Nerve growth factor-induced neurite outgrowth in PC12 cells involves the coordinate induction of microtubule assembly and assembly-promoting factors

Nerve growth factor (NGF) regulates the microtubule-dependent extension and maintenance of axons by some peripheral neurons. We show here that one effect of NGF is to promote microtubule assembly during neurite outgrowth in PC12 cells. Though NGF causes an increase in total tubulin levels, the formation of neurites and the assembly of microtubules follow a time course completely distinct from that of the tubulin induction. The increases in microtubule mass and neurite extension closely parallel 10- and 20-fold inductions of tau and MAP1, proteins shown previously to promote microtubule assembly in vitro. When NGF is removed from PC12 cells, neurites disappear, microtubule mass decreases, and both microtubule-associated proteins return to undifferentiated levels. These data suggest that the induction of tau and MAP1 in response to NGF promotes microtubule assembly and that these factors are therefore key regulators of neurite outgrowth.     

Cytoplasmic dynein-mediated assembly of pericentrin and gamma tubulin onto centrosomes

Centrosome assembly is important for mitotic spindle formation and if defective may contribute to genomic instability in cancer. Here we show that in somatic cells centrosome assembly of two proteins involved in microtubule nucleation, pericentrin and gamma tubulin, is inhibited in the absence of microtubules. A more potent inhibitory effect on centrosome assembly of these proteins is observed after specific disruption of the microtubule motor cytoplasmic dynein by microinjection of dynein antibodies or by overexpression of the dynamitin subunit of the dynein binding complex dynactin. Consistent with these observations is the ability of pericentrin to cosediment with taxol-stabilized microtubules in a dynein- and dynactin-dependent manner. Centrosomes in cells with reduced levels of pericentrin and gamma tubulin have a diminished capacity to nucleate microtubules. In living cells expressing a green fluorescent protein-pericentrin fusion protein, green fluorescent protein particles containing endogenous pericentrin and gamma tubulin move along microtubules at speeds of dynein and dock at centrosomes. In Xenopus extracts where gamma tubulin assembly onto centrioles can occur without microtubules, we find that assembly is enhanced in the presence of microtubules and inhibited by dynein antibodies. From these studies we conclude that pericentrin and gamma tubulin are novel dynein cargoes that can be transported to centrosomes on microtubules and whose assembly contributes to microtubule nucleation.     

Isolation of microtubule protein from chicken erythrocytes and determination of the critical concentration for tubulin polymerization in vitro and in vivo

Microtubule protein isolated from nucleated chicken erythrocytes was examined with respect to composition and assembly properties to determine its significance in a microtubule bundle called the marginal band. 1) The protein contains greater than 95% tubulin with small amounts of tau polypeptides and no high molecular weight polypeptides. 2) Microtubule assembly in vitro at 37 degrees C is characterized by low levels of nucleation, despite an abundance of ring oligomers at 5 degrees C, as indicated by long lag times, slow assembly rates, and microtubules that are twice as long as brain microtubules assembled under the same conditions. 3) By radioimmunoassay and sodium dodecyl sulfate gel analysis we determined that 0.6% of erythrocyte protein is tubulin of which three-quarters is in a nonextractable form and is associated with the microtubule bundle and the cell cortex. From these values the in vivo concentrations of total tubulin and tubulin dimer subunits are 2.4 and 0.7 mg/ml, respectively. The value of 0.7 mg/ml is close to the range of values of 0.1-0.6 mg/ml for the critical concentration of erythrocyte microtubule protein in vitro, suggesting that the assembly properties of tubulin in vitro and in vivo are similar.     

