, 14 and 15 protofilaments are observed to coexist in specimens prepared under various assembly conditions. Confirmation of these results is obtained by observations of thin sections of pelleted samples fixed and stained using the glutaraldehyde/tannic acid technique. Images of individual microtubules show both characteristic contrast profiles across their width and typical variations of these profiles along their length. The profiles across the images indicate the protofilament number of the microtubule. The lengthwise variations indicate how the protofilaments are aligned with respect to the microtubule axis giving what has previously been called a supertwist. In 13 protofilament microtubules the protofilaments are paraxial. In 14 and 15 protofilament microtubules, the protofilaments are skewed with respect to the microtubule axis. The skew is greater for the 15 protofilament case than for 14 protofilaments. The skew allows the extra protofilaments to be accommodated by the surface lattice. These results should also be relevant to situations in vivo.

The crystal structure of the minus-end-directed microtubule motor protein ncd reveals variable dimer conformations

BACKGROUND The kinesin superfamily of microtubule-associated motor proteins are important for intracellular transport and for cell division in eukaryotes. Conventional kinesins have the motor domain at the N terminus of the heavy chain and move towards the plus end of microtubules. The ncd protein is necessary for chromosome segregation in meiosis. It belongs to a subfamily of kinesins that have the motor domain at the C terminus and move towards the minus end of microtubules. RESULTS The crystal structure of dimeric ncd has been obtained at 2.9 A resolution from crystals with the C222(1) space group, with two independent dimers per asymmetric unit. The motor domains in these dimers are not related by crystallographic symmetry and the two ncd dimers have significantly different conformations. An alpha-helical coiled coil connects, and interacts with, the motor domains. CONCLUSIONS The ncd protein has a very compact structure, largely due to extended interactions of the coiled coil with the head domains. Despite this, we find that the overall conformation of the ncd dimer can be rotated by as much as 10 degrees away from that of the twofold-symmetric archetypal ncd. The crystal structures of conventional kinesin and of ncd suggest a structural rationale for the reversal of the direction of movement in chimeric kinesins.

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.

The network structure of a steroid/cyclohexane physical gel

Abstract Steroid/cyclohexane gels have been observed by the freeze-etching replication electron microscopy technique. Stereo images reveal clearly the three-dimensional gel network which has a mesh size of about 300 nm and consists of long filaments mostly 9.1 nm in diameter in agreement with previous SANS results. A 4.6-nm-diameter protofilament is observed. The 9.1-nm filaments appear to be made from two or more twisted protofilaments. There are two principal types of contact zone between the main filaments in which the filaments are either in parallel juxtaposition or fuse to form a filament of diameter less than the sum of the two incoming filaments.

Structural links to kinesin directionality and movement.

The kinesin motor proteins generate directional movement along microtubules and are involved in many vital processes, including cell division, in eukaryotes. The kinesin superfamily is characterized by a conserved motor domain of ∼320 residues. Dimeric constructs of N and C class kinesins, with the motor domains at opposite ends of the heavy chain, move towards microtubule plus and minus ends, respectively. Their crystal structures differ mainly in the region linking the motor domain core to the α-helical coiled coil dimerization domain. Chimeric kinesins show that regions outside of the motor domain core determine the direction of movement and mutations in the linker region have a strong effect on motility. Recent work on chimeras and mutants is discussed in a structural context giving insights to possible molecular mechanisms of kinesin directionality and motility.

Electron microscope structural study of modified fibrin and a related modified fibrinogen aggregate.

The structure of proteolytically modified fibrin and a closely related modified fibrinogen aggregate have been studied by analysis of electron microscope images. For both structures, we propose a model that consists of double-stranded, 2-fold helical protofibrils, which are associated laterally to form ordered fibrils, with a C222 space group: a = 44.0 nm, b = c = 9.4 nm. Each fibril is 80 nm or less in diameter, and twists along its length in a right-handed sense, with a pitch from 7 to 12 times the molecular length. The fibrils associate laterally to form bundles, which tend to twist in a left-handed sense, with a pitch of the order of 40 times the molecular length. The specific volume of modified fibrin calculated from this model is 3.9 A3 per dalton, which is comparable to the specific volume of 3.6 A3 per dalton for modified fibrinogen crystals but is lower than the 6 A3 per dalton determined for fibrin from light-scattering experiments. Comparison of our electron microscope results with X-ray and neutron diffraction data suggest a similar, but less well-ordered, structure for native fibrin, with a smaller fibril, approximately 18.4 nm wide, consisting of eight protofibrils.

Monotonic versus oscillating microtubule assembly: a cryo‐electron microscope study

Depending on the free GTP concentration, microtubules can assemble following either a monotonic or an oscillatory mode. We have used cryoelectron microscopy to compare the tubulin assemblies characteristic of each polymerization pathway. We focus on the first assembly peak. At this particular time point, despite their strikingly different subsequent evolution, both systems are similar with regard to the extent of tubulin polymerization and to the microtubule length distribution. The present study shows that whilst the observed microtubule structures are the same in both systems, the oscillatory system shows quantities of closed ring‐like tubulin oligomers, far in excess of those observed in the monotonic system. Furthermore, the conversion of the oscillating system to a monotonic one by GTP addition during the first oscillation is accompanied by a marked decrease in the number of rings. Based on these results we propose that the GTP dependent step which governs microtubule oscillations is the opening of inactive tubulin oligomers.