GTP hydrolysis during microtubule assembly

The GTP cap model of dynamic instability [Mitchison, T., & Kirschner, M.W. (1984) Nature (London) 312, 237] postulates that a GTP cap at the end of most microtubules stabilizes the polymer and allows continuing assembly of GTP-tubulin subunits while microtubules without a cap rapidly disassemble. This attractive explanation for observed microtubule behavior is based on the suggestion that hydrolysis of GTP is not coupled to assembly but rather takes place as a first-order reaction after a subunit is assembled onto a polymer end. Carlier and Pantaloni [Carlier, M., & Pantaloni, D. (1981) Biochemistry 20, 1918] reported a lag of hydrolysis behind microtubule assembly and a first-order rate constant for hydrolysis (kh) of 0.25/min. A lag has not been demonstrated by other investigators, and a kh value that specifies such a slow rate of hydrolysis is difficult to reconcile with reported steady-state microtubule growth rates and frequencies of disassembly. We have looked for a lag using tubulin free of microtubule-associated protein at concentrations of 18.5-74 microM, assembly with and without glycerol, and two independent assays of GTP hydrolysis. No lag was observed under any of the conditions employed, with initial rates of hydrolysis increasing in proportion to rates of assembly. If hydrolysis is uncoupled from assembly, we estimate that kh must be at least 2.5/min and could be much greater, a result that we argue may be advantageous to the GTP cap model. We also describe a preliminary model of assembly coupled to hydrolysis that specifies formation and loss of a GTP cap, thus allowing dynamic instability.     

Equilibrium and kinetic analysis of microtubule assembly in the presence of guanosine diphosphate

In this paper we expand upon a previously reported observation of the effects of GDP on microtubule assembly. A ratio of GDP to GTP of ten (1 mm-GDP and 0.1 mm-GTP) is generally sufficient to completely block microtubule assembly, but only limited depolymerization is induced if GDP is added after assembly has reached a plateau in the presence of GTP. When added during polymerization, GDP arrests further elongation, and greater steady-state levels of assembly are obtained the later the time of addition of GDP. To explain this behavior we examined the rates of assembly and disassembly and the apparent critical concentration (C₀) of tubulin in the presence of GDP. GDP-tubulin polymerizes very slowly as compared to GTP-tubulin, while depolymerization rates, as determined by dilution, are nearly identical in GTP and GDP. The C₀ value calculated from the assembly and disassembly rates in GTP is within experimental error of the C₀ value at steady-state determined directly. In the presence of GDP, however, the C₀ value calculated from rate measurements is at least 60 times greater than that determined by equilibrium analysis. Our results indicate that the net assembly rate in GDP is not a valid measure of the reaction occurring at steady-state. A limited amount of depolymerization may occur upon addition of GDP to microtubules, and this appears to be due to a decrease in the fraction of protein able to participate in the polymerization reaction. The amount of tubulin “inactivated” by GDP is increased by the removal of microtubule-associated proteins. GDP-tubulin will stabilize existing microtubules, even when its polymerization cannot be demonstrated. These results are inconsistent with present models of microtubule assembly, and a new model involving co-operative interaction of microtubule-associated protein-tubulin oligomers at microtubule ends is proposed.     

Light-induced phosphorylation of rod outer segments by guanosine triphosphate.

Significant GTP-dependent protein kinase activity is present in isolated photoreceptors of the retina. No transfer of the γ-³²P group from GTP-γ-³²P to ATP was detected. Although no stimulation of GTP-kinase activity by cyclic GMP or cyclic AMP was observed, exposure of dark-adapted photoreceptors to light resulted in a 46-fold increase in protein phosphorylation.     