Possible regulation of the in vitro assembly of bovine brain tubulin by the bovine thioredoxin system

Microtubule assembly in vitro and in vivo is highly sensitive to a variety of sulfhydryl-reactive reagents, raising the question of the possible existence of a physiological sulfhydryl-mediated system for regulating microtubule assembly. However, the specific reagents which have previously been used to inhibit microtubule assembly in vitro are either nonphysiological or, if physiological, effective only at concentrations much higher than their physiological ones. Because of reports of association in vivo between microtubules and the sulfhydryl-reactive proteins thioredoxin and thioredoxin reductase, we decided to examine the interaction in vitro between microtubules and the thioredoxin system, comprising thioredoxin, thioredoxin reductase and NADPH. At pH 6.8, both the mammalian and the Escherichia coli thioredoxin systems inhibited microtubule assembly by 4-35% (19 +/- 9%) by reducing one intra-subunit disulfide bond in the tubulin dimer. The thioredoxin-reducible disulfide of the tubulin dimer remains protected from thioredoxin in the assembled microtubules. Thioredoxin or thioredoxin reductase alone, or together in the absence of NADPH, were incapable of either reducing tubulin or inhibiting microtubule assembly. Microtubules formed from reduced tubulin were found to be stable and morphologically identical to those obtained from native tubulin dimers. Since the components of the thioredoxin system were used at concentrations similar to their physiological ones, our results suggest a potential role of the thioredoxin system in regulation of microtubule assembly in vivo.     

Human chromosomes and centrioles as nucleating sites for the in vitro assembly of microtubules from bovine brain tubulin

Treatment of HeLa cells with Colcemid at concentrations of 0.06-0.10 mug/ml leads to irreversible arrest in mitosis. Colcemid-arrested cells contained few microtubules, and many kinetochores and centrioles were free of microtubule association. When these cells were exposed to microtubule reassembly buffer containing Triton X-100 and bovine brain tubulin at 37 degrees C, numerous microtubules were reassembled at all kinetochores of metaphase chromosomes and in association with centriole pairs. When bovine brain tubulin was eliminated from the reassembly system, microtubules failed to assemble at these sites. Similarly, when EGTA was eliminated from the reassembly system, microtubules failed to polymerize. These results are consistent with other investigations of in vitro microtubule assembly and indicate that HeLa chromosomes and centrioles can serve as nucleating sites for the assembly of microtubules from brain tubulin. Both chromosomes and centrioles became displaced from their C-metaphase configurations during tubulin reassembly, indicating that their movements were a direct result of microtubule formation. Although both kinetochore- and centriole- associated microtubules were assembled and movement occurred, we did not observe direct extension of microtubules from kinetochores to centrioles. This system should prove useful for experimental studies of spindle microtubule formation and chromosome movement in mammalian cells.     

Interactions of tobacco microtubule-associated protein MAP65-1b with microtubules

Tobacco microtubule associated protein (MAP65) (NtMAP65s) constitute a family of microtubule-associated proteins with apparent molecular weight around 65 kDa that collectively induce microtubule bundling and promote microtubule assembly in vitro. They are associated with most of the tobacco microtubule arrays in situ. Recently, three NtMAP65s belonging to the NtMAP65-1 subfamily have been cloned. Here we investigated in vitro the biochemical properties of one member of this family, the tobacco NtMAP65-1b. We demonstrated that recombinant NtMAP65-1b is a microtubule-binding and a microtubule-bundling protein. NtMAP65-1b has no effect on microtubule polymerization rate and binds microtubules with an estimated equilibrium constant of dissociation (K(d)) of 0.57 micro m. Binding of NtMAP65-1b to microtubules occurs through the carboxy-terminus of tubulin, as NtMAP65-1b was no longer able to bind subtilisin-digested tubulin. In vitro, NtMAP65-1b stabilizes microtubules against depolymerization induced by cold, but not against katanin-induced destabilization. The biological implications of these results are discussed.     

Microtubule nucleation from stable tubulin oligomers

Microtubule assembly from purified tubulin preparations involves both microtubule nucleation and elongation. Whereas elongation is well documented, microtubule nucleation remains poorly understood because of difficulties in isolating molecular intermediates between tubulin dimers and microtubules. Based on kinetic studies, we have previously proposed that the basic building blocks of microtubule nuclei are persistent tubulin oligomers, present at the onset of tubulin assembly. Here we have tested this model directly by isolating nucleation-competent cross-linked tubulin oligomers. We show that such oligomers are composed of 10-15 laterally associated tubulin dimers. In the presence of added free tubulin dimers, several oligomers combine to form microtubule nuclei competent for elongation. We provide evidence that these nuclei have heterogeneous structures, indicating unexpected flexibility in nucleation pathways. Our results suggest that microtubule nucleation in purified tubulin solution is mechanistically similar to that templated by gamma-tubulin ring complexes with the exception that in the absence of gamma-tubulin complexes the production of productive microtubule seeds from tubulin oligomers involves trial and error and a selection process.     

Purification, characterization, and assembly properties of tubulin from unfertilized eggs of the sea urchin Strongylocentrotus purpuratus

Tubulin was purified from unfertilized eggs of the sea urchin Strongylocentrotus purpuratus by chromatography of an egg supernatant fraction on DEAE-Sephacel or DEAE-cellulose followed by cycles of temperature-dependent microtubule assembly and disassembly in vitro. After two assembly cycles, the microtubule protein consisted of the alpha- and beta-tubulins (greater than 98% of the protein) and trace quantities of seven proteins with molecular weights less than 55 000; no associated proteins with molecular weights greater than tubulin were observed. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on urea-polyacrylamide gradient gels, the alpha- and beta-tubulins did not precisely comigrate with their counterparts from bovine brain. Two-dimensional electrophoresis revealed that urchin egg tubulin contained two major alpha-tubulins and a single major beta species. No oligomeric structures were observed in tubulin preparations maintained at 0 degrees C. Purified egg tubulin assembled efficiently into microtubules when warmed to 37 degrees C in a glycerol-free polymerization buffer containing guanosine 5'-triphosphate. The critical concentration for assembly of once- or twice-cycled egg tubulin was 0.12-0.15 mg/mL. Morphologically normal microtubules were observed by electron microscopy, and these microtubules were depolymerized by exposure to low temperature or to podophyllotoxin. Chromatography of a twice-cycled egg tubulin preparation on phosphocellulose did not alter its protein composition and did not affect its subsequent assembly into microtubules. At concentrations above 0.5-0.6 mg/mL, a concentration-dependent "overshoot" in turbidity was observed during the assembly reaction. These results suggest that egg tubulin assembles into microtubules in the absence of the ring-shaped oligomers and microtubule-associated proteins that characterize microtubule protein from vertebrate brain.     

Assembly of pure tubulin in the absence of free GTP: effect of magnesium, glycerol, ATP, and the nonhydrolyzable GTP analogues

We describe in vitro microtubule assembly that exhibits, in bulk solution, behavior consistent with the GTP cap model of dynamic instability. Microtubules assembled from pure tubulin in the absence of free nucleotides could undergo one cycle of assembly, but could not sustain an assembly plateau. After the initial peak of assembly was reached and bound E-site GTP hydrolyzed to GDP, the microtubules gradually disassembled. We studied buffer conditions that maximized this disassembly while still allowing robust assembly to take place. While both glycerol and glutamate increased the rate of initial assembly and then slowed disassembly, magnesium promoted initial assembly and, surprisingly, enhanced disassembly. After cooling, a second cycle of assembly was unsuccessful unless GTP or the hydrolyzable GTP analogue GMPCPOP was readded. The nonhydrolyzable GTP analogues GMPPNP and GMPPCP could not support the second assembly cycle in the absence of E-site GTP. Analysis using HPLC found no evidence that GMPPNP, GMPPCP, or ATP could bind to free tubulin, and these nucleotides did not compete with GTP for the E-site. We have, however, demonstrated that the nonhydrolyzable GTP analogues and ATP do have an important effect on microtubule assembly. GMPPNP, GMPPCP, and ATP could each enhance the rate of assembly and stabilize the plateau of assembled microtubules against disassembly, while not binding appreciably to free tubulin. We conclude that these nucleotides, as well as GTP itself, enhance assembly by binding to a site on microtubules that is not present on free, unpolymerized tubulin. We estimate the affinity (KD) of the polymeric site for nucleotide triphosphates to be approximately 10(-4)M.     