Repetitive recycling of guanosine triphosphate cyclohydrolase I for synthesis of dihydroneopterin triphosphate

A procedure for enzymatic production of dihydroneopterin triphosphate is described that allows GTP cyclohydrolase I to be reused repetitively. The reaction takes place in an ultrafiltration cell, and the product is collected in the filtrate, whereas the enzyme remains in the cell to be reused with additional substrate. This is repeated until the enzyme activity drops below a desirable level. The purity of the dihydroneopterin triphosphate is satisfactory for utilization of this compound for studies on enzymes involved in the synthesis of tetrahydrobiopterin and drosopterin. A procedure for purification of dihydroneopterin triphosphate is described that uses C18-silica and silica cartridges.     

Magnesium requirements for guanosine 5'-O-(3-thiotriphosphate) induced assembly of microtubule protein and tubulin

Two conflicting interpretations on the role of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) in microtubule protein and tubulin assembly have been previously reported. One study finds that GTP gamma S promotes assembly while another study reports that GTP gamma S is a potent inhibitor of microtubule assembly. We have examined the potential role of Mg2+ to learn if the conflicting interpretations are due to a metal effect. Turbidity, electron microscopy, and nucleotide binding and hydrolysis were used to analyze the effect of the Mg2+ concentration on GTP gamma S-induced assembly of microtubule protein (tubulin + microtubule-associated proteins) in the presence of buffer +/- 30% glycerol and in buffer with GTP added before or after GTP gamma S. GTP gamma S substantially lowers the Mg2+ concentration required to induce cross-linked or clustered rings of tubulin. These cross-linked rings do not assemble well into microtubules, and GTP only partially restores microtubule assembly. However, taxol will promote GTP gamma S-induced cross-linked rings of microtubule protein to assemble into microtubules. The effect of GTP gamma S on microtubule protein assembly in the presence of Zn2+ with and without added Mg2+ suggests that GTP gamma S also effects the formation of Zn2+-induced sheet aggregates. Purified tubulin was used in assembly experiments with Mg2+, Zn2+, and taxol to better understand GTP gamma S interactions with tubulin. The optimal Mg2+ concentration for assembly of tubulin is lower with GTP gamma S than with GTP.(ABSTRACT TRUNCATED AT 250 WORDS)     

An oscillatory mode for microtubule assembly.

Depending upon the conditions under which polymerization takes place, pure tubulin can assemble into microtubules following either the usual monotonic kinetics or a more complex oscillatory mechanism. When present, these oscillations involve large cyclic changes in the extent of polymer formed before a steady-state is reached. Analysis of the microtubules formed at different times shows that these oscillations involve marked redistribution in both the length and number of microtubules. No significant difference is found between two populations of microtubules corresponding to the same level of assembly, one for which the extent of polymerization will remain stable with time and one for which it will decrease by as much as 90% in the next oscillation. The amplitude of these oscillations is sensitive to changes in the concentrations of protein, nucleotide (GTP, GDP or GMPpNp), magnesium ion or GTP regenerating system. A complete shift from an oscillatory to a monotonic polymerization can be induced by a minor increase in the concentration of free nucleotide, GTP or GDP.     

Mechanism of microtubule assembly. Changes in polymer structure and organization during assembly of sea urchin egg tubulin

Assembly of tubulin, purified from eggs of the sea urchin Stronglyocentrotus purpuratus, was examined at physiological (18 degrees C) and nonphysiological (37 degrees C) temperatures. Critical concentrations for assembly were 0.71 mg/ml at 18 degrees C and 0.21 mg/ml at 37 degrees C. At tubulin concentrations above 1.2 mg/ml at 18 degrees C and 0.5 mg/ml at 37 degrees C, a concentration-dependent "overshoot" in turbidity and in small-angle light scattering was observed; turbidity and scattering increased rapidly to a peak, then decreased asymptotically toward a steady-state value. Quantitative sedimentation analysis revealed that the mass of assembled polymer reached and maintained a constant level during overshoot of turbidity. Changes in the wavelength dependence of turbidity were consistent with the initial formation of sheets of tubulin, followed by conversion of the sheets to microtubules, both at 18 and 37 degrees C. Examination by negative-stain electron microscopy showed that sheetlike structures predominated during the early stages of overshoot assembly, while complete microtubules were present at steady state. Furthermore, measurements of average polymer length revealed that the overshoots in turbidity and in light scattering are unlikely to be caused by polymer length redistribution. Qualitative observations of solution birefringence suggested that the polymer became progressively more aligned during assembly. These results suggest that the turbidity/light-scattering overshoots reflect changes in the form or in the organization of the assembling polymer, or both.     