Microtubule assembly in cytoplasmic extracts of Xenopus oocytes and eggs

We have investigated the differences in microtubule assembly in cytoplasm from Xenopus oocytes and eggs in vitro. Extracts of activated eggs could be prepared that assembled extensive microtubule networks in vitro using Tetrahymena axonemes or mammalian centrosomes as nucleation centers. Assembly occurred predominantly from the plus-end of the microtubule with a rate constant of 2 microns.min-1.microM-1 (57 s-1.microM-1). At the in vivo tubulin concentration, this corresponds to the extraordinarily high rate of 40-50 microns.min-1. Microtubule disassembly rates in these extracts were -4.5 microns.min-1 (128 s-1) at the plus-end and -6.9 microns.min-1 (196 s-1) at the minus-end. The critical concentration for plus-end microtubule assembly was 0.4 microM. These extracts also promoted the plus-end assembly of microtubules from bovine brain tubulin, suggesting the presence of an assembly promoting factor in the egg. In contrast to activated eggs, assembly was never observed in extracts prepared from oocytes, even at tubulin concentrations as high as 20 microM. Addition of oocyte extract to egg extracts or to purified brain tubulin inhibited microtubule assembly. These results suggest that there is a plus-end-specific inhibitor of microtubule assembly in the oocyte and a plus-end-specific promoter of assembly in the eggs. These factors may serve to regulate microtubule assembly during early development in Xenopus.     

In vivo and in vitro studies on the role of HMW-MAPs in taxol-induced microtubule bundling

The involvement of high molecular weight microtubule-associated proteins (HMW-MAPs) in the process of taxol-induced microtubule bundling has been studied using immunofluorescence and electron microscopy. Immunofluorescence microscopy shows that HMW-MAPs are released from microtubules in granulosa cells which have been extracted in a Triton X-100 microtubule-stabilizing buffer (T-MTSB), unless the cells are pretreated with taxol. 1.0 microM taxol treatment for 48 h results in microtubule bundle formation and the retention of HMW-MAPs in these cells upon extraction with T-MTSB. Electron microscopy demonstrates that microtubules in control cytoskeletons are devoid of surface structures whereas the microtubules in taxol-treated cytoskeletons are decorated by globular particles of a mean diameter of 19.5 nm. The assembly of 3 X cycled whole microtubule protein (tubulin plus associated proteins) in vitro in the presence of 1.0 microM taxol, results in the formation of closely packed microtubules decorated with irregularly spaced globular particles, similar in size to those observed in cytoskeletons of taxol-treated granulosa cells. Microtubules assembled in vitro in the absence of taxol display prominent filamentous extensions from the microtubule surface and center-to-center spacings greater than that observed for microtubules assembled in the presence of taxol. Brain microtubule protein was purified into 6 s and HMW-MAP-enriched fractions, and the effects of taxol on the assembly and morphology of these fractions, separately or in combination, were examined. Microtubules assembled from 6 s tubulin alone or 6 s tubulin plus taxol (without HMW-MAPs) were short, free structures whereas those formed in the presence of taxol from 6 s tubulin and a HMW-MAP-enriched fraction were extensively crosslinked into aggregates. These data suggest that taxol induces microtubule bundling by stabilizing the association of HMW-MAPs with the microtubule surface which promotes lateral aggregation.     

Formation of double-walled microtubules and multilayered tubulin sheets by basic proteins

Some basic proteins enable microtubule protein to form special assembly products in vitro, known as double-walled microtubules. Using histones (H1, core histones) as well as the human encephalitogenic protein to induce the formation of double-walled microtubules, we made the following electron microscopic observations: (1) Double-walled microtubules consist of an "inner" microtubule which is covered by electron-dense material, apparently formed from the basic protein, and by a second tubulin wall. (2) The tubulin of the second wall seems to be arranged as protofilaments, surrounding the inner microtubule in a helical or ring-like manner. (3) The surface of double-walled microtubules lacks the projections of microtubule-associated proteins, usually found on microtubules. (4) In the case of protofilament ribbons (incomplete microtubules), H1 binds exclusively to their convex sides that correspond to the surface of microtubules. Zn2+-induced tubulin sheets, consisting in contrast to microtubules of alternately arranged protofilaments, are covered by H1 on both surfaces. Furthermore, multilayered sheet aggregates appeared. The results indicate that the basic proteins used interact only with that protofilament side which represents the microtubule surface. In accordance with this general principle, models on the structure of double-walled microtubules and multilayered tubulin sheets were derived.     

CLIP-170/tubulin-curved oligomers coassemble at microtubule ends and promote rescues

BACKGROUND: CLIP-170 is a microtubule binding protein specifically located at microtubule plus ends, where it modulates their dynamic properties and their interactions with intracellular organelles. The mechanism by which CLIP-170 is targeted to microtubule ends remains unclear today, as well as its precise effect on microtubule dynamics. RESULTS: We used the N-terminal part of CLIP-170 (named H2), which contains the microtubule binding domains, to investigate how it modulates in vitro microtubule dynamics and structure. We found that H2 primarily promoted rescues (transitions from shrinkage to growth) of microtubules nucleated from pure tubulin and isolated centrosomes, and stimulated microtubule nucleation. Electron cryomicroscopy revealed that H2 induced the formation of tubulin rings in solution and curved oligomers at the extremities of microtubules in assembly conditions. CONCLUSIONS: These results suggest that CLIP-170 targets specifically at microtubule plus ends by copolymerizing with tubulin and modulates microtubule nucleation, polymerization, and rescues by the same basic mechanism with tubulin oligomers as intermediates.     

Mechanical stress induced mechanism of microtubule catastrophes

Microtubules assembled in vitro from pure tubulin can switch occasionally from growing to shrinking states or resume assembly, an unusual behavior termed "dynamic instability of microtubule growth". Its origin remains unclear and several models have been proposed, including occasional switching of the microtubules into energetically unfavorable configurations during assembly. In this study, we have asked whether the excess energy accumulated in these configurations would be of sufficient magnitude to destabilize the capping region that must exist at the end of growing microtubules. For this purpose, we have analyzed the frequency distribution of microtubules assembled in vitro from pure tubulin, and modeled the different mechanical constraints accumulated in their wall. We find that the maximal excess energy that the microtubule lattice can store is in the order of 11 kBT per dimer. Configurations that require distortions up to approximately 20 kBT are allowed at the expense of a slight conformational change, and larger distortions are not observed. Modeling of the different elastic deformations suggests that the excess energy is essentially induced by protofilament skewing, microtubule radial curvature change and inter-subunit shearing, distortions that must destabilize further the tubulin subunits interactions. These results are consistent with the hypothesis that unfavorable closure events may trigger the catastrophes observed at low tubulin concentration in vitro. In addition, we propose a novel type of representation that describes the stability of microtubule assembly systems, and which might be of considerable interest to study the effects of stabilizing and destabilizing factors on microtubule structure and dynamics.     