Purification and properties of guanosine triphosphate cyclohydrolase II from Escherichia coli.

An enzyme that uses GTP as substrate for the formation in stoichiometric quantities of formate, inorganic pyrophosphate, and 2,5-diamino-6-hydroxy-4-(ribosylamino)pyrimidine-5'-phosphate has been purified 2200-fold from extracts of Escherichia coli B. This enzyme is named GTP cyclohydrolase II to distinguish it from a previously studied E. coli enzyme, named GTP cyclohydrolase (and called GTP cyclohydrolase I in this paper), that catalyzes the first of a series of enzymatic reactions leading to the biosynthesis of the pteridine portion of folic acid (Burg, A. W., and Brown, G. M. (1968) J. Biol. Chem. 243, 2349-2358). Some of the properties of GTP cyclohydrolase II are: (a) divalent cations are required for activity (Mg2+ is most effective); (b) its molecular weight, estimated by filtration on Sephadex G-200, is 44,000; (c) the K-m for GTP is 41 mum; (d) its pH optimum is 8.5; and (e) its activity is inhibited by inorganic pyrophosphate, one of the products of the reaction. Compounds not used as substrate are: GDP, GMP, guanosine, dGTP, ATP, ITP, and XTP. Properties a, b, c, and e (above), as well as the nature of the products, distinguish this enzyme from GTP cyclohydrolase I. Since GTP cyclohydrolase II apparently is not concerned with the biosynthesis of folic acid, the possible physiological role of this enzyme in the biosynthesis of riboflavin is considered in the light of the present investigations and the previously published work on riboflavin biosynthesis by other investigators.     

Spatial regulation improves antiparallel microtubule overlap during mitotic spindle assembly

The mitotic spindle plays an essential role in chromosome segregation during cell division. Spindle formation and proper function require that microtubules with opposite polarity overlap and interact. Previous computational simulations have demonstrated that these antiparallel interactions could be created by complexes combining plus- and minus-end-directed motors. The resulting spindles, however, exhibit sparse antiparallel microtubule overlap with motor complexes linking only a nominal number of antiparallel microtubules. Here we investigate the role that spatial differences in the regulation of microtubule interactions can have on spindle morphology. We show that the spatial regulation of microtubule catastrophe parameters can lead to significantly better spindle morphology and spindles with greater antiparallel MT overlap. We also demonstrate that antiparallel microtubule overlap can be increased by having new microtubules nucleated along the length of existing astral microtubules, but this increase negatively affects spindle morphology. Finally, we show that limiting the diffusion of motor complexes within the spindle region increases antiparallel microtubule interaction.     

Inhibition of bacteriophage-Q beta ribonucleic acid polymerase by guanosine triphosphate analogues.

Sixteen compounds related to GTP were evaluated as inhibitors of bacteriophage-Q beta poly(C)-dependent poly(G) polymerase. Non-phosphorylated compounds, including guanine, guanosine and deoxyguanosine, were inactive. Phosphorylated compounds gave significant inhibition at millimolar concentrations. For nucleotides the feature important for inhibition was the 5'-phosphate chain. Four triphosphates, XTP, ITP, 7-methyl-GTP and 2'-O-methyl-GTP, gave 50% inhibition of both the poly(C)- and poly(U2,C)-dependent reactions at concentrations from 0.1 to 5 mM. XTP was 10-fold more potent an inhibitor of the reaction with poly(U2,C) as template. None of these four compounds was able to substitute for GTP as substrate to a significant extent. The most active compound, 2'-O-methyl-GTP, was a competitive inhibitor (Ki = 0.4 mM) of GTP in the poly(C)-dependent reaction.     