Taxol binds to polymerized tubulin in vitro

Taxol, a natural plant product that enhances the rate and extent of microtubule assembly in vitro and stabilizes microtubules in vitro and in cells, was labeled with tritium by catalytic exchange with (3)H(2)O. The binding of [(3)H]taxol to microtubule protein was studied by a sedimentation assay. Microtubules assembled in the presence of [(3)H]taxol bind drug specifically with an apparent binding constant, K(app), of 8.7 x 19(-7) M and binding saturates with a calculated maximal binding ration, B(max), of 0.6 mol taxol bound/mol tubulin dimer. [(3)H]Taxol also binds and assembles phosphocellulose-purified tubulin, and we suggest that taxol stabilizes interactions between dimers that lead to microtubule polymer formation. With both microtubule protein and phosphocellulose- purified tubulin, binding saturation occurs at approximate stoichiometry with the tubulin dimmer concentration. Under assembly conditions, podophyllotoxin and vinblastine inhibit the binding of [(3)H]taxol to microtubule protein in a complex manner which we believe reflects a competition between these drugs, not for a single binding site, but for different forms (dimer and polymer) of tubulin. Steady-state microtubules assembled with GTP or with 5’-guanylyl-α,β-methylene diphosphonate (GPCPP), a GTP analog reported to inhibit microtubule treadmilling (I.V. Sandoval and K. Weber. 1980. J. Biol. Chem. 255:6966-6974), bind [(3)H]taxol with approximately the same stoichiometry as microtubules assembled in the presence of [(3)H]taxol. Such data indicate that a taxol binding site exists on the intact microtubule. Unlabeled taxol competitively displaces [(3)H]taxol from microtubules, while podophyllotoxin, vinblastine, and CaCl(2) do not. Podophyllotoxin and vinblastine, however, reduce the mass of sedimented taxol-stabilized microtubules, but the specific activity of bound [(3)H]taxol in the pellet remains constant. We conclude that taxol binds specifically and reversibly to a polymerized form of tubulin with a stoichiometry approaching unity.     

A model for the microtubule organizing activity of the centrosomes and kinetochores in mammalian cells

In this report I review shortly recent evidence on the role of centrosomes and kinetochores in organized microtubule assembly in cells. I arrive at the conclusion that models for these organizing centres must provide an explanation for the following observations:

• 1.1. Both centrosomes and kinetochores induce microtubule assembly in their immediate vicinity at a tubulin concentration below the cytoplasmic critical concentration.
• 2.2. Initially, the newly assembled microtubules are not necessarily anchored to the MTOC's.
• 3.3. The assembly initiating and microtubule stabilizing activity of the MTOC's is abrogated by lowering the critical tubulin concentration in the cytoplasm.
• 4.4. Microtubules attach to the centrosomes with their minus end and to the kinetochores with their plus end.
• 5.5. Interactions between centrosomes and kinetochores or microtubules derived from them are important in guiding microtubule elongation and stabilizing kinetically unfavored microtubule sets (kinetochore microtubules).

Models that are based on the presence of seeds or templates in the MTOC's do not predict observations 2 and 3. Models which conceive MTOC's as sites where microtubules are anchored at their minus end which is consequently capped do not predict observations 3 and 4. We propose a model that explains all the observations summarized above. Both centrosomes and kinetochores induce assembly at low tubulin concentrations by being domains where the critical tubulin concentration is lower than elsewhere in the cytoplasm. Once formed, microtubules may become more or less securely fixed to the MTOC by their minus (centrosome) or plus end (kinetochore). Because this anchoring may occur through lateral bonds between the microtubule surface and a component of the MTOC the end can remain free to add or loose subunits. The model allows cells to build microtubule sets of different polarity and stability. Unlike the seed and minus end capping models it is compatible with mechanisms of intracellular motility based on microtubule treadmilling.     

Microtubule structure at improved resolution.

Microtubule architecture can vary with eukaryotic species, with different cell types, and with the presence of stabilizing agents. For in vitro assembled microtubules, the average number of protofilaments is reduced by the presence of sarcodictyin A, epothilone B, and eleutherobin (similarly to taxol) but increased by taxotere. Assembly with a slowly hydrolyzable GTP analogue GMPCPP is known to give 96% 14 protofilament microtubules. We have used electron cryomicroscopy and helical reconstruction techniques to obtain three-dimensional maps of taxotere and GMPCPP microtubules incorporating data to 14 A resolution. The dimer packing within the microtubule wall is examined by docking the tubulin crystal structure into these improved microtubule maps. The docked tubulin and simulated images calculated from "atomic resolution" microtubule models show tubulin heterodimers are aligned head to tail along the protofilaments with the beta subunit capping the microtubule plus end. The relative positions of tubulin dimers in neighboring protofilaments are the same for both types of microtubule, confirming that conserved lateral interactions between tubulin subunits are responsible for the surface lattice accommodation observed for different microtubule architectures. Microtubules with unconventional protofilament numbers that exist in vivo are likely to have the same surface lattice organizations found in vitro. A curved "GDP" tubulin conformation induced by stathmin-like proteins appears to weaken lateral contacts between tubulin subunits and could block microtubule assembly or favor disassembly. We conclude that lateral contacts between tubulin subunits in neighboring protofilaments have a decisive role for microtubule stability, rigidity, and architecture.     

A microtubule-associated protein from Xenopus eggs that specifically promotes assembly at the plus-end

We have isolated a protein factor from Xenopus eggs that promotes microtubule assembly in vitro. Assembly promotion was associated with a 215-kD protein after a 1,000-3,000-fold enrichment of activity. The 215-kD protein, termed Xenopus microtubule assembly protein (XMAP), binds to microtubules with a stoichiometry of 0.06 mol/mol tubulin dimer. XMAP is immunologically distinct from the Xenopus homologues to mammalian brain microtubule-associated proteins; however, protein species immunologically related to XMAP with different molecular masses are found in Xenopus neuronal tissues and testis. XMAP is unusual in that it specifically promotes microtubule assembly at the plus-end. At a molar ratio of 0.01 mol XMAP/mol tubulin the assembly rate of the microtubule plus-end is accelerated 8-fold while the assembly rate of the minus-end is increased only 1.8-fold. Under these conditions XMAP promotes a 10-fold increase in the on-rate constant (from 1.4 s-1.microM-1 for microtubules assembled from pure tubulin to 15 s-1.microM-1), and a 10-fold decrease in off-rate constant (from 340 to 34 s-1). Given its stoichiometry in vivo, XMAP must be the major microtubule assembly factor in the Xenopus egg. XMAP is phosphorylated during M-phase of both meiotic and mitotic cycles, suggesting that its activity may be regulated during the cell cycle.     

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.     

Comparative studies of microtubule mechanics with two competing models suggest functional roles of alternative tubulin lateral interactions

The dynamic assembly and disassembly of microtubules and the mechanical properties of these polymers are essential for many key cellular processes. Mathematical and computational modeling, especially coupled mechanochemical modeling, has contributed significantly to our understanding of microtubule dynamics. However, critical discrepancies exist between experimental observations and modeling results that need to be resolved before further progress toward a complete model can be made. Open sheet structures ranging in length from several hundred nanometers to one micron have often been observed at the growing ends of microtubules in in vitro studies. Existing modeling studies predict these sheet structures to be short and rare intermediates of microtubule disassembly rather than important components of the assembly process. Atomic force microscopy (AFM) studies also reveal interesting step-like gaps of the force-indentation curve that cannot yet be explained by existing theoretical models. We have carried out computational studies to compare the mechanical properties of two alternative models: a more conventional model where tubulin dimers are added directly into a microtubule lattice, and one that considers an additional type of tubulin lateral interaction proposed to exist in intermediate sheet structures during the microtubule assembly process. The first model involves a single type of lateral interactions between tubulin subunits, whereas the latter considers a second type that can convert to the canonical lateral contact during microtubule closure into a cylinder. Our analysis shows that only the second model can reproduce the AFM results over a broad parameter range. We propose additional studies using different sizes of AFM tips that would allow to unambiguously distinguish the relative validity of the two models.