Ran-GTP coordinates regulation of microtubule nucleation and dynamics during mitotic-spindle assembly

It was recently reported that GTP-bound Ran induces microtubule and pseudo-spindle assembly in mitotic egg extracts in the absence of chromosomes and centrosomes, and that chromosomes induce the assembly of spindle microtubules in these extracts through generation of Ran-GTP. Here we examine the effects of Ran-GTP on microtubule nucleation and dynamics and show that Ran-GTP has independent effects on both the nucleation activity of centrosomes and the stability of centrosomal microtubules. We also show that inhibition of Ran-GTP production, even in the presence of duplicated centrosomes and kinetochores, prevents assembly of a bipolar spindle in M-phase extracts.     

Copolymerization of two distinct tubulin isotypes during microtubule assembly in vitro

Cells contain multiple tubulin isotypes that are the products of different genes and posttranslational modifications. It has been proposed that tubulin isotypes become segregated into different classes of microtubules each adapted to specific activities and functions. To determine if mixtures of tubulin isotypes segregate into different classes of polymers in vitro, we used immunoelectron microscopy to examine the composition of microtubule copolymers that assembled from mixtures of purified tubulin subunits from chicken brain and erythrocytes, each of which has been shown to exhibit distinct assembly properties in vitro. We observed that (a) the two isotypes coassemble rapidly and efficiently despite the fact that each isotype exhibits its own unique biochemical and assembly properties; (b) at low monomer concentrations the ratio of tubulin isotypes changes along the lengths of elongating copolymers resulting in gradients in immuno-gold labeling; (c) two distinct classes of copolymers each containing a distinct ratio of isotypes assemble simultaneously in the same subunit mixture; and (d) subunits and polymers of different isotypes associate nearly equally well with each other, there being only a slight bias favoring interactions among subunits and polymers of the same isotype. The observations agree with previous studies on the homogeneous distribution of multiple isotypes within cells and suggest that if segregation of isotypes does occur in vivo, it is most likely directed by cell-specific microtubule-associated proteins (MAPs) or specialized intracellular conditions.     

Estimation of the diffusion-limited rate of microtubule assembly.

Microtubule assembly is a complex process with individual microtubules alternating stochastically between extended periods of assembly and disassembly, a phenomenon known as dynamic instability. Since the discovery of dynamic instability, molecular models of assembly have generally assumed that tubulin incorporation into the microtubule lattice is primarily reaction-limited. Recently this assumption has been challenged and the importance of diffusion in microtubule assembly dynamics asserted on the basis of scaling arguments, with tubulin gradients predicted to extend over length scales exceeding a cell diameter, approximately 50 microns. To assess whether individual microtubules in vivo assemble at diffusion-limited rates and to predict the theoretical upper limit on the assembly rate, a steady-state mean-field model for the concentration of tubulin about a growing microtubule tip was developed. Using published parameter values for microtubule assembly in vivo (growth rate = 7 microns/min, diffusivity = 6 x 10(-12) m2/s, tubulin concentration = 10 microM), the model predicted that the tubulin concentration at the microtubule tip was approximately 89% of the concentration far from the tip, indicating that microtubule self-assembly is not diffusion-limited. Furthermore, the gradients extended less than approximately 50 nm (the equivalent of about two microtubule diameters) from the microtubule tip, a distance much less than a cell diameter. In addition, a general relation was developed to predict the diffusion-limited assembly rate from the diffusivity and bulk tubulin concentration. Using this relation, it was estimated that the maximum theoretical assembly rate is approximately 65 microns/min, above which tubulin can no longer diffuse rapidly enough to support faster growth